Research Article

Antifungal Compounds of Tribulus terrestris Root for the Control of Pyricularia oryzae, the Cause of Rice Blast Disease

Arshad Javaid1*, Freeha Anjum1, Aneela Anwar2, Mahrukh Asif1, Sadia Ahmad1 and Malik F.H. Ferdosi3

1Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan; 2Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan; 3Department of Horticulture, Faculty of Agricultural Sciences, University of the Punjab Lahore, Pakistan.

Received | August 02, 2024; Accepted | September 15, 2024; Published | September 27, 2024

*Correspondence | Arshad Javaid, Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan; Email: [email protected]

Citation | Javaid, A., Anjum, F., Anwar, A., Asif, M., Ahmad, S. and Ferdosi, M.F.H., 2024. Antifungal compounds of Tribulus terrestris root for the control of Pyricularia oryzae, the cause of rice blast disease. Pakistan Journal of Weed Science Research, 30(3): 131-137.

DOI | https://dx.doi.org/10.17582/journal.PJWSR/2024/30.3.131.137

Keywords | Antifungal activity, Natural fungicides, Pyricularia oryzae, Rice blast, Root extract, Tribulus terrestris

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Rice serves as a staple food for nearly 50% of the global population. It is primarily consumed by 2.5 to 3.5 billion individuals across the globe particularly living in low-income countries (Asibi et al., 2019). It is a significant cash crop and ranks as the second most essential staple food after wheat. It is highly susceptible to numerous diseases, with rice blast being the most destructive. It is caused by the fungus Pyricularia oryzae (Wang et al., 2024). The pathogen has the ability to attack rice crop at different growth stages. It produces lesion of varying colors on different aboveground parts of rice plants such as leaves, culms and panicles (Asibi et al., 2019). This filamentous fungus belongs to Ascomycete. The disease is polycyclic in nature and spreads by means of conidia, which have the ability to spread in an area of 230 m from the source. Darkness together with high relative humidity and a wind velocity of 3.5 ms−1 favor the dispersal of conidia (Kingsolver et al., 1984). Yield losses due to rice blast generally range from 10–30% in different rice-growing countries (Skamnioti and Gurr, 2009). The losses may reach up to 50% when there are severe disease outbreaks (Ashkani et al., 2014).

Fungal plant diseases are an increasing concern with significant economic impacts (Khan and Javaid, 2022, 2023a). Nature provides a vast array of chemical diversity, making natural products a vital source for developing new antifungal agents (Ali et al., 2017). While the synthetic fungicides application is an effective method for controlling rice blast, excessive reliance on these chemicals can negatively impact beneficial microorganisms, and also harms the environment consumer’s health risks (Awla et al., 2017). Plants produce a variety of medicinal components capable of inhibiting pathogen growth, and numerous studies have been conducted recently to evaluate the antifungal activity of plant extracts against plant pathogens (Ferdosi et al., 2021; Naqvi et al., 2023; Rafiq et al., 2024).

Tribulus terrestris, a natural herb from the Zygophyllaceae family, is used to treat different diseases, including hypertension. It is found in many tropical and temperate regions worldwide, such as the US, Mexico, the Mediterranean, and throughout Asia (Hussain et al., 2009). Its different parts contain medicinally important chemical constituents such as alkaloids, flavonoids, saponins and flavonol glycosides (Chhatre et al., 2014). T. terrestris is used to treat asthma, urinary problems and ophthalmia (Qureshi et al., 2010). In addition, it exhibits antitumor, vasodilatory, antihelminthic, antihypertensive and cytotoxic properties (Hashim et al., 2014). The roots of Tribulus terrestris contain several bioactive compounds such as saponins, flavonoids, alkaloids, and glycosides, most of which are also antifungal in nature. Some of these root compounds can inhibit essential enzymes for fungal growth and metabolism, thereby preventing their proliferation. Additionally, the plant antioxidant properties can induce oxidative stress in fungal cells, causing damage and reducing their viability (Negri et al., 2014). Results of an earlier study revealed that the stem extract of T. terrestis can control P. oryzae growth in vitro (Javaid et al., 2019). There is a need to conduct similar studies using extracts of other parts of this plant. This study was, therefore, carried out to evaluate the antifungal potential of root extract of T. terrestis and identification of possible antifungal constituents in it.

Materials and Methods

Preparation of root extract

Roots of mature T. terrestis plants were collected from Lahore, Pakistan. After washing, these roots were dried in shade and crushed. A weighed amount (200 g) of root material was soaked in 1.0 L methanol for two weeks followed by successive filtration through muslin cloth and filter papers. Methanol was evaporated on a rotary evaporator and 5.45 g methanolic extract was obtained (Javaid et al., 2023).

Antifungal bioassay

Antifungal bioassays with methanolic root extract of the weed was carried out following procedure of Khan and Javaid (2023b) with some modifications. For preparation of stock solutions, an amount of 4.725 g of root extract was dissolved in 4 mL of dimethyl sulfoxide (DMSO) and raised the volume to 10.5 mL by adding distilled water. Same amount of DMSO was mixed in 6.5 mL of distilled water to get 10.5 mL of control solution. For preparation of different concentrations of the extract in growth medium, appropriate volumes of the two solutions (stock and control) were added to 42 mL autoclaved malt extract broth to acquire a final volume of medium up to 45 mL. This amount was divided into three equal portions to have 3 replicates of each concentration. Actively growing discs (5 mm) of P. oryzae were used as inoculum. After inoculation, the flasks were incubated at 27 oC and the fungal biomass was harvested on pre-weighed filter papers after 10 days. Fungal biomass along with filter papers was dried in an electric oven at 60 oC and weighed. Fungal dry weight was calculated by subtracting the weight of filter paper from the total weight.

GC-MS analysis

Methanolic root extract of the test weed species was analyzed by GC-MS on an Agilent 7890A GC system with 5975C mass spectrometer. Details of various conditions set for GC-MS analysis are presented in Javaid et al. (2022).

Statistical analysis

Antifungal bioassay was carried out using three replicates of each treatment. Standard errors of means of the four replicates were calculated using MS Excel software. The data were analyzed by one-way ANOVA followed by LSD test at P= 0.05 for calculation of significant differences among the treatment means using software Statistix 8.1.

Results and Discussion

Antifungal activity of methanolic root extract

Root extract of T. terrestris showed remarkable antifungal activity and reduced the biomass of P. oryzae by 45–56% over control. Antifungal activity of the methanolic extract was increased gradually as the concentration was increased from 1% to 5% (Figure 1). Earlier, Al-Bayati and Al-Mola (2008) reported that root of T. terrestris collected from Iraq were extracted in water, ethanol and chloroform and their antifungal activity was evaluated against Candida albicans. All the extracts showed insignificant antifungal activities. Information regarding antifungal activity of root extract of this weed is rare. Previous studies have shown that extract of aerial parts of this weed such as stem extract suppressed the growth of P. oryzae by 35–43% when same concentrations of the extract were used (Javaid et al., 2019). Previous studies on T. terrestris were generally carried to assess its antibacterial activity. Root extract of T. terrestris showed antibacterial activity against Escherichia coli and various other bacterial species (Chhatre et al., 2014). Likewise, Sasikala et al. (2014) reported that acetone root extract (80 µL) of T. terrestris caused wide zones of inhibition against the growth of Escherichia coli (33.6 mm) and Bacillus cereus (23.6 mm).

GC-MS analysis

Sixteen compounds were detected in GC-MS analysis in the root extract of T. terrestris as shown in Table 1 and Figure 2. The most abundant compound was neotigogenin (29.30%) followed by hexadecanoic acid, methyl ester (19.70%) and cis-13-octadecenoic acid, methyl ester (18.10%). Neotigogenin was previously identified in T. terrestris and Solanum paniculatum with antibacterial and anticancer activities, respectively (Valadares et al., 2009; Batoei et al., 2016). Hexadecanoic acid, methyl ester and cis-13-octadecenoic acid, methyl ester belong to fatty acid methyl esters group. Many studies have shown that fatty acid methyl esters showed antifungal activity against a variety of fungal species including Aspergillus flavus, A. ochraceus, A. fumigatus and A. niger (Shen et al., 2012; Abdelillah et al., 2013), Candida albicans and C. parapsilosis (Agoramoorthy et al., 2007).

 

Table 1: Compounds identified methanolic root extract of Tribulus terrestris through GC-MS analysis.

Comp No.	Names of compounds	Molecular formula	Molecular weight	Retention time (min)	Peak area (%)
1	3-Methoxyacetophenone	C9H10O2	150.17	12.696	0.99
2	Methyl tetradecanoate	C15H30O2	242.39	17.227	1.10
3	Pentadecanoic acid, methyl ester	C16H32O2	256.42	18.885	0.94
4	Hexadecanoic acid, methyl ester	C17H34O2	270.45	20.516	19.70
5	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	C19H34O2	294.47	23.099	7.11
6	cis-13-Octadecenoic acid, methyl ester	C19H36O2	296.48	23.206	18.10
7	9-Octadecenoic acid, methyl ester, (E)-	C19H36O2	296.48	23.281	0.95
8	Methyl stearate	C19H38O2	298.50	23.602	6.65
9	Tetracosamethyl-cyclododecasiloxane	C24H72O12Si12	889.84	26.533	1.22
10	Phenol, 2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl	C23H32O2	340.49	27.705	1.19
11	Tetracosanoic acid, methyl ester	C25H50O2	382.66	33.738	1.03
12	Squalene	C30H50	410.71	35.455	1.71
13	Cholesterol	C27H46O	386.65	42.328	2.21
14	Stigmasterol	C29H48O	412.69	45.714	6.05
15	Tigogenin	C27H44O3	416.63	46.730	1.30
16	Neotigogenin	C27H44O3	416.63	47.276	29.30

 

 

 

Moderately abundant compounds included methyl stearate (6.65%), stigmasterol (6.05%) and 9,12-octadecadienoic acid (Z, Z)-, methyl ester (7.11%) (Table 1). Methyl stearate possess antifungal activity in addition to various other biological activities (Adnan et al., 2019; Mazumder et al., 2020). Stigmasterol isolated from aerial parts of Neocarya macrophylla and Spillanthes acmella presented antifungal activity against different Candida spp. that was comparable with the fungicide fluconazole (Yinusa et al., 2014; Yusuf et al., 2018). 9,12-Octadecadienoic acid (Z, Z)-, methyl ester is a natural compound with a number of biological activities such as antimicrobial, wound healing, skin repair and anti-inflammatory (Salem et al., 2022).

Ten constituents were ranked as less abundant, which included cholesterol (2.21%), squalene (1.71%), tigogenin (1.30%), tetracosamethyl-cyclododecasiloxane (1.22%), phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4-methyl- (1.19%), methyl tetradecanoate (1.10%), tetracosanoic acid, methyl ester (1.03%), 3-methoxyacetophenone (0.99%), 9-octadecenoic acid, methyl ester, (E)- (0.95%), and pentadecanoic acid, methyl ester (0.94%) (Table 1). Most of these compounds are fatty acid methyl esters which are famous for inhibitory activity against fungi (Abdelillah et al., 2013).

Conclusions and Recommendations

This study concludes that methanolic root extract of T. terrestris is effective in controlling growth of rice blast pathogen P. oryzae. GC-MS analysis showed that it contains a number of antifungal compounds such as fatty acid methyl esters, stigmasterol and neotigogenin.

Novelty Statement

Tribulus terrestris is a weed plant whose medicinal and pharmacological importance is well-documented. A previous study showed that stem extract of this weed has the ability to control a highly damaging fungal pathogen of rice, Pyricularia oryzae. However, such studies regarding antifungal activity of root extract of this weed are lacking. Therefore, the present study was carried out to explore the potential of methanolic root extract of this weed in the management of rice blast pathogen, P. oryzae.

Author’s Contribution

Arshad Javaid supervised the whole study, carried our statistical analysis, prepared graphs and did final editing of the mnauscript. Freeha Anjum conducted the antifungal bioassay. Aneela Anwar supervised the GC-MS study. Sadia Ahmad and Mahrukh Asif contributed in paper writing. Malik F.H. Ferdosi collected and processed the plant material, and also helped in GC-MS analysis.

Conflict of interest

The authors have declared no conflict of interest.

References

Abdelillah, A., B. Houcine, D. Halima, C. Meriem, Z. Imane, S.D. Eddine, M. Abdallah and C. Daoudi. 2013. Evaluation of antifungal activity of free fatty acids methyl esters fraction isolated from Algerian Linum usitatissimum L. seeds against toxigenic Aspergillus. Asian Pac. J. Trop. Biomed., 3(6): 443-448. https://doi.org/10.1016/S2221-1691(13)60094-5

Adnan, M., C.M.N. Uddin, A.T.M.M. Kamal, M.O.K. Azad, A. Paul, S.B. Uddin, J.W. Barlow, M.O. Faruque, C.H. Park and D.H. Cho. 2019. Investigation of the biological activities and characterization of bioactive constituents of Ophiorrhiza rugosa var. Prostrata (D. Don) and Mondal leaves through in vivo, in vitro, and in silico approaches. Molecules, 24: 1367. https://doi.org/10.3390/molecules24071367

Agoramoorthy, G., M. Chandrasekaran, V. Venkatesalu and M.J. Hsu. 2007. Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from India. Braz. J. Microbiol., 38: 739-742. https://doi.org/10.1590/S1517-83822007000400028

Al-Bayati, F.A. and H.F. Al-Mola. 2008. Antibacterial and antifungal activities of different parts of Tribulus terrestris L. growing in Iraq. J. Zhejiang Univ. Sci. B, 9(2): 154-159. https://doi.org/10.1631/jzus.B0720251

Ali, A., A. Javaid and A. Shoaib. 2017. GC-MS analysis and antifungal activity of methanolic root extract of Chenopodium album against Sclerotium rolfsii. Planta Daninha, 35: 1-8. https://doi.org/10.1590/s0100-83582017350100046

Asibi, A.E., Q. Chai and J.A. Coulter. 2019. Rice blast: A disease with implications for global food security. Agronomy, 9(8): 451. https://doi.org/10.3390/agronomy9080451

Ashkani, S., M.Y. Rafii, M. Shabanimofrad, A.R. Harun, 2014. Genetic Analysis of resistance to rice blast: A study on the inheritance of resistance to the blast disease pathogen in an F3 population of rice. J. Phytopathol., 163(4): 300-309.

Awla, H.K., J. Kadir, R. Othman, T.S. Rashid, S. Hamid and M.Y. Wong. 2017. Plant growth-promoting abilities and biocontrol efficacy of Streptomyces sp. UPMRS4 against Pyricularia oryzae. Biol. Contr., 112: 55-63. https://doi.org/10.1016/j.biocontrol.2017.05.011

Batoei, S., M. Mahboubi and R. Yar. 2016. Antibacterial activity of Tribulus terrestris methanol extract against clinical isolates of Escherichia coli. Herb. Pol., 62: 57-66. https://doi.org/10.1515/hepo-2016-0011

Chhatre, S., T. Nesari, G. Somani, D. Kanchan and S. Sathaye. 2014. Phytopharmacological overview of Tribulus terrestris. Pharmacogn. Rev., 8(15): 45-51. https://doi.org/10.4103/0973-7847.125530

Ferdosi, M.F.H., I.H. Khan, A. Javaid and T. Sattar. 2021. Antibacterial activity of essential oil of Paulownia fortunei (Seem.) Hemsl. flowers. J. Plant., 3(1): 27-32.

Hashim, S., T. Bakht, K.B. Marwat and A. Jan, 2014. Medicinal properties, phytochemistry and pharmacology of Tribulus terrestris L. (Zygophyllaceae). Pak. J. Bot., 46: 399-404.

Hussain, F., M. Abid, S.S. Shaukat, S. Farzana and M. Akbar. 2015. Antifungal activity of some medicinal plants on different pathogenic fungi. Pak. J. Bot., 47(5): 2009-2013.

Hussain, A.A., A.A. Mohammed, H..H. Ibrahim, and A.H. Abbas. 2009. Study the biological activities of Tribulus terrestris extracts. World Acad. Sci. ENg. Technol., 57: 433-435.

Javaid, A., A. Ali, I.H. Khan and M.F.H. Ferdosi. 2023. Leaves of Chenopodium album as source of natural fungicides against Sclerotium rolfsii. Arab. J. Chem., 16(5): 104677. https://doi.org/10.1016/j.arabjc.2023.104677

Javaid, A., F. Anjum and N. Akhtar. 2019. Molecular characterization of Pyricularia oryzae and its management by stem extract of Tribulus terrestris. Int. J. Agric. Biol., 21(6): 1256-1262.

Javaid, A., F. Anjum and N. Akhtar. 2019. Molecular characterization of Pyricularia oryzae and its management by stem extract of Tribulus terrestris. Int. J. Agric. Biol., 21(6): 1256-1262.

Javaid, A., H. Qudsia, I.H. Khan, A. Anwar and M.F.H. Ferdosi. 2022. Antifungal activity of Senna occidentalis root extract against Macrophomina phaseolina and its GC-MS analysis. Pak. J. Weed Sci. Res., 28(1): 115-122. https://doi.org/10.28941/pjwsr.v28i1.1033

Khan, I.H. and A. Javaid. 2022. Histopathological changes in root and stem of mungbean exposed to Macrophomina phaseolina and dry biomass of Chenopodium quinoa. Microsc. Res. Tech., 85(7): 2596-2606. https://doi.org/10.1002/jemt.24114

Khan, I.H. and A. Javaid. 2023a. Penicillium citrinum causing postharvest decay on stored garlic cloves in Pakistan. J. Plant Pathol., 105(1): 337. https://doi.org/10.1007/s42161-022-01254-4

Khan, I.H. and A. Javaid. 2023b. Antifungal potential of Chenopodium quinoa root extract against Macrophomina phaseolina (Tassi) Goid. Allelopathy J., 58(1): 61-72. https://doi.org/10.26651/allelo.j/2023-58-1-1420

Kingsolver, C.H., T.H. Barkside and M.A. Marchetti. 1984. Rice blast epidemiology: Bulletin of the pennsylvania agricultural experiment station; No.853; Pennsylvania State College, Agricultural Experiment Station: State College, PA, USA, pp. 29–40.

Mazumder, K., A. Nabila, A. Aktar and A. Farahnaky. 2020. Bioactive variability and in vitro and in vivo antioxidant activity of unprocessed and processed flour of nine cultivars of Australian lupin species: A comprehensive substantiation. Antioxidants, 9(4): 282. https://doi.org/10.3390/antiox9040282

Naqvi, S.F., A. Javaid and I.H. Khan. 2023. Fungicidal activity of stem extract of Chenopodium murale L. against pathogen of Fusarium wilt of tomato. Allelopathy J., 59(1): 69-80. https://doi.org/10.26651/allelo.j/2023-59-1-1432

Negri, M., T.P. Salci, C.S. Shinobu-Mesquita, I.R. Capoci, T.I. Svidzinski and E.S. Kioshima. 2014. Early state research on antifungal natural products. Molecules, 19(3): 2925-2956. https://doi.org/10.3390/molecules19032925

Pooja, K. and A. Katoch. 2014. Past, present and future of rice blast management. Plant Sci. Today, 1: 165-173. https://doi.org/10.14719/pst.2014.1.3.24

Qureshi, R., G.R. Bhatti and R.A. Memon. 2010. Ethnomedicinal uses of herbs from northern part of Nara desert, Pakistan. Pak. J. Bot., 42: 839-851.

Rafiq, M., A. Javaid, A. Kanwal, A. Anwar, I.H. Khan, Q. Kanwal and C. Cheng. 2024. GC-MS analysis and antifungal potential of flower extract of Acacia nilotica subsp. indica against Macrophomina phaseolina. Microb. Pathogen., 194: 106819. https://doi.org/10.1016/j.micpath.2024.106819

Salem, S.H., S.S. El-Maraghy, A.Y. Abdel-Mallek, M.A.A. Abdel-Rahman, E.H.M. Hassanein, O.A. Al-Bedak and F.E.A.A. El-Aziz. 2022. The antimicrobial, antibiofilm, and wound healing properties of ethyl acetate crude extract of an endophytic fungus Paecilomyces sp. (AUMC 15510) in earthworm model. Sci. Rep., 12(1): 19239. https://doi.org/10.1038/s41598-022-23831-4

Sasikala, T, R. Prabakaran and S. Sabitha. 2014. Antimicrobial activities of Tribulus terrestris L. on selected pathogenic microorganisms. Int. J. Pharm. Phytopharm. Res., 4(3): 182-186.

Shen, Y., Y.F. Sun, Z. Sang, C. Sun, Y. Dai and Y. Deng. 2012. Synthesis characterization antibacterial and antifungal evaluation of novel monosaccharide esters. Molecules, 17: 8661-8673. https://doi.org/10.3390/molecules17078661

Skamnioti, P. and S.J. Gurr. 2009. Against the grain: safeguarding rice from rice blast disease. Trend. Biotechnol., 27(3): 141-150.

Valadaresa, Y.M., G.C. Brandãoa, E.G. Kroonb, J.D. S. Filhoc, A.B. Oliveiraa and F.C. Braga. 2009. Antiviral activity of Solanum paniculatum extract and constituents. Z. Naturforsch., 64: 813-818. https://doi.org/10.1515/znc-2009-11-1210

Wang, D, F. Zhu, J. Wang, H. Ju, Y. Yan, S. Qi, Y. Ou and C. Tian. 2024. Pathogenicity analyses of rice blast fungus (Pyricularia oryzae) from Japonica rice area of Northeast China. Pathogens, 13(3): 211. https://doi.org/10.3390/pathogens13030211

Wong, M.Y., S. Hamid, I.S.N. Afifah and A.R.N. Husna. 2020. Botanical extracts as biofungicides against fungal pathogens of rice. Pertanika J. Trop. Agric. Sci., 43: 457-466. https://doi.org/10.47836/pjtas.43.4.03

Yinusa, I., N.I. George, U.O.A. Shuaibu and R.G. Ayo. 2014. Bioactivity of stigmasterol isolated from the aerial part of Spillanthes acmella Murr. on selected microorganism. Int. J. Curr. Microbiol. Appl. Sci., 3: 475-479.

Yusuf, A.I., M. I. Abdullahi, G.A. Aleku, I.A.A. Ibrahim, C.O. Alebiosu, M. Yahaya, H.W. Adamu, A. Sanusi, M.M. Mailafiya and H. Abubakar. 2018. Antimicrobial activity of stigmasterol from the stem bark of Neocarya macrophylla. J. Med. Plant. Econ. Dev., 2(1): 38. https://doi.org/10.4102/jomped.v2i1.38