Fungal disease is one of the major problems in agriculture. Fungal pathogens are accountable for approximately 85% of plant diseases. Apart from these, public health conditions are also influenced by consequential fungal infection as well as approximately 1.5 million killed and more than a billion people were affected by fungal disease. Our present exploration has been conducted to assess the antifungal efficiency of Azadirachta indica, Ocimum tenuiflorum, and Murraya paniculata leaf extract against three phytopathogenic fungi viz. Pichia kudriavzevii, Lasiodiplodia theobromae and Fusarium oxysporum, at the concentration of 300 µg/disc by food poisoned technique. The result showed that all of these three extracts have significant antifungal efficiency against all of the tested fungus. Maximum antifungal activity was recorded in Murraya paniculata with an inhibition percentage of 100% (0.00±0.000 mm) against three fungi. In addition, Lasiodiplodia theobromae and Fusarium oxysporum, growth was totally suppressed in terms of Ocimum tenuiflorum and Murraya paniculata extract. The lowest antifungal effect was 47.18% (34.33±0.272 mm) revealed in Azadirachta indica extract against Pichia kudriavzevii. Among these three extracts, the order of antifungal effect was Murraya paniculata˃Ocimum tenuiflorum˃Azadirachta indica. Amis of this screening to focus antifungal effects of three experimental medicinal plants. These findings indicate leaf of these three plants may be useful for the treatment of various diseases associated with these fungi and could be useful to develop novel, secure and fecund bio-fertilizer for pest control. Further phytochemicals analysis is required to evaluate the compounds responsible for their antifungal effects.
Although asthma, AIDS, cancer, organ transplantation, and many other health conditions are influenced by consequential fungal infection as well as approxima-tely 1.5 million killed and more than a billion people were affected by fungal disease yet it is a forlorn topic by public health specialists (Bongomin et al., 2017). Apart from, the fungal pathogen is one of the biggest problems in agriculture. Fungus is the most devastating pathogen for plants and is responsible for 85% of plants disease.
Pichia kudriavzevii infects immunodeficient patients considered as opportunistic pathogen induce mastitis in mammals (Hurst, 2016). Additionally, Trunk fungi Lasiodiplodia theobromae is responsible for Grapevine trunk diseases (GTDs) are the most destructive dis-eases of vineyards worldwide (Rusin et al., 2020). Moreover, the pathogenic Fusarium oxysporum strains are liable to cause Fusarium wilt disease, conducted a major threat for agriculture, and ranked 10th most demolishing plant pathogenic fungus (Dean et al., 2012; Fisher et al., 2012).
The drugs are frequently used as antifungal have a side effect, hence there is exclusive need for the discovery of novel, secure and fecund antifungal drugs, for instance, medicinal plants could play a significant role in the field of drug discovery. Azadirachta indica ext-racts inhibited the growth of some fungus, bacteria, viruses, insecticides and also exposed antioxidant, anti-ulcer, anti-carcinogenic, and anti-malarial properties (Subapriya and Nagini, 2005). Alongside, extract from neem pledges and significantly abate fungal conidial germination (Jabeen et al., 2013). Because of these antimicrobials and pharmaethological properties, Aza-dirachta indica could be used for pest control along with health benefits (Rony et al., 2019). Ocimum tenuiflorum belonging to the family of Lamiaceae also known as Ocimum sanctum, Tulsi or Holy Basil. Euge-nol, one of the key phytochemical of Ocimum tenui-florum oil act against fungi leading to bio-deterioration of foodstuff during storage (Subramanian, 2014).
Murraya paniculata L family of Rutaceae, commonly used as herbal therapy in India, China, Indonesia, and Bangladesh (Zhang et al., 2011). It contains an exu-berant source of phyto compounds, liable for anti-fungal, antibacterial, analgesic, antispasmodic, bron-chodilator, vasodilating actions, anti-thyperglycemic and antioxidant action(Fazal-ur-Rehman et al., 2014; P et al., 2015; Saqib et al., 2015). The above plants are widely used as medicinal plants in Bangladesh. Our present investigation deals with the evaluation antifungal efficiency of Azadirachta indica, Ocimum tenuiflorum, and Murraya paniculata leaf extract against Pichia kudriavzevii, Lasiodiplodia theobromae and Fusarium oxysporum to find out effective agents that could be useful to develop new, secure and effective bio-fertilizer to pest control and as well as treatment of various disease associated with these fungi.
Plant materials - Plant leaves of Azadirachata indica, Ocimum teniflorum, and Murraya paniculata were collected from Rajshahi University Campus, Rajshahi-6205, Bangladesh. These plant leaves were used as plant material in our current study.
Extract preparation - The samples were washed indi-vidually under running tap water to remove any traces of soil particles and other dirt, cut into small pieces and air-dried for seven days. 20 gm of powdered dried leaves soaked with 60 mL methanol and allowed to macerate at room temperature for 15 days with an orbital shaker. Then, strained through sterile mark in cloth and finally filtered through sterile Whatman No.1 filter paper. The filtrates were then set for 3 days to evaporate the methanol and afford a blackish mass. The output extracts were collected to glass vials and preserved separately in a refrigerator at 4°C.
Test microorganisms - Methanol leaf extract of Aza-dirachta indica, Ocimum tenuiflorum, and Murraya paniculata were investigated for their antifungal efficiency against three fungi including Pichia kudri-avzevii, Lasiodiplodia theobromae, and Fusarium oxy-sporum. Investigated fungi were collected from Prof. Joarder DNA and Chromosome Research Lab. Dept. of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.
Determination of antifungal activity - In-vitro anti-fungal activity was accomplished by food poisoned technique against three pathogenic fungi Pichia kudri-avzevii, Lasiodiplodia theobromae and Fusarium oxy-sporum at the concentration of 300 µg/ml. 20 ml quantities of potato dextrose agar (PDA) media were plated in the petri dish with fungal culture. In this investigation, 300 µg/ml concentrations of plant ext-racts were spread with a sterile spreader on test orga-nism-seeded plates. A plate containing only fungal culture was used as a control. The activity of the plant extract was determined after seven days of incubation at 37ºC temperature. After 7 days solid dry weight of the fungus was taken using an oven. The antifungal activity of these plants extract was assessed on the basis of inhibition of mycelial growth (%) and cal-culated using the formula (Habib et al., 2019).
Inhibition of mycelial growth (%) = [(Gc-Gt)/Gc]*100
Where, ‘C is the average diameter of the fungal colony in control plates and, ‘T is the average dia-meter of the fungal colony in poisoned plates (Gupta and Tripathi, 2011).
In-vitro antifungal efficiency of three medicinal plants Azadirachta indica, Ocimum tenuiflorum and Murraya paniculata methanol leaf extract were exhibited strong antifungal activity against Pichia kudriavzevii, Lasio-diplodia theobromae and Fusarium oxysporum at the concentration of 300 µg/disc (Fig 1). The diameter zone of inhibition of Azadirachta indica, Ocimum tenuiflorum and Murraya paniculata leaf extract are presented in Table 1.
three biological replicates.
Khan et al. explained that Azadirachta indica leaf extract had a characteristic effect on dermatophytes, leading to the exclusion in protease action of derma-tophytes due to maybe it flavonoid quercetin in the extract (Matthews, 1989). Our present finding exhi-bited three fungi growth was significantly inhibited by our investigated extracts at 300µg/disc, whereas Shiv-puri et al. found Azadirachta indica ethanol extract had antifungal efficiency to five pathogenic fungi at the concentration from 500 to 1000 µg/ml (Shivpuri et al., 1997). In addition, Kishore et al. also reported that Azadirachta indica inhibited about 90% Phaeoi-sariopsis personata conidial germination (Kishore et al., 2001).
In addition, Ocimum tenuiflorum leaf extract revealed remarkable antifungal efficiency at 300 µg/disc. Among the three fungi, Pichia kudriavzevii and Lasio-diplodia theobromae growth were fully inhibited by Ocimum tenuiflorum methanol leaf extract with an inhibition percentage of 100% (0.00±0.00 mm) and 100% (0.00±0.00 mm) (Fig 2, B).
Moreover, Ocimum tenuiflorum also showed an anti-fungal effect on Fusarium oxysporum with an inhi-bition percentage of 69.93% (15.33±0.544 mm). Gupta et al. found that Ocimum Sanctum Linn aqueous extract reducing the fungal growth of A. niger, Cladpsporium sp. and Rizopous, at the concentration of 40% (Gupta et al., 2013). Besides these many scientific studies indicated that Ocimum sanctum has antifungal activity and these studies validation our present investigation (Shahen et al., 2019)
In this investigation, Azadirachta indica, Ocimum tenuiflorum and Murraya paniculata methanol leaf extract has shown potential antifungal properties. We also found the maximum antifungal effect in Murraya paniculata against all tested fungus. These findings indicate leaf of these three plants could be useful to treat various diseases associated with these fungi. Further phytochemicals analysis is required to assess the compounds responsible for their antifungal effects.
The authors want to thank Prof. Joarder DNA & Chromosome Research Lab, Dept. of Genetic Engin-eering and Biotechnology, University of Rajshahi, Rajshahi-6205, for providing financial support and other facilities during the whole research work.
The authors declared no potential conflicts of the interest with the present experiment.
Md. Ekhlas Uddin Dipu, Department of Biochemistry and Molecular Biology Gono Bishwabidalay, Dhaka, Bangladesh.
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.
Nesa M, Hosen ME, Khan MAI, Kabir MH, and Zaman R. (2021). In-vitro antifungal activity of Azadirachta indica, Ocimum tenuiflorum and Murraya paniculata leaf extract against three phytopathogenic fungi. Am. J. Pure Appl. Sci., 3(5), 113-118. https://doi.org/10.34104/ajpab.021.01130118