Antifungal Effects of some Plant Essential Oils against Alternaria alternata (Fr.) Keissl. and Aspergillus niger van Tiegh. from Grapes

Author: Bina Bhattarai and Sanjay Kumar Jha

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Abstract

The essential oil of six plants viz. Murraya koenigii (L.) Spreng. Eucalyptus citriodora Hook. Artemisia indica Willd. Cinnamomum camphora (L.) J. Presl., Cinnamomum tamala (Buch.-Ham.) Nees and Eberm and Lantana camara L. were assessed in vitro for their antifungal activity against Alternaria alternata (Fr.) Keissl. and Aspergillus niger van Tiegh., causing postharvest rots in grapes. The test fungi were isolated from infected grapes. The essential oils were extracted through hydro distillation process using Clevenger’s apparatus. For screening of antifungal activity, treatments at concentration of 20, 40, 80, 160 and 320 µl/ml and controls were set to determine percentage inhibition of mycelial growth of two test fungi using poisoned food technique. All the tested oils exhibited significant antifungal effect (p<0.05) over tested fungi. Among six essential oils, Cinnamomum camphora showed the most effective antifungal activity against Aspergillus niger which inhibited the mycelial growth by 81.58 % and 100 % at 20 and 80 µl/ml oil concentration respectively. Cinnamomum tamala showed the best antifungal effect in controlling Alternaria alternata among all six oils which inhibited the mycelial growth by 93.11 % at 20 µl/ml and by 100 % at 80 µl/ml oil concentrations. With appropriate formulation, these EOs can replace synthetic preservatives used in increasing selflife of grapes.

Keywords

Antifungal activity, hydro distillation, mycelial growth, Percentage inhibition, Poisoned food technique.

Conclusion

This study concludes that six different EOs extracted from six different plants can be promising in management of post-harvest diseases of grapes especially in controlling two rots fungi namely Aspergillus niger and Alternaria alternata. The oil of Cinnamomum camphora and Cinnamomum tamala showed the most effective antifungal activity against Aspergillus niger and Alternaria alternata respectively. The results suggest their possible use as an alternative or complements to synthetic compounds. Further studies on isolation and characterization of the active (antifungal) compound are needed.

References

Eurasian grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world (Mattia et al. 2008). Its global production in the year 2012 was 691 Mql (OIV 2013). Numerous studies have indicated that grape consumption may be beneficial in reducing the plasma concentration of cholesterol and preventing atherosclerosis (Hashemi 2014). They are useful in fighting dyspepsia, hemorrhoids, stones in the urinary tract, bile ducts, activating liver functions, ease digestion, help reduce the cholesterol level of the blood and eliminate uric acid (FAO 2005). Essential oils are non-water based phytochemicals made up of volatile aromatic compounds (Lawless 2013). Essential oil bearing plants constitute a rich source of bioactive chemicals, which have been reported to have various antifungal properties. These chemicals are often active against a limited number of species, including the specific target species. They are also biodegradable and non-toxic (Adebayo et al. 2013). Naturally occurring biologically active compounds from plants are generally assumed to be more acceptable and less hazardous than synthetic compounds and represent a rich source of potential disease-control agents (Tripathi et al. 2008). Post-harvest disease account for substantial losses of fruits, vegetables and other plant products during the transit and storage process (Mahmoudi et al. 2012). The primary cause of post-harvest loss in table grapes is grey mould disease or Botrytis cinerea (Pearson & Goheen 1988, Snowdon 1990). Some other important fungal post-harvest diseases of table grapes include Aspergillus rot (Aspergillus niger) which doesn’t grow below 5ºC, Alternaria rot (Alternaria alternata), blue mold rot (Penicillium spp.), Rhizopus rot (Rhizopus oryzae; R. stolonifer), anthracnose (Elsinoeampelina, Glomerella cingulata) and others (Snowdon 1990). MATERIALS AND METHOD A. Collection of plant samples and extraction Leaves of Cinnamomum tamala, Cinnamomum camphora and Murraya koenigii were collected from garden of CDB, TU. Similarly leaves of Eucalyptus citriodora, Artemisia indica and Lantana camara were collected from around TU area, Kirtipur and were air-dried and stored at room temperature in darkness until distillation. Bhattarai and Jha 260 The air-dried materials were subjected to hydro distillation for 6-8 h using Clevenger’s apparatus. The essential oils were collected, dehydrated using anhydrous sodium sulphate (Na2SO4) and stored at temperature >10ºC until use and analysis. B. Isolation and identification of test fungi (Aspergillus niger and Alternaria alternata) Some pieces of fungal colony from infected grapes were transferred aseptically on PDA plates. A week later, the observed fungal colony were identified using standard literature (Ellis 1971, Watanabe 2010). C. Assessment of fungi toxicity The fungi toxicity of the essential oils were assessed by poisoned food technique (Grover & Moore 1962). Oils were diluted into different concentrations of 20, 40, 80, 160 and 320 µl/ml with 60 % Acetone (Rao & Srivastava 1994). At first, 1ml of each concentration of essential oil was poured into sterilized petriplates followed by addition of 9 ml of melted PDA. Each petriplates were then inoculated by a 4 mm diameter of the test fungus. In control sets, Fungicide namely Mancozeb 75 % WP and 60 % Acetone were used instead of essential oil. Observations were recorded on 7th day. Five replications were maintained and fungi toxicity was measured in terms of percent inhibition of mycelial growth calculated as; Inhibition of MG (%) = [(gc–gt)/ gc] × 100 [Where; gc = mean colony diameter in control sets and gt= mean colony diameter in treatment sets]. D. Statistical analysis Excel 2013 was used for entering data, drawing charts and required graphs. The data were analysed with the help of ANOVA followed by Post-Hoc; Bonferroni test at (p<0.05) using Software statistical package for social science (SPSS) version 20. RESULTS AND DISCUSSION A. Percentage yield of essential oils Among six plant samples, Eucalyptus citriodora has the highest yield (3%) followed by Cinnamomum camphora (2%), Cinnamomum tamala (1%), Artemisia indica (0.6%), Murraya koenigii (0.5%) and Lantana camara (0.1%), respectively. B. Antifungal bioassay of essential oils The results shows that all six essential oils has significant antifungal effect (p<0.05) over mycelial growth of both test fungi. Artemisia indica oil has better effects over Alternaria alternata than Aspergillus niger. As, it completely inhibits the mycelial growth of Alternaria alternata at 80 µl/ml concentration (Fig. 1). Meanwhile, camphor oil has better effects over Aspergillus niger than Alternaria alternata because it completely inhibits the mycelial growth of Aspergillus niger (Fig. 1B). Similarly, tejpaat oil exhibited better effects over Alternaria alternata than Aspergillus niger (Fig. 1C). (A) (B) (C) (D) Bhattarai and Jha 261 (E) (F) Fig. 1. (A). Antifungal effect of Artemisia indica against Aspergillus niger and Alternaria alternata (B). Cinnamomum camphora (C). Cinnamomum tamala (D). Eucalyptus citriodora (E). Lantana camara (F). Murraya koenigii. [The mean values sharing same alphabet are not significantly different.“*” indicates the significant difference between the mycelial growth of two fungi at different concentration]. Eucalyptus citriodora oil has greater effects over Aspergillus niger than Alternaria alternata (Fig. 1D). Similarly, Lantana camara oil has better effects over Alternaria alternata than Aspergillus niger. As, it completely inhibits the mycelial growth of Alternaria alternata at 160 µl/ml concentration (Fig. 1E). Murraya koenigii oil has better effects over Aspergillus niger. But, exceptionally at 160 µl/ml concentration oil completely inhibits the mycelial growth of Alternaria alternata (Fig. 1F). Similarly, two way ANOVA results for both the test fungi illustrates that plant species (oil type), concentration of oil and their interaction all have significant effect on mycelial growth of the test fungi (Table 1 and 2). Table 1. ANOVA for mycelial growth of Aspergillus niger at different oil concentrations. Source Sum of square Degree of freedom (df) Mean square F Sig. Plant(Oil type) 143.222 5 28.644 4.200 0.005 Concentration of oil 1155.513 6 192.586 28.240 0.000 Interaction 204.590 30 6.820 873.915 0.000 Table 2: ANOVA for mycelial growth of Alternaria alternata at different oil concentrations. Source Sum of square Degree of freedom (df) Mean square F Sig. Plant(Oil type) 105.550 5 21.110 3.979 0.007 Concentration of oil 1198.935 6 199.823 37.664 0.000 Interaction 159.160 30 5.305 685.613 0.000 Fig. 2. Fungi toxicities of different essential oils at different concentrations against Aspergillus niger. Bhattarai and Jha 262 Among all six essential oils,Cinnamomum camphora showed the most effective antifungal activity against Aspergillus niger. The presence of camphor as main component (68%) and linalool, the second most important (9%) may be the reason for their effective antifungal activity (Frizzoet al., 2000; Chen et al., 2013). At 20 µl/ml concentration, C. camphora showed the highest inhibition (81.58%), followed by C. tamala (72.2%), Murraya koenigii (66.87%), Lantana camara (47.25%), Artemisia indica (29.35%) and Eucalyptus citriodora (5.7%) respectively. Similarly, at 40 µl/ml concentration, C. camphora showed the highest inhibition (93.17%), followed by C. Tamala (85.21%), Artemisia indica (78.22%), Murraya koenigii (75.2%), Lantana camara (64.51%), and Eucalyptus ciriodora (15.57%) respectively. Meanwhile, at 80 µl/ml oilconcentration, C. camphora and C. tamala 100% inhibition of mycelial growth followed by Artemisia indica (92.58%), Murraya koenigii (84.37%), Lantana camara (83.06%) and Eucalyptus citriodora (62.88%) respectively. At 320 µl/ml oil concentration rest of the oils also showed 100% inhibition of the mycelialcolony (Fig. 2). Fig. 3. Fungi toxicities of different essential oils at different concentrations against Alternari aalternata. Fig. 4. Antifungal activity of Cinnamomum camphora Fig. 5. Antifungal activity of Cinnamomum tamala against Aspergillus niger. against Alternaria alternata. Cinnamomum tamala showed best activity whereas Eucalyptus citriodorashowed least antifungal effect in controlling Alternaria alternata among all six oils (Fig. 3). It contains eugenol, cinnamaldehyde, cinnamyl alcohol, cinnamylacetate and cinnamic acid and many other responsible for the observed antifungal properties (Pandey et al., 2012). At 20µl/ml oil concentration, C. tamala showed highest inhibition (93.11%) followed by Artemisia indica (72.8%), Lantana camara (69.02%), C. camphora (60.91%), Murraya koenigii (33.16%) and Eucalyptus citriodora (10.01%) respectively. C. tamala showed highest inhibition (95.71%) followed by Artemisia indica (87.36%), C. camphora (83.35%), Lantana camara (74.62%), Murraya koenigii (51.19%) and Eucalyptus citriodora (13.19%) at 40 µl/ml oil concentration respectively. Similarly, C. tamala and Artemisia indica showed the highest inhibition (100%) followed by C. camphora (94.35%), Lantana camara (91.43%), Murraya koenigii (53.23%) and Eucalyptus citriodora (28.93%) at 80 µl/ml oil concentration respectively. And, at 160 µl/ml oil concentration, rest of the oils exhibited 100% inhibition of mycelial growth of Alternaria alternata. Bhattarai and Jha 263 The difference in fungi toxicity at same concentration in different essential oils may be due to different chemical composition of the oils (Singh et al.1983).

How to cite this article

Bina Bhattarai and Sanjay Kumar Jha (2016). Antifungal Effects of some Plant Essential Oils against Alternaria alternata (Fr.) Keissl. and Aspergillus niger van Tiegh. from Grapes. Biological Forum – An International Journal 8(2): 259-263.