Research Article | Volume: 8, Issue: 6, Nov-Dec, 2020

An effective and eco-friendly technique for control of post-harvest fungal pathogens of orange (Citrus sinensis) isolated from the distribution chain of Delhi NCR

Gayatri Krishna Geethu Gopinath Anupama Sharma Avasthi   

Open Access   

Published:  Nov 25, 2020

DOI: 10.7324/JABB.2020.80609
Abstract

Orange (Citrus sinensis) is an important horticultural crop of India which is prone to post-harvest damage during storage, transport, and distribution due to fungal pathogens. These phytopathogenic fungi are conventionally controlled through synthetic fungicides. However, with increasing concern for health hazards and environmental pollution due to rampant usage of synthetic fungicides has forced us to devise alternative natural and eco-friendly strategies for their effective control. Natural plant-derived extracts and compounds have evoked immense interest toward their antibacterial and antifungal activities. The present investigation envisages the isolation and characterization of predominant fungal pathogens from C. sinensis obtained from 56 samples collected from the distribution chain of Delhi NCR. The isolated fungi included Aspergillus niger, Alternaria citri, and Penicillium digitatum. In vitro assays were conducted to assess the fungicidal/fungistatic potential of 11 bioactive plant extracts on these isolates by determination of minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC). Out of 11 bioactive extracts tested 10 had fungistatic, 4 had fungicidal, and 1 had no activity on these isolates. n-Hex fraction of Nepeta longibracteata and Actinocarya tibetica and MeOH extract of Zingiber officinale and Glycyrrhiza glabra was found to be most active against A. niger with MIC and MFC values of 125 μg/mL. MeOH extract of G. glabra was most active against A. citri and n-Hex fraction of N. longibracteata and A. tibetica and MeOH extract of Z. officinale and G. glabra was most active against P. digitatum. The results obtained from the present study indicated that these extracts demonstrate antifungal effects against post-harvest phytopathogen of C. sinensis and can offer effective bioactive control. However, more advanced experimental studies are required to identify and pinpoint the exact bioactive compound/molecule responsible for the antifungal activities observed and further work is required to develop appropriate formulations which can be effectively integrated and applied commercially.


Keyword:     Citrus sinensis Phytopathogenic fungi Aspergillus niger Alternaria citri Penicillium digitatum Minimum


Citation:

Krishna G, Gopinath G, Avasthi AS. An effective and eco-friendly technique for control of post-harvest fungal pathogens of orange (Citrus
sinensis
) isolated from the distribution chain of Delhi NCR. J App Biol Biotech. 2020;8(6):54-59. https://dx.doi.org/10.7324/JABB.2020.80609

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Horticultural crops, particularly fruits and vegetables, play an important role in improving the nutritional status of an individual and economy of a region or country. The varied agro-climatic conditions prevalent in India are favorable for the cultivation of a variety of such fruits and vegetables. Citrus varieties, in particular, are cultivated in 0.62 million hectares area with a total production of 4.79 million tonnes [1]. The cultivation is mainly in the states of Maharashtra, Madhya Pradesh, Tamil Nadu, Assam, Orissa, West Bengal, Rajasthan, Nagaland, Mizoram, and Arunachal Pradesh. However, since these varieties are high in both water as well as nutrient content, they are very prone to attack by microbial pathogens throughout the distribution chain, including various stages of picking, packing, storage, and transportation [2]. These post-harvest losses cause a reduction in yield as well as the quality of these fruits. In addition, they also result in causing heavy losses to producers as well as consumers. Further, at the macro level, crores of rupees are also lost. In fact, India loses fruits worth Rs. 12,700–15,876 crores (20–25% loss at an average price of Rs.10,000/t) and vegetables worth Rs. 12,588 crores (a loss of about 20% at an average price of Rs. 5000/t) per year [3].

Since the harvested citrus fruits are acidic, most of the pathogenic infection is caused by fungi and not bacteria. This infection and subsequent decay by phytopathogenic fungi can occur at any level of the distribution chain beginning right after the harvest. The infection usually follows any type mechanical injury of the fruit, disrupting the outer protective layer which facilitates the entry of the fungi [4]. Some post-harvest decays of fruit can also be due to latent infections occurring in the farms which typically include infections caused by Alternaria alternate pv. citri and Phytophthora citrophthora causing black and brown rots. Green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, and anthracnose caused by Colletotrichum gloeosporioides. Apart from this Geotrichum candidum, Diplodia natalensis, Penicillium sp., Trichoderma viride, Fusarium sp., Alternaria alternata, Aspergillus niger, Aspergillus fumigatus, and Aspergillus ochraceus have also been implicated for their involvements in causing infection [5]. Most of these fungi are classified as wound pathogens meaning that although these might be always present on the fruit surface, they only under mechanical damage to the outer cover or favorable environmental conditions multiply to attain damaging levels and this can occur anytime during their handling and transport in the distribution chain posing serious threat [6-8]. Further deterioration in fruit quality is induced as fruit maturation occurs. Favorable temperature and humidity further enhance the process of decay [9]. Practicing hygienic packaging and sanitization methods can only to a certain extent restrict their further growth and multiplication [7].

The present regime to currently regulate post-harvest citrus diseases involves various synthetic fungicides such as imazalil, thiabendazole, and sodium ortho-phenyl phenate, fludioxonil, pyrimethanil [10]. However, recent concerns about pesticide residues, environmental pollution, and development of pathogens resistance [11] have necessitated the need to develop an alternate strategy for the management of these post-harvest losses.

Due to the aforesaid limitations of synthetic fungicides, it becomes imperative to explore other safe and eco-friendly approaches to control citrus post-harvest diseases, which present lower risks to both human health as well as the environment [12]. In recent times various promising alternatives like the use of antagonistic organisms, induction of plant’s natural resistance through signaling pathways have been proposed, but these are not of much significance for the control of post-harvest phytopathogenic fungi. A more suitable approach is to explore the possibility of application of naturally derived bioactive compounds and indeed, this strategy has gained much popularity and scientific interest [2,8,13,14] due to their recently reported antifungal activities non-phytotoxicity and biodegradability [15]. To cite an example, Askarne et al. have reported antifungal activities of 50 different species of plants collected from regions of southern Morocco with demonstrated high antifungal activity of Anvillea radiata and Thymus leptobotrys against P. italicum even at concentrations of 10% m/v [16]. Various other medicinal plants such as garlic [17], neem [18], Withania somnifera L., and Acacia seyal L. [19] are also reported inhibitors P. digitatum. Very recently, Zhu et al. have reported in vivo antifungal activity of tannic acid on P. digitatum by significantly decreasing mycelial growth and spore germination by as much as 70% [20]. Extracts of Capsicum annuum and Zingiber officinale have also been reported to control or inhibit post-harvest diseases in citrus [21].

Previous studies in our lab have demonstrated significant activity of various Indian medicinal plant extracts and fractions against standard fungal strains Aspergillus, Penicillium, and Fusarium [22-24]. In the present study, we are extrapolating the effect of these extracts, specifically on the post-harvest fungal pathogens of citrus fruits in our region.

Present investigation involves the isolation and identification of post-harvest fungal pathogens of citrus varieties from the distribution chain of Delhi NCR and to develop a strategy to control the growth of the phytopathogenic fungi using a variety of natural plant extracts. Further, a methodology is developed to determine the optimum concentrations for fungicide applications based upon minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) values. All these investigations will be ultimately very helpful for the management of post-harvest diseases of citrus and minimize post-harvest losses. An additional step of covering the fruit surface with bioactive extracts can be a good step for citrus fruit preservation.


2. MATERIALS AND METHODS

2.1. Sample Collection

A survey was conducted in the Mother Dairy Safal stores and domestic markets of Delhi NCR and orange samples which showed visible signs of fungal infection were picked from wooden box or pile of fruits. Fifty-six such samples were collected from 15 locations in paper bags as described by Rasool et al. [5]. These were brought to the Chemical Biology Lab of Amity Institute of Biotechnology, Amity University, Noida, India, and categorized according to the visible symptoms of post-harvest diseases.

2.2. Isolation of Fungi

Fungi present in the diseased fruits and vegetables were isolated on potato dextrose agar (PDA) medium. Samples with visible disease symptoms were removed by a sterilized knife in such a manner as to contain the lesion edges too. These were then immersed in HgCl2 (1:10,000) for 2–3 mins for surface sterilization and washed thoroughly in sterile distilled water until all the HgCl2 was washed away. The surface-sterilized tissues were further cut into smaller pieces using a sterile blade and placed on PDA medium using disinfected forceps. The plates were incubated at 28°C for 4–5 days. To obtain pure culture of the fungal isolate, repeated subculturing was performed on PDA plates with incubation at 28°C for 5 days. The fungi were sub-cultured on fresh agar plates and identified by their morphological and cultural characteristics. The purified isolates were maintained on PDA slants for further studies.

2.3. Identification of Phytopathogenic Fungi

Phenotypic identification was performed on standard growth conditions, as described previously. Genus level identification was carried out based upon macro as well as microscopic characteristics. These included visible colonial morphology, color, texture, shape and appearance, and microscopic characteristics such as conidia shape, hyphal color, septation, pigmentation, fruiting bodies, or any other visible structures by observing lactophenol cotton blue-stained slides under the compound microscope at the magnification of ×10, ×45, and ×100 [25]. For this purpose, a small quantity of the aerial mycelia with representative spores was placed on a drop of lactophenol cotton blue stain on a clean slide. A mounting needle was used to evenly spread the mycelia and the spores and subsequently a coverslip was gently placed with little pressure to eliminate air bubbles. The slide was then observed under a binocular compound light microscope with ×10 and ×40 objective lenses. The morphological characteristics and appearance of the fungal organisms seen were identified as per Onuorah et al. [26].

2.4. Preparation of Extracts

Medicinal plants Orchis latifolia and Alpinia galanga were purchased from authorized vendor and Nepeta longibracteata, Actinocarya tibetica, Rhodiola imbricata, Lepidium latifolium, Perovskia abrotanoides, and Echinacea purpurea were collected from different regions of Ladakh as described in our previous works [24]. Ginger rhizome dry (Z. officinale) and Mulethi or Licorice (Glycyrrhiza glabra) were also procured locally. The extractions were carried out as per standard protocol developed in our lab and four fractions of each extract were obtained. Only those fractions which demonstrated substantial antifungal activity in our previous studies [22-24] against Aspergillus, Penicillium, and Alternaria were used in the present investigation for determination of antifungal activity against the fungal isolates.

2.5. Effects of Plant Extracts on Fungal Growth in vitro

The fungicidal activity of the bioactive fractions of various extracts was determined by 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) based colorimetric method which measured the formation of purple formazan by oxidation by mitochondrial oxygenases of the viable fungi [27]. The tests were performed in 96-well flat-bottom microtiter plates containing 100 mL of PDB where the final inocula in each well were in the range 1 × 104–5 × 104 CFU/mL. Varying concentrations of different extracts (31.25–1000 mg/mL) were added to the wells with appropriate positive and negative controls. After the plate was prepared and sealed, it was incubated for 2–3 days. After the appropriate incubation time, 25 mL of 5 mg of MTT/mL was added and the plate was further incubated for 3 h. After this period, 200 mL of isopropanol containing 5% 1 M HCl was added for dye extraction. Further incubation of 30 min was done with gentle agitation at room temperature and appearance of purple formazan crystals was observed with viable cells. The MIC of the extract was defined as the minimum concentration that completely inhibited growth in all the experiments carried out.

MFC was determined by agar dilution method using PDB and various concentrations of plant extracts in concentrations ranging from 31.25 to 1000 mg/mL. For this assay, negative control was taken as blank PDB alone and PDB with spore suspension and without the extract served as a positive control. All samples in which no visible growth was present were further subcultured on extract free PDA plates for 7 days at 27°C. The highest dilution of the extract that could prevent the fungal growth on agar plate was considered as MFC [28].


3. RESULTS AND DISCUSSION

In India, citrus fruits are mostly harvested manually which can cause physical damage, injuries, and bruising as well as unavailability of optimum temperature and dumping of the wooden boxes irregularly in the market. Packing in the wooden boxes forcibly itself adds to the issues of bruising from where the pathogen of post-harvest rots easily enter and spoil a whole lot of fruits. The transports of citrus in vans and trucks without a controlled environment have also contributed toward the diseases. The transport time was another factor affecting the incidence of diseases.

The results from this study have shown that many fungi can cause citrus fruit spoilage during post-harvest period. Symptoms were observed on the diseased citrus fruits and greenish/brownish-black and necrotic patches were present on the skin of the citrus fruit. A mass of mycelia growing on the surface of the fruits was also observed, suggesting the decay or spoilage may be caused by these fungal organisms.

Upon isolation of pure culture, the isolates were characterized and based upon morphological and microscopic examinations were identified as A. niger, P. digitatum, and Alternaria citri. The characteristic features of the isolates are described in Table 1.

Table 1: Characterization of isolated fungal pathogens from diseased citrus fruits.

Causative fungusDiseaseColony characteristicsSpore characteristicsSize of spore
Aspergillus nigerBlack rot moldColonies are initially white plane, but later turn black from center as conidia are producedConidia are globose in shape4–5 µm in diameter
Penicillium digitatumCitrus green moldColonies are white, which later become olive green as conidia are producedConidia are usually oval in shape6–15 μm long
Alternaria citriCitrus black rotColonies are initially white plane, but later turn black or gray as conidia are producedConidia are oval, with vertical and transverse walls20–50 µm long

Table 2 shows the frequency of various fungi in our tested samples. It was seen that A. niger and A. citri (41.1 and 37.5%, respectively) had higher prevalence as compared to P. digitatum which had only 21.4% prevalence.

Table 2: Frequency of occurrence of fungi in the various fruits.

FungiNo. of samplesFrequency of occurrence (%)
Aspergillus niger2341.1
Penicillium digitatum1221.4
Alternaria citri2137.5

There are previous reports of isolation of pathogenic fungi of genera Aspergillus, Fusarium, Mucor, Penicillium, and Rhizopus from many varieties of non-specific ripe fruits. However, P. digitatum and A. citri were found only in Citrus species causing rotting and our results are in confirmation of the same [11,21]. Our results are in agreement with previous studies where A. niger, A. citri, and P. digitatum have been isolated from infected oranges and other citrus fruits and are responsible for brown/black rot [17,21]. The dominance of Aspergillus and Alternaria species along with Penicillium in citrus fruits of the distribution chain confirms their prevalence in fruits exposed to tropical humid climate of India. These three geniuses are potential health risks to consumers of this fruit and it is by products. However, P. digitatum are common fungi in the environment that demonstrate no pathogenicity toward human beings [28].

During various previous studies conducted and published from our lab, we have reported antifungal activities of various plant extracts and fractions. Ethyl acetate fraction of A. galanga and O. latifolia had demonstrated a significant zone of inhibition MTCC 872 A. niger [22,23]. We have also reported strong antifungal activities of n-Hex and DCM fraction of N. longibracteata, n-Hex fraction of A. tibetica, and E. purpurea, DCM fraction of E. purpurea and L. latifolium, and aqueous fraction of E. purpura [24]. In addition, we also tested methanolic extracts of Z. officinale and G. glabra both of which reportedly have strong antimicrobial activities [29]. The MIC values of these 11 extracts/fractions against fungal isolates A. niger, A. citri, and P. digitatum are provided in Table 3.

Table 3: MICa values of bioactive extracts/fractions against fungal isolates.

Plant extract/fractionMICa against A. nigerMICa against A. citriMICa against P. digitatum
EtOAc fraction of
A. galanga
250500250
EtOAc fraction of
O. latifolia
250250250
n-Hex fraction of
N. longibracteata
125250125
DCM fraction of
N. longibracteata
250500500
n-Hex fraction of
A. tibetica
125250250
n-Hex fraction of
E. purpurea
125500125
DCM fraction of
E. purpurea
250500500
DCM fraction of
L. latifolium
5001000500
Aq. fraction of
E. purpurea
1000NDND
MeOH extract of
Z. officinale
125250125
MeOH extract of
G. glabra
250125125

MIC determined by microdilution method and expressed in μg/mL. ND- no antifungal activity detected. A. galangal: Alpinia galangal, O. latifolia: Orchis latifolia, N. longibracteata: Nepeta longibracteata, A. tibetica: Actinocarya tibetica, E. purpurea: Echinacea purpurea, L. latifolium: Lepidium latifolium, Z. officinale: Zingiber officinale, G. glabra: Glycyrrhiza glabra, A. niger: Aspergillus niger, A. citri: Alternaria citri, P. digitatum: Penicillium digitatum

Here, it is important to state that an extract or fraction is considered antimicrobial as per the criteria proposed by Morales et al. [30], with MIC values of <100 mg/mL being considered as strong/good activity and MIC values ranging from 100 to 500 mg/mL as moderate activity. MIC values higher than 500 mg/mL are considered to have very weak antimicrobial activity. Based on the above criteria moderate to good activities were exhibited by all extracts except DCM fraction of E. purpurea against A. niger and P. digitatum. Amongst all the tested fractions A. citri was found to be most resistant and had highest MIC. The results are in agreement with previous studies of Behbahani et al., 2016 [28], where the efficacy of plant extracts has been demonstrated against A. citri and P. digitatum. All these extracts/fractions have reported the presence of secondary metabolites by qualitative phytochemical analysis in our previous studies. Biological activities have been associated with all classes of secondary metabolites, including flavonoids, saponins, cardiac glycosides, tannins, triterpenes, and alkaloids. The observed wide range of antifungal properties for the methanolic extracts and fractions can be described by the presence of bioactive secondary metabolites. The EtOAc fraction of A. galanga was reported to contain triterpenoids and cardiac glycosides in significant amounts. The total phenolic content of this fraction was also high (110 mg GAE/g of the extract [22]. Similarly, EtOAc fraction of O. latifolia had the presence of flavonoids, steroids, and tannins [23]. Similarly, high-altitude plants have reported a high concentration of phenolics and flavonoids as exhibited by high total phenolics and flavonoids content [24].

The application of these bioactive extracts can be used for protection against fungal infection in the distribution chain. However, more mechanistic insights are required to develop a protocol for protection. Hence, to further study whether the mode of action of these extracts/fraction is fungistatic or fungicidal, MFC values were determined and are presented in Table 4.

Table 4: MFCb values of bioactive extracts/fractions against fungal isolates.

Plant extract/fractionMFC against A. nigerMFC against A. citriMFC against P. digitatum
EtOAc fraction of
A. galanga
5001000500
EtOAc fraction of
O. latifolia
250500250
n-Hex fraction of
N. longibracteata
250250250
DCM fraction of
N. longibracteata
500500500
n-Hex fraction of
A. tibetica
250250250
n-Hex fraction of
E. purpurea
250500250
DCM fraction of
E. purpurea
500500500
DCM fraction of
L. latifolium
500NDND
Aq. fraction of
E. purpurea
NDNDND
MeOH extract of
Z. officinale
125250125
MeOH extract of
G. glabra
125250125

MFC expressed in μg/mL. ND: No activity detected in this concentration range. A. galangal: Alpinia galangal, O. latifolia: Orchis latifolia, N. longibracteata: Nepeta longibracteata, A. tibetica: Actinocarya tibetica, E. purpurea: Echinacea purpurea, L. latifolium: Lepidium latifolium, Z. officinale: Zingiber officinale, G. glabra: Glycyrrhiza glabra, A. niger: Aspergillus niger, A. citri: Alternaria citri, P. digitatum: Penicillium digitatum

It is clear from the above data that all fractions showed fungistatic activity against A. niger and P. digitatum. However, fungicidal activities were demonstrated only by six fractions against A. niger and P. digitatum. These included EtOAc fraction of O. latifolia, n-Hex fraction of N. longibracteata, A. tibetica, and E. purpurea, and MeOH extract of Z. officinale and G. glabra. Against A. citri only four fractions demonstrated fungicidal activity (n-Hex fraction of N. longibracteata and A. tibetica and MeOH extract of Z. officinale and G. glabra). Combining both the results n-Hex fraction of N. longibracteata and A. tibetica and MeOH extract of Z. officinale and G. glabra are the most bioactive and promising fractions that need further exploration for their application in control of phytopathogenic fungi. It was also evident from our study that non-polar extracts exhibited better fungicidal activities. The n-Hex and DCM fractions had lower MIC and MFC values as compared to aqueous fractions. Similar results have been obtained in a previous study by Pinelo et al. [31], who advocated that the chemical nature of the solvent used for extraction along with the methodology adopted impacts the chemical nature of bioactive compounds that can be isolated. The reason attributed for this is the solubility of the extractives in various solvents as per the polarity which accounts for variations in the degree of bioactivities. The nature of the compounds in extracts responsible for antifungal activity must be non-polar and hence better extracted with non-polar solvents. It is indeed corroborated in our previous studies where GCMS analysis of n-Hex fraction of N. longibracteata has shown the presence of a large percentage of long-chain fatty acids and their methyl esters [24]. Lower activities of the aqueous extract could be reasoned with the observation that during maceration and grinding of plant material during extract preparation, phenolases and hydrolases are released which may modulate the activity of the bioactive present in the extract. Another reason for the reduced activity may be the incomplete extraction of these bioactives. The results of our study demonstrate that the MFC of the extracts evaluated was similar or higher concentrations than in the MIC assays, but not at lower concentrations [Table 4]. It is also imperative to state at this stage that the antifungal activity of bioactive fractions or extracts is not due to the action of a single active compound but the synergistic effect of several compounds that might be available in the extract.

Since the application of plant extracts and fractions in the prevention of post-harvest losses in the distribution chain is still in the early stages, the mechanism of mode of action of these extracts is not very well characterized. Nevertheless, some initial and recent studies have attributed the antifungal action to a combination of mechanisms. These may include signaling mechanisms, including salicylate and jasmonic acid pathways and induction of host resistance responses [32]. Essential oils can also play an effective role by creating a barrier and also through synergistic effects of their components. This synergism has been known to delay the development of resistance in the phytopathogenic fungi [2]. In addition, components of essential oils seem to have no specific cellular targets they can be used for broad-spectrum control of fungal pathogens. The volatility, ephemeral nature, and biodegradability of essential oil compounds also add to the advantages for disease management. This has been demonstrated by the methanolic extracts of W. somnifera and A. seyal. A high total phenolic content has been shown to disrupt the membrane functionality and this may be another reason for our observed antifungal activities as all our fractions have high TPC and TFC values [22-24].

However, these studies are still in infancy stages and much more investigations on the mode of action of plant extracts are required before these can be integrated into citrus crop management [4]. To make this technology commercially viable additional studies are required to formulate a product which is effective during in vivo treatments, is stable over a longer duration of time to cover the entire distribution chain, and most importantly does not alter the quality parameters of citrus fruits such as flavor and aroma.


4. CONCLUSION

The results of the present investigations demonstrate the effectiveness of plant extracts and fractions against phytopathogenic fungi of Citrus sinensis isolated from the distribution chain of Delhi NCR. In particular, n-Hex fraction of N. longibracteata, A. tibetica, and E. purpurea and MeOH extract of Z. officinale and G. glabra was most active against the fungal isolates A. niger, P. digitatum, and A. citri with significant MIC and MFC values. Additional studies are required to identify the chemical identities of the bioactive responsible for the observed antifungal activity which may offer new alternatives to post-harvest fungal pest management of citrus fruits with minimum health hazards. The major challenge will be the commercially viable production of these alternate and eco-friendly methods.


5. ACKNOWLEDGMENTS

Authors acknowledge Dr. A.K. Chauhan, Founder President, Amity University, for his continuous motivation and encouragement and Dr. Chanderdeep Tandon, Director, Amity Institute of Biotechnology, Amity University, for providing the infrastructure to carry out the investigations and Dr. Sabari Ghosal for her unrelenting support and encouragement.


6. CONFLICT OF INTEREST

Authors declared that they do not have any conflicts of interest.


7. FINANCIAL SUPPORT AND SPONSORSHIP

None.

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28. Behbahani BA, Yazdi FT, Shahidi F, Riazi F. Antifungal effect of the aqueous and ethanolic Avicennia marina extracts on Alternaria citri and Penicillium digitatum. Zahedan J Res Med Sci 2016;18:e5992.

29. Sharma H, Yunus GY, Agrawal R, Kalra M, Verma S, Bhattar S. Antifungal efficacy of three medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major against oral Candida albicans: A comparative analysis. Indian J Dent Res 2016;27:433. [CrossRef]

30. Nascimento GG, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol 2000;31:247-56. [CrossRef]

31. Pinelo M, Rubilar M, Jerez M, Sineiro J, Núñez MJ. Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace. J Agric Food Chem 2005;53:2111-7. [CrossRef]

32. Leite MC, Bezerra AP, Sousa JP, Guerra FQ, Lima ED. Evaluation of antifungal activity and mechanism of action of citral against Candida albicans. Evid Based Complement Alternat Med 2014;2014:378280. [CrossRef]

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27. Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva Á. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochem 2012;114:785-96. https://doi.org/10.1016/j.acthis.2012.01.006

28. Behbahani BA, Yazdi FT, Shahidi F, Riazi F. Antifungal effect of the aqueous and ethanolic Avicennia marina extracts on Alternaria citri and Penicillium digitatum. Zahedan J Res Med Sci 2016;18:e5992.

29. Sharma H, Yunus GY, Agrawal R, Kalra M, Verma S, Bhattar S. Antifungal efficacy of three medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major against oral Candida albicans: A comparative analysis. Indian J Dent Res 2016;27:433. https://doi.org/10.4103/0970-9290.183135

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31. Pinelo M, Rubilar M, Jerez M, Sineiro J, Núñez MJ. Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace. J Agric Food Chem 2005;53:2111-7. https://doi.org/10.1021/jf0488110

32. Leite MC, Bezerra AP, Sousa JP, Guerra FQ, Lima ED. Evaluation of antifungal activity and mechanism of action of citral against Candida albicans. Evid Based Complement Alternat Med 2014;2014:378280. https://doi.org/10.1155/2014/378280

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