Screening plants extracts for Antifungal activity against Rhizoctonia solani

 

Shweta Singh1, Jaiganesh Rengarajan2, Iyappan Sellamuthu1*

1Department of Genetic Engineering, College of Engineering and Technology,

Faculty of Engineering and Technology, SRM Institute of Science and Technology,

SRM Nagar, Kattankulathur, 603203 Kanchipuram, Chennai, Tamil Nadu, India.

2Department of Biotechnology, College of Engineering and Technology,

Faculty of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar,

Kattankulathur, 603203 Kanchipuram, Chennai, Tamil Nadu, India.

*Corresponding Author E-mail: iyappans@srmist.edu.in

 

ABSTRACT:

The adverse effect of pesticides used for controlling pests and the diseases caused by them is extensively rising. To overcome this harmful environmental impact, alternative methods are being studied and developed. Out of many possible methods, one effective method is to use plant extracts which embodies natural substances having antifungal properties. In an attempt towards  enhancement of sustainable antifungal approach, four different concentrations of four plant extracts Citrus limon, Azadirachta indica, Ocimum gratissimum and Acalypha indica, were tested for their antifungal activity against Rhizoctonia solani, which is a serious threat for plants, and is responsible for considerable crop and yield losses. The aim of this study is to minimize yield  losses and to regain the attention of formers towards exploiting natural resources for diseases control.  From the results, it can be concluded that different concentrations of plant extracts have caused substantial inhibition in the mycelial growth of R. solani. The leaf extract of Ocimum gratissimum was highly effective in inhibiting mycelial growth, at the  concentration of 500mg/ml of extract followed by Citrus limon, Azadirachta indica, and Acalypha indica. These plants might have potential for the development of natural fungicide for the management of diseases caused by fungal pathogens. Also, fungicides developed from these botanical extracts can prove to be highly effective for the management of plant diseases and would be simply obtainable, nonpolluting, biodegradable and economical.

 

KEYWORDS: Antifungal activity, disk diffusion method, medicinal plants, rhizoctonia solani, zones of inhibition.

 

 


1. INTRODUCTION:

Plants are infected by an extensive range of pathogens among which fungi are considered to instigate varieties of rot diseases. Many pathogens including Rhizoctonia solani, one of the most recognized species of genus Rhizoctonia is responsible for the destruction of crops worldwide, reducing the market values of crops and making them unsuitable for our consumption.

 

R. solani belongs to phylum Basidiomycota is a plant pathogenic fungus with wide host range including economically important agricultural crops such as rice, wheet, tomato, potato and pepper etc.1-6.  Chemical fungicides are frequently used for fungal rots and are very common for the management of these diseases.  But the use of these compounds has ensued the debut of new pathogens which are resilient to these fungicides and are also adverse for human health and natural environment. Consequently, plant beneficial microbes and plant extracts shown attractive features of inhibiting growth of plant pathogens. Among the bacterial groups, Pseudomonas sp. and Bacillus sp. are demonstrated as most potent fungal inhibitors7,8,9.  In the other side,  the extracts, which are obtained from the plants are thought to be extra adequate and less precarious as compared to the manufactured compounds and hence can be used as a substitute for antifungal compounds. Extracts obtained from numerous botanicals have gotten immense recognition and also gained scientific attention for their microbial activity such as antibacterial, antiviral, and antifungal activity. 

 

Solvent extraction is the most preferred one for the extraction of secondary metabolites from plants and also successfully demonstrated for antimicrobial compounds.  Methanolic extracts from Ammannia baccifera Linn.10 and ethanolic extract from Centella asiatica11 shows growth inhibition for human pathogen Candida albicans.  Some of Family Fabaceae plant’s methanolic extract were also shows good inhibition against Aspergillus niger12. Similarly, ethanolic extract of Cleome rutidosperma13, Cyclamen hederifolum14 and Wattakaka volubilis15 were demonstrated as a potent source of antifungal compounds.  Curcumin shows inhibitory as well as synergistic effect with known antifungal drugs against Candida albicans16.  Antibacterial activity of C.limon17, Azadirachta indic18, Ocimum gratissimum19 20 and Acalypha indica21,22 were found to be a good source of antimicrobial agents against various pathogens.  In the present study, considering the tremendous future of plants as sources for antimicrobial medicine pertaining to bactericidal and antifungal agents, a scientific investigation was initiated to screen the native flora for antifungal activity from Citrus limon, Azadirachta indica, Ocimum gratissimum and Acalypha indica against R. solani which is one of the major pathogenic fungi infecting wide range of plants. 

 

2. MATERIALS AND METHODS:

2.1 Sample Collection:

Fresh leaves of Citrus Citrus limon, Azadirachta indica, Ocimum gratissimum and Acalypha indica were collected from Potheri, Chengalpattu district, Tamil nadu. The samples were collected in separate plastic bags on sunny days in order to avoid any moisture content on the samples.

 

2.2 Microbial culture and growth conditions:

The fungal culture of R. solani MTCC4633 was ordered from (Microbial Type Culture Collection and Gene Bank), CSIR Institute of Microbial Technology, Chandigarh. The fungus was grown on Potato Dextrose Agar (PDA) medium at room temperature and was maintained with periodic sub culturing.

 

2.3 Preparation of plant extracts:

The disease-free healthy leaves were selected and washed from all the sides with running tap water to take off the dust particles. After washing with tap water for three times, and once with sterile water, the leaves were kept for shade drying for two weeks.  The dried leaves were powdered using an electric blender and stored in different zip lock covers. Ten grams of leaf powder was dissolved in 100ml of  methanol, in 250ml conical flasks for each plant sample. The samples were kept in an upright position in the shaker incubator for 48 hours, maintained at room temperature at 120rpm. The solvent was changed after 24 hours for effective extraction. After running for 48 hours, the colorless solvent was filtered through Whatman No. 1 filter paper, to get the supernatant. The supernatant was collected in separate petri plates and was allowed to air dry for solvent to evaporate. Then the extract was stored in the eppendorf tubes in -20 degrees for further analysis of antifungal activity18.

 

2.4 Assessment of Antifungal activity:

Antifungal activity of the extracts was screened by disk diffusion method. The disk diffusion test also known as agar diffusion test, or Kirby–Bauer test is a test of the antibiotic sensitivity of bacteria or fungus (Magaldi et al., 2004). DMSO was used as negative control and the solution of clotrimazole and lignocaine hydrochloride was used as a positive control.  Different concentrations of plant extracts were dissolved in DMSO to different concentrations such as 50mg/ml, 100mg/ml, 200mg/ml and 500mg/ml for each plant sample. Discs were impregnated with 25µl of plant extracts from each concentration using micropipette. Then the discs were left to dry for drying at RT for 2 to 3hrs.  In a PDA plate, mycelium of fungus was placed in the center of the plate. Then, the impregnated discs containing the diffused extracts were placed  separately on the PDA plate. The plates were incubated for 48 hours at RT. After the incubation, the plates were examined for clear zone of inhibition in the vicinity of the disc and recorded18.

 

3. RESULTS AND DISCUSSION:

For antifungal screening of plant extracts from four plants belonging to different families as mentioned were evaluated against R. solani, one of the most pathogenic fungus responsible for major crop loss worldwide.  The plant leaf samples for selected plants were collected, air dried and powdered. The powder were extracted with ethanol for bioactive compounds against R. solani.  Among the 4 plants screened, Ocimum gratissimum and Citrus Limon shows better inhibition.  Zone of inhibition by the plant extracts were observed after 48 hours and measured using ruler (Table 1).  It was observed that different concentration of extracts showed substantial inhibition of fungus. After screening diffusates for their antifungal potential, Ocimum gratissimum appeared the most effective inhibition in radial mycelial growth of pathogen and followed by Citrus limon (Fig. 1).  The other  plant extracts were found to be less effective.  These report were supported by the studies of O. gratissimum to have  antimicrobial, antifungal23, and the hexane fraction of this inhibits dermatophytes24.  Similarly, the other species of Ocimum also reported as a potent source of a antibacterial and antifungal compounds25,26.

 

 

Table 1: Zone of inhibition of plant extracts against R. solani

Name of the plants

Zone of inhibition(mm)

50 mg/ml

100 mg/ml

200 mg/ml

500 mg/ml

Citrus limon

-

4.0

5.8

7.5

Azadirachta indica

-

-

3.0

5.0

Ocimum gratissimum

3.0

6.0

7.0

9.0

Acalypha indica

-

-

4.0

5.0

 


 

Fig. 1. Disk diffusion method results after 48 hours of incubation.  1. Citrus limon  2. Azadirachta indica  3. Ocmium gratissimum 4. Acalypha indica  and 5. Contro (DMSO) .  Four different concentration  like i. 50mg/ml, ii. 100mg/ml, iii. 200mg/ml and iv. 500mg/ml were used.

 

 

Other plants selected for the present study C.limon were also demonstrated to posses potent medicinal property. Ethanolic extract of C.limon were studied against Candida glabrata, Aspergillus niger, Saccharomyces cerevisiae and Candida parapsilosis27,28,29,30 The bark and leaf extract of Azadirachta indica were demonstrated to antibacterial activity31. It is used as traditional medicine for treating several diseases like infections of the upper tract, cold, flu and pneumonia.  In a previous study, It was also demonstrated that the aqueous extracts of neem cake shows inhbition against sporulation of C. lunata, H. pennisetti, and C. gloeosporioides f. sp. Mangiferae32 and methonolic extract inhibit   Aspergillus flavus, Alternaria solani, and Cladosporium33 A. indica  contain acalyphin and also flavonoids, and the extracts of this herb are frequently utilized asthma and pneumonia34.

 

4. CONCLUSIONS:

The results of this study have shown that the methanol extracts of Citrus limon, Azadirachta indica, Ocimum gratissimum, and Acalypha indica have boundless possibility as antimicrobial agents in the management of risky and destructive pathogens. The study unconcealed that escalating the concentration of botanical diffusates lessens the mycelial growth of fungal pathogenic agents radially when performed in vitro. The plant extracts are more adequate and less risky as compared to the manufactured compounds and hence might be used as a substitute for manmade antifungal compounds.  The results from this initial research to look at the potential use of these botanical diffusates for controlling the diseases induced by R. solani perceived to be encouraging with regards the mycelial growth of the plant.  Additional detailed investigation of the bioactive constituents of such plants for similar mechanism of action will support the development of new complementary and reasonable antifungal chemicals to a great extent which will be economical and highly effective in reduction of crop losses worldwide.

 

5. ACKNOWLEDGEMENT:

The authors are grateful to the SRM Institute of Science and Technology, Kattankulathur, India, for their support in conducting this work.

 

6. REFERENCES:

1.      Taheri P, Gnanamanickam S, Höfte M. Characterization, Genetic Structure, and Pathogenicity of Rhizoctonia sp. Associated with Rice Sheath Diseases in India. Phytopathology. 2007; 97(3): 373-383.

2.      Sturrock CJ, Woodhall J, Brown M, Walker C, Mooney SJ, Ray RV. Effects of damping-off caused by Rhizoctonia solani anastomosis group 2-1 on roots of wheat and oil seed rape quantified using X-ray Computed Tomography and real-time PCR. Frontiers in Plant Science. 2015; 6: 461. 

3.      Gondal AS, Rauf A, Naz, F. Anastomosis Groups of Rhizoctonia solani associated with tomato foot rot in Pothohar Region of Pakistan. Scientific Reports. 2019; 9: 3910.

4.      Read PJ, Hide, G.A., Firmager, J.P. et al. Growth and yield of potatoes as affected by severity of stem canker (Rhizoctonia solani). Potato Research. 1989; 32:9–15. 

5.      Tsror L. Biology, epidemiology and management of Rhizoctonia solani on potato. Journal of  Phytopathology. 2010; 158(10): 649–58.

6.      Sid Ahmed A, Ezziyyani M, Pérez Sánchez C, Candela ME. Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants. European Journal of  Plant Pathology. 2003; 109(6): 633–7.

7.      Vinoth P,  Subramani K, Natarajan RK, Subaiya SV, Mahesh S, Usha B and Sellamuthu I. Screening of plant growth promoting rhizobacteria for antifungal activity against Fusarium oxysporum.  Journal of Environmental Biology. 2019; 40: 235-239.

8.      León M, Yaryura PM, Montecchia MS, Hernández AI, Correa OS, Pucheu NL, Kerber NL, García AF. Antifungal Activity of Selected Indigenous Pseudomonas and Bacillus from the Soybean Rhizosphere. International Journal of Microbiology. 2009; 2009:1-9.

9.      Ramanpreet Kaur, Mainam Anamika Devi, Pratibha Vyas. Endophytic Pseudomonas sp. TCA1 from Tinospora cordifolia Stem with Antagonistic and Plant growth-promoting Potential. Research Journal of Pharmacy and Technoloty. 2017; 10(2): 456-460.

10.   Das SK, Dhake AS, Nayak A, Das NB, Pandeya SN. Antibacterial and Antifungal Activity of Aerial Part of Plant Ammannia baccifera Linn. Research Journal of Pharmacy and Technology. 2011;4(3): 430-432.

11.   Jagtap NS, Khadabadi SS, Ghorpade DS, Banarase NB, Naphade SS. Antimicrobial and Antifungal Activity of Centella asiatica (L.)Urban, Umbeliferae. Research J. Pharm. and Tech.2009;2(2): 328-330.

12.   Saranya Shankar, Mythili Sathiavelu. A Comparative Study on the Biological Activities of Medicinal Plants Leaf Extracts Belongs to the Family Fabaceae. Research Journal of Pharmacy and Technology.2019;12(3): 1129-1136.

13.   Khuntia A, Mohanty SK. Antifungal Activity of Cleome rutidosperma Aerial Parts. Research Journal of Pharmacy and Technology. 2011;4(7): 1103-1105.

14.   Nour Hassan Boissa, Maysa Yaziji, Rim Salame. Screening of antifungal activity in vitro of Cyclamen hederifolum tubers extracts. Research Journal of Pharmacy and Technology. 2016; 9(10): 1677-1680.

15.   Sangavi R., Anuradha R.. Antibacterial and Antifungal Activity of Wattakaka volubilis. Research Journal of Pharmacy and Technology. 2017; 10(11): 3775-3778.

16.   Ananta Choudhury, Suman Saha, Sanjib Bahadur, Amit Roy. Synergistic Antifungal Activity of Bioactive Phytochemical in Combination with Standard Antifungal Drugs. Research Journal of Pharmacy and Technology. 2019; 12(5): 2346-2352

17.   Okeke MI, Okoli AS, Eze EN, Ekwume GC, Okosa EU, Iroegbu CU. Antibacterial activity of Citrus limonum fruit juice extract. Pakistan Journal of Pharmaceutical Sciences. 2015; 28(5): 1567-1571.

18.   Mistry KS, Sanghvi Z, Parmar G, Shah S. The antimicrobial activity of Azadirachta indica, Mimusops elengi, Tinospora cardifolia, Ocimum sanctum and 2% chlorhexidine gluconate on common endodontic pathogens: An in vitro study. European Journal of Dentistry. 2014;8(2): 172-177.

19.   Matasyoh LG, Matasyoh JC, Wachira FN, Kinyua MG, Muigai AW, Mukiama TK. Antimicrobial activity of essential oils of Ocimum gratissimum L. From different populations of Kenya. African journal of Traditional Complement Alternative Medicines. 2008; 5(2): 187-193.

20.   Melo RS, Albuquerque Azevedo ÁM, Gomes Pereira AM, et al. Chemical Composition and Antimicrobial Effectiveness of Ocimum gratissimum L. Essential Oil Against Multidrug-Resistant Isolates of Staphylococcus aureus and Escherichia coli. Molecules. 2019; 24(21): 3864.

21.   Govindarajan M, Jebanesan A, Reetha D, Amsath R, Pushpanathan T, Samidurai K. Antibacterial activity of Acalypha indica L. Europian Review for Medical and  Pharmacological Sciences. 2008; 12(5): 299-302.

22.   Noumedem JA, Tamokou Jde D, Teke GN, Momo RC, Kuete V, Kuiate JR. Phytochemical analysis, antimicrobial and radical-scavenging properties of Acalypha manniana leaves. Springerplus. 2013; 2: 503. 

23.   Pandey S. Antibacterial and antifungal activities of Ocimum Gratissimum L. International Journal of Pharmacy and Pharmaceutical Sciences. 2017; 9(12): 26-31.

24.   Silva MR, Oliveira JG Jr, Fernandes OF, et al. Antifungal activity of Ocimum gratissimum towards dermatophytes. Mycoses. 2005; 48(3): 172-175.  

25.   Hayat M. Mukhtar, Mohit  Mangla, Mohit Sanduja. Meticulous Approach towards Pharmacognosy and Socio-economic Impact of Ethnomedicinal Plant: Ocimum kilimandscharicum. Research Journal of Pharmacy and Technology. 2020; 13(10): 4751-4764

26.   Saftarasmi, Lakshmi T. Ocimum Sanctum in Dental Care -A Mini Review. Research Journal of Pharmacy and Technology. 2014; 7(1): 101-103.

27.   Otang WM, Afolayan AJ. Antimicrobial and antioxidant efficacy of Citrus limon L. peel extracts used for skin diseases by Xhosa tribe of Amathole District, Eastern Cape, South Africa. South African Journal of Botany. 2016; 102:46–49.

28.   Hamdan D, Ashour ML, Mulyaningsih S, El-Shazly A, Wink M. Chemical composition of the essential oils of variegated pink-fleshed lemon (Citrus x limon L. Burm. f.) and their anti-inflammatory and antimicrobial activities. Zeitschrift für Naturforschung - Section C Journal of Biosciences. 2013; 68C: 275–284.

29.   Lamine M, Rahali FZ, Hammami M, Mliki A. Correlative metabolite profiling approach to understand antioxidant and antimicrobial activities from citrus essential oils. International Journal of Food Science and Technology. 2019; 54: 2615–2623.

30.   Guerrini A, Rossi D, Grandini A, Scalvenzi L, Rivera PFN, Andreotti E, Tacchini M, Spagnoletti A, Poppi I, Maietti S. Biological and chemo-diverse characterization of Amazonian (Ecuador) Citrus petitgrains. Journal of Applied Botony and Food Quality. 2014; 87: 108–116.

31.   Yerima M B, Jodi S M, Oyinbo K, Maishanu H M, Farouq AA, Junaidu AU. Effect of neem extracts (Azadirachta indica) on bacteria isolated from adult mouth. Journal of Basic and Applied Sciences. 2012; 20: 64–67

32.   Anjali K, Ritesh K, Sudarshan M, Jaipal SC, Kumar S. Antifungal efficacy of aqueous extracts of neem cake, karanj cake and vermicompost against some phytopathogenic fungi. The Bioscan. 2013; 8: 671–674.

33.   Shrivastava DK, Swarnkar K. Antifungal activity of leaf extract of neem (Azadirachta indica Linn). International Journal of Current Microbiology and Applied Sciences. 2014; 3(5): 305–308.

34.   Chekuri S, Arun Jyoti B, Sompaga SJ, Panjala S, Anupalli RR. Evaluation of AntiMicrobial and Anti-Fungal Activity of Acalyphaindica L., Leaf Extract. International Journal of Pharmacognosy and Phytochemical Research. 2018; 10(1); 48-51.

 

 

 

 

Received on 10.09.2020            Modified on 01.12.2020

Accepted on 08.01.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(12):6545-6548.

DOI: 10.52711/0974-360X.2021.01132