Introduction

Macrophomina phaseolina (Tassi) Goid. is a necrotrophic fungal pathogen responsible for soil- and seed-borne diseases in up to 500 economically important host plants including mungbean, mashbean, soybean, sunflower, sorghum, maize, linseed, chickpea and alfalfa (Pawlowski et al. 2015). At initial stages of infection, symptom is not visible but later on they can appear as black lesions on plant stem, peduncle and branches which ultimately invade the vascular bundles and causes root rot, collar rot, seedling blight and damping off disease in plants (Arora 2017). It forms hard sclerotia, which can survive for long time in the soil and upon favorable environmental conditions become primary source of infection. The pathogen becomes more destructive under dry and humid environmental conditions. As the disease progresses, it provokes the root system destruction along with chlorosis, growth losses, withering, and ultimately death of a plant (Ullah et al. 2019).

Many of the fungicides are in practice for the control of fungal pathogens but no registered fungicide is available in market for the management of M. phaseolina due to the production of sclerotia. The fungicides have also hazardous effects on environment that makes their usage restricted (Kalsoom et al. 2019). In this regard scientists are making efforts to find out environment friendly and cheap bio-products derived from plants to control the soil-borne pathogens as they are non-phytotoxic and an excellent substitute to synthetic fungicides. Plants contain a large number of secondary metabolites such as tannin, terpenoids, alkaloids, flavonoids, phenols, volatile oils, glycosides and steroids, which manifest antifungal properties (Khan et al. 2019). Recently, various reports on different parts of plant extracts exhibited strong antifungal potential against plant pathogenic fungi under in vitro conditions. In this regard, the extracts of Sisymbrium irio, Senna occidentalis, Azadirachta indica, Kochia indica and Sonchus oleraceous have proved very effective to control M. phaseolina (Javed et al. 2018; Munir et al. 2018).

Members of Chenopodiaceae family such as Chenopodium ambrosioides, C. album and C. murale are known to possess several allelochemicals found very useful in obstructing the growth of phytopathogens especially M. phaseolina (Javaid and Amin 2009). C. quinoa also belongs to this family and is known as a pseudo-cereal. It gained worldwide importance due to its diverse genetic characteristics and recently introduce in North America, Asia, Africa and Europe. It is commonly known as quinoa, became an excellent food crop for humans as an alternate to wheat because of its high nutritious values. In recent years, its cultivation has also been started in Pakistan on a large scale due to its remarkable tolerance to salinity, drought and heat (Hernandez-Ledesma 2019). Therefore, keeping in view that it is a member of family Chenopodiaceae, it was hypothesized that C. quinoa may also contain antifungal compounds with potent efficacies against plant pathogens. Therefore, stem extracts of four varieties of quinoa were explored for their potential to control M. phaseolina and the detection of antifungal compounds through GC-MS analysis.

Materials and Methods

Antifungal bioassays

For the collection of plant stem in appropriate quantity, four quinoa verities namely V1, V2, V7 and V9 were cultivated in winter 2017 in Lahore. Seeds of the four varieties were obtained from University of Agriculture, Faisalabad, Pakistan. Details regarding origin of these varieties are given in Table 1. At the time of maturity, the stems of each variety were collected, dried and thoroughly crushed. Methanolic extracts were prepared by macerating 200 g of crushed stems of each variety in methanol (1 L) and kept for two weeks at room temperature. Thereafter, the mixture was coarse filtrated by muslin cloth and the extract was concentrated by recovering the solvents on rotary evaporator at 45°C. Stock solution of 15 mL of each extract was prepared in dimethyl sulphoxide (5 mL) by dissolving 9 g of crude methanolic extracts with subsequent addition of autoclaved distilled water. Similarly, control solution was prepared without the addition of plant extract. Five concentration viz. 1, 2, 3, 4 and 5% were formulated by mixing control and stock solutions in suitable amounts with four replicates of each as reported by Javaid et al. (2017). M. phaseolina was procured from Biofertilizer and Biopesticide Lab, IAGS, Punjab University Lahore. Five-millimeter diameter mycelial plugs of 7-day-old M. phaseolina culture were added to each conical flask and left to stand at 28°C. After 7 days, fungal mats were collected on filter papers and dried in an electric oven at 70°C for data collection.

The variety named V7 was selected and fractionated with different solvents with increase in their polarities. For this, 3 kg of shade dried, powdered plant stem was dipped into 10 L of methanol for 15 days and filtered through a muslin cloth. After that, the thick gummy extract was suspended in autoclaved distilled water 200 mL and kept for 4 h. The mixture was successively fractionated beginning with n-hexane (5 × 500 mL) followed by chloroform (500 mL), ethyl acetate (500 mL) and n-butanol (500 mL) into a separating funnel. Among these solvents, chloroform and n-hexane fractions were evaporated to obtain their crude extract. The in vitro biological activity of n-hexane and chloroform fraction was assessed against M. phaseolina. Out of the selected extracts, 1 mL of dimethyl sulphoxide was added to each 1.2 g of the extract to dissolve in followed by the addition of malt extract 5 mL in order to prepare the sequential concentrations starting with 200 mg mL^-1 and then it was divided into two aliquots. One aliquot was used for further serial dilution to make the lower concentrations viz. 100, 50, …, 1.562 mg mL^-1 and the other one was used to evaluate extract bio-efficacy. A control was also prepared similarly in a series without extract addition to maintain the amount of dimethyl sulphoxide. Inoculum of M. phaseolina was prepared from 8-day-old culture in autoclaved distilled water. The assay was performed by adding 50 µL aliquots of the inoculum in each test tube and left to stand at 28°C for 7 days. The obtained fungal mats were filtered and weighed after seven days of incubation (Shafique et al. 2016). Three replicates of each treatment were run simultaneously.

GC-MS analysis

GC-MS analysis of n-hexane and chloroform fractions was carried out for compounds identification. Ten milligrams of each of the two fractions were dissolved in 1 mL of their respective solvents and filtered through Whatman® glass microfiber filters grade GF/C. Analysis was performed by using a Shimadzu GC-2010plus system coupled with an auto sampler AOC-20s, an auto injector AOC-20i, and a gas chromatograph. Using helium as a carrier gas, a volume of 1.0 µL sample was injected by setting injector temperature at 250°C. The interface temperature was adjusted at 320°C. After injection of sample, the initial column temperature was 100°C for 60 s that was enhanced from 100 to 200°C at 20°C min^-1 and hold for 2.0 min, finally from 200°C to 300°C at 40°C min^-1. The total run time was 10.9 min.

Statistical analysis

Completely randomized design was selected for both the laboratory experiments and all the data were analyzed by ANOVA and LSD test (P≤0.05) using Statistix 8.1.

Results

Antifungal activity

ANOVA presented in Table 2 indicates that the effect of extract concentration (C), quinoa varieties (V) and V×C was found to be very effective (P≤0.001) for the production of fungal biomass. Among the quinoa varieties, V7 methanolic extract showed a remarkable antifungal activity causing 80–89% suppression of fungal biomass. V9 extract was ranked as the second most effective antifungal source against M. phaseolina where it reduced its growth by 69–88% over control by using different concentrations. Although methanolic leaf extracts of other two varieties significantly declined fungal growth but their antifungal potentials were less pronounced than V7 and V9 as extracts of V1 and V2 inhibited fungal growth by 45–71% and 45–80%, respectively (Fig. 1).

Table 1: Details of four varieties used in the present study

Quinoa lines	Origin	Plant name
V1	Colorado, USA	Colorado 407D
V2	New Mexico, USA	IESP
V7	New Mexico, USA	2WANT
V9	Chile	Pichaman

Fig. 1: Effect of different concentrations of methanolic stem extract of four varieties of C. quinoa on biomass of M. phaseolina. Vertical bars show standard errors of means of four replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD test.

Fig. 2: Effect of different concentrations of n-hexane, chloroform and ethyl acetate fractions of methanolic stem extract of C. quinoa on biomass of M. phaseolina. Vertical bars show standard errors of means of four replicates. Values with different letters at their top show significant difference (P≤0.05) as determined by LSD test.

Table 2: Analysis of variance (ANOVA) for the effect of different concentrations of methanolic stem extracts of four varieties of C. quinoa on biomass of M. phaseolina

Sources of variation	df	SS	MS	F values
Varieties (V)	3	143547	47849	328^*
Concentration (C)	5	1305852	261170	1790^*
V ´ C	15	47138	3143	21.5^*
Error	72	10505	146
Total	95	1507042

*, Significant at P≤0.001

The highest activity was shown by V7 methanolic extract and was thus selected for further studies. Different fractions of the extract were effective against M. phaseolina (Fig. 2). The selected non-polar fraction n-hexane, and less polar fraction chloroform completely arrested the growth of fungal pathogen with 1.562 mg mL^-1 the lowest MIC value (Fig. 2A–B). In comparison to the others, two more polar fractions viz. n-butanol and ethyl acetate were relatively less inhibitory in nature with MIC values of 25 and 12.5 mg mL^-1, respectively. There was 52–100% and 66–100% reduction in M. phaseolina biomass over control due to the n-butanol and ethyl acetate fractions (Fig. 2C–D). The aqueous fraction with the highest polarity showed the least antifungal efficacy by suppressing 46–100% fungal growth (Fig. 2E).

GC-MS analysis

GC-MS chromatogram of n-hexane indicates the presence of 15 constituents as given in Fig. 3A. The most prevailing compounds were 9,12-octadecadien-1-ol, (Z,Z)- (22.23%) followed by 9,12-octadecadienoic acid-(Z,Z)-, methyl ester (16.84%) and 1-(+)-ascorbic acid 2,6-dihexadecanoate (15.18%). Moderately abundant compounds were hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)-ethyl ester (10.99%) and hexadecenoic-acid, methyl ester (7.37%). Whereas, the least abundant compounds were 1,2-benzedicarboxylic acid, diisooctyl-ester, ar-tumerone, 6-hexadecenoic-acid, 7-methyl, methyl ester (Z), octadecanoic acid, phytol, tetradecanoic acid, curlone, 2-propenoic-acid,3-[4-(acetyloxy)-3-methoxyphenyl]-, methyl ester, octadecanoic acid, methyl ester and benzoic-acid,4-hydroxy-3,5-dimethoxy-, hydrazide with peak areas ranging from 4.62 to 1.18% (Table 3; Fig. 4).

Table 3: List of compounds in n-hexane fraction of methanolic stem extract of C. quinoa identified by GC-MS analysis

Names of compounds	Molecular formula	Molecular weight	Retention time (min)	Peak area (%)
Ar-tumerone	C₁₅H₂₀O	216	5.797	3.75
Curlone	C₁₅H₂₂O	218	5.996	2.50
Tetradecanoic Acid	C₁₄H₂₈O₂	228	6.177	2.53
Benzoic acid,4-hydroxy-3,5-dimethoxy-,hydrazide	C₉H₁₂N₂O₄	212	6.258	1.18
2-Propenoic acid,3-[4-(acetyloxy)-3-methoxyphenyl]-,methyl ester	C₁₃H₁₄O₅	250	6.608	2.07
Hexadecanoic acid, methyl ester	C₁₇H₃₄O₂	270	6.911	7.37
1-(+)-Ascorbic acid 2,6-dihexadecanoate	C₃₈H₆₈O₈	652	7.104	15.18
9,12-Octadecadienoic acid(z,z)-,methyl ester	C₁₉H₃₄O₂	294	7.637	16.84
Phytol	C₂₀H₄₀O	296	7.698	2.97
Octadecanoic acid, methyl ester	C₁₉H₃₈O₂	298	7.735	1.54
9,12-Octadecadien-1-ol,(z,z)-	C₁₈H₃₄O	266	7.837	22.23
Octadecanoic acid	C₁₈H₃₆O2	284	7.908	3.05
6-Hexadecenoic acid,7-methyl,methyl ester (z)	C₁₈H₃₄O2	282	8.492	3.18
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester	C₁₉H₃₈O4	330	9.645	10.99
1,2-Benzedicarboxylic acid, diisooctyl ester	C₂₄H₃₈O₄	390	9.753	4.62

Table 4: List of compounds in chloroform fraction of methanolic stem extract of C. quinoa identified by GC-MS analysis

Names of compounds	Molecular formula	Molecular weight	Retention time (min)	Peak area (%)
Benzene, nitro-	C₆H₅NO₂	123	2.861	1.23
3-Acetoxy-3-hydroxypropionic acid,methyl ester	C₆H₁₀O₅	162	2.918	1.94
4-((1E)-3-Hydroxy-1-Propenyl)-2-methoxyphenol	C₁₀H₁₂O₃	180	6.168	3.28
1-Tetracosanol	C₂₄H₅₀O	354	6.208	3.36
1-Pentacosanol	C₂₅H₅₂O	368	6.255	6.28
2,4-Hexadienedioic acid,3,4-diethyl-,dimethyl ester,(z,z)-	C₁₂H₁₈O₄	226	6.436	1.81
3-Isopropoxy-4-methoxybenzamide	C₁₁H₁₅NO₃	209	6.538	2.01
Hexadecanoic acid, methyl ester	C₁₇H₃₄O₂	270	6.906	4.274
Piperine	C₁₇H₁₉NO₃	285	7.020	8.43
n-Hexadecanoic acid	C₁₆H₃₂O₂	256	7.066	3.95
4,8-Ethano-4H-1,3-benzodioxin,hexahydro-	C₁₀H₁₆O₂	168	7.142	1.99
Benzenemethanol,2,5-dimethoxy acetate	C₁₁H₁₄O₄	210	7.260	6.195
Dimethyl 1-(2-methoxyethyl)-5-methylpyrazole-3,4-dicarboxylate	C₁₁H₁₆N₂O₅	256	7.390	1.71
8,11-Octadecadienoic acid, methyl ester	C₁₉H₃₄O₂	294	7.629	16.68
9,12-Octadecadienoic acid(z,z)-	C₁₈H₃₂O₂	280	7.795	9.14
Cis-9-hexadecenal	C₁₆H₃₀O	238	8.617	1.08
gamma-Sitosterol	C₂₉H₅₀O	414	9.343	3.49
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester	C₁₉H₃₈O4	330	9.624	2.07
1,2-Benzedicarboxylic acid, diisooctyl ester	C₂₄H₃₈O₄	390	9.752	12.48
1-Triacontanol	C₃₀H₆₂O	438	10.335	8.60

Table 5: Potential antifungal constituents in n-hexane and chloroform fractions of Chenopodium quinoa stem extract

Names of compounds	Property	Reference
9,12-Octadecadien-1-ol,(Z,Z)-	Antifungal	Wang et al. (2008)
Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester	Antifungal	Al-Marzoqi et al. (2015)
1,2-Benzedicarboxylic acid, diisooctyl ester	Antifungal	Rahman and Anwar (2006)
8,11-Octadecadienoic acid, methyl ester	Antifungal	Kianinia and Farjam (2018)
2,4-Hexadienedioic acid,3,4-diethyl-,dimethyl ester,(Z,Z)-	Antifungal	Chhouk et al. (2018)

Chloroform fraction revealed the presence of 20 compounds (Table 4; Fig. 3B). 8,11-Octadecadienoic acid, methyl ester (16.68%) was present abunduntly followed by 1,2-benzedicarboxylic acid, diisooctyl ester (12.48%) and 9,12-octadecadienoic_acid_(Z,Z)- (9.14%). The moderately abundant compounds were 1-triacontanol (8.60%), piperine (8.43%), 1-pentacosanol (6.28%) and benzenemethanol,2,5-dimethoxy acetate (6.195%). However, the least abundant compounds were hexadecanoic_acid, methyl-ester; n-hexadecanoic acid; gamma-sitosterol; 1-tetracosanol; 4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol; hexadecanoic_acid,2-hydroxy-1-(hydroxymethyl) ethyl_ester; 3-isopropoxy-4-methoxybenzamide; 4,8-ethano-4H-1,3-benzodioxin,hexahydro-; 3-acetoxy-3-hydroxypropionic acid, methyl ester; 2,4-hexadienedioic acid, 3,4-diethyl, dimethyl ester (Z,Z)-; dimethyl 1-(2-methoxyethyl)-5-methylpyrazole-3,4-

Fig. 3: GC-MS chromatograms of n-hexane and chloroform fractions of methanolic stem extract of C. quinoa

Fig. 4: Structures of potential antifungal compounds identified in n-hexane and chloroform fractions of stem extract of C. quinoa through GC-MS

dicarboxylate; benzene, nitro- and cis-9-hexadecenal with peak areas ranges from 4.274 to 1.08% (Table 5).

Discussion

In general, methanolic stem extracts of all the four quinoa varieties significantly reduced growth of M. phaseolina. Previously, literature regarding antifungal activity of C. quinoa is very limited. Woldemichael and Wink (2001) reported antifungal activity of C. quinoa against Candida albicans. Saponins, a diverse group of natural compounds containing steroid aglycone or triterpene and one or more chains of sugar in their structure (Guçlu-Ustundag and Mazza 2007), are well known for their antifungal activity (Tsuzuki et al. 2007). The plant contains at least 26 saponins (Madl et al. 2006), which might be the reason of its antifungal activity against M. phseolina (Woldemichael and Wink 2001). Besides saponins, a number of other components like eugenol, thymol, carvacrol, phenolics, linalool and flavonoides are also reported in quinoa that are known for their antimicrobial properties (Juneja et al. 2012).

Methanolic extracts showed a marked variation in different verities towards their antifungal potential. V7 possessed the greatest antifungal potential followed by V9. Similar varietals differences in antifungal activity have also been recorded among the extracts of varieties of Vitis vinifera, Allium sativum and Cupressus arizonica against a wide range of fungal pathogens (Fratianni et al. 2016; Jediyi et al. 2019). Varietals antifungal activity differences could be attributed to the difference in chemical composition among the varieties (Khouadja et al. 2015). Jediyi et al. (2019) reported that V. vinifera varieties were also different in phenols and flavonoids contents so provided a marked variation in antifungal activities among the selected varieties.

Chloroform and n-hexane fractions were highly antifungal and completely retarded the growth of the pathogen even at lower concentrations. To reveal the chemical composition of these fractions a GC-MS analysis was performed to identify the known antifungal compounds. Literature survey showed that these compounds might be responsive in inhibiting the growth of M. phaseolina. Wang et al. (2008) isolated 9,12-octadecadien-1-ol,(Z,Z)- as a major component from Digitaria sanguinalis and found it to be very effective against Curvularia eragrostidis. Similarly, Al-Marzoqi et al. (2015) stated that hexadecenoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is antifungal in nature against Aspergillus flavus and A. niger. Likewise, diisooctyl phthalate also known as 1,2-benzedicarboxylic_acid, diisooctyl ester was previously isolated from the leaves of Hugonia mystax and Plumbago zeylanica roots as a major chemical constituent. This compound was very effective against M. phaseolina, Alternaria alternata, Botryodiplodia theobromae and Fusarium equiseti (Rahman and Anwar 2006). Moreover, a compound namely 8,11-octadecadienoic acid, methyl ester was also isolated from a medicinal plant Arum maculatum which was found to be effective in arresting the growth of Penicillium digitatum and A. niger (Kianinia and Farjam 2018). Similarly, Chhouk et al. (2018) identified 2,4-hexadienedioic_acid,3,4-diethyl-, dimethyl_ester (Z,Z)- from Khmer a medicinal plant and reported that this compound possessed antifungal activity against many pathogentic fungi (Table 5).

Conclusion

There were large differences among the four selected varieties of quinoa towards their antifungal potential. M. phaseolina was very susceptible to extracts of V7. The pathogenic growth was completely controlled when treated with chloroform and n-hexane fractions of methanic extract of this variety. Various possible antifungal compounds were identified through GC-MS.

Acknowledgements

Seeds of four varieties of quinoa were provided by Prof. Dr. Shahzad Maqsood Ahmed Basra, Department of Agronomy, University of Agriculture, Faisalabad that is highly acknowledged.

Author Contributions

IHK did experimental work and wrote the paper. AJ supervised the work and contributed in writing and finalizing the paper.

References

Al-Marzoqi AH, IH Hameed, SA Idan (2015). Analysis of bioactive chemical components of two medicinal plants (Coriandrum sativum and Melia azedarach) leaves using gas chromatography-mass spectrometry (GC-MS). Afr J Biotechnol 14:2812‒2830

Arora MK (2017). Plant maturity in relation to plant development in sorghum bicolor effected by charcoal rot. Intl J Adv Multidiscip Res 4:74‒77

Chhouk K, W Diono, H Kanda, M Goto (2018). Efficacy of supercritical carbon dioxide integrated hydrothermal extraction of Khmer medicinal plants with potential pharmaceutical activity. J Environ Chem Eng 6:2944‒2956

Fratianni F, R Riccardi, P Spigno, MN Ombra, A Cozzolino, P Tremonte, R Coppola, F Nazzaro (2016). Biochemical characterization and antimicrobial and antifungal activity of two endemic varieties of garlic (Allium sativum L.) of the Campania Region, Southern Italy. J Med Food 19:686‒691

Guçlu-Ustundag O, G Mazza (2017). Saponins: Properties, applications and processing. Crit Rev Food Sci Nutr 47:231‒58

Hernandez-Ledesma B (2019). Quinoa (Chenopodium quinoa Willd.) as source of bioactive compounds: A review. Bioactive Comp Health Dis 2:27‒47

Javaid A, L Afzal, A Shoaib (2017). Biological control of charcoal rot of mungbean by Trichoderma harzianum and shoot dry biomass of Sisymbrium irio. Planta Danin 35; Article e017165756

Javaid A, M Amin (2009). Antifungal activity of methanol and n-hexane extracts of three Chenopodium species against Macrophomina phaseolina. Nat Prod Res 23:1120‒1127

Javed S, A Javaid, AH Al-Taei, MZ Qureshi (2018). Identification of antimicrobial compounds from n-hexane stem extract of Kochia indica by GC-MS analysis. Mycopathology 16:51‒55

Jediyi H, K Naamani, AA Elkoch, A Dihazi, N Lemjiber (2019). A comparative study of phenols composition, antioxidant, and antifungal potency of leaves extract from five Moroccan Vitis vinifera L. varieties. J Food Saf 2019; Article e12756

Juneja VK, HP Dwivedi, X Yan (2012). Novel natural food antimicrobials. Ann Rev Food Sci Technol 3:381‒403

Kalsoom R, S Chohan, MS Haider, M Abid (2019). Synergistic effect of plant extracts and fungicide against purple blotch disease of onion. Plant Prot 3:53‒57

Khan MMM, H Firdous, Z Tahir, K Zahid, MA Haider, A Qadus, MS Mushtaq (2019). Response of commercial and candidate lines of soybean (Glycine max L.) against root rot disease and its chemicals management. Discovery Agric 5:29‒40

Khouadja W, R Oliveira, A Raies, ACP Dias (2015). Oxidant and genotoxic-mediated strong antifungal activity of the essential oils from Cupressus arizonica var. arizonica and var. glabra. Indust Crops Prod 77:614‒623

Kianinia S, MH Farjam (2018). Chemical and biological evaluation of essential oil of Arum maculatum. Iran J Sci Technol 42:395‒399

Madl T, H Sterk, M Mittelbach (2006). Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa. J Amer Soc Mass Spectrom 17:795‒806

Munir M, A Shoaib, A Javaid, Z Arshad, M Rafiq (2018). Anti-mycotic potential of Trichoderma spp. and leaf biomass of Azadaricta indica against the charcoal rot pathogen Macrophomina phaseolina (Tassi) Goid in cowpea. Egypt J Biol Pest Cont 28; Article 26

Pawlowski ML, CB Hill, GL Hartman (2015). Resistance to charcoal rot identified in ancestral soybean germplasm. Crop Sci 55:1230‒1235

Rahman MS, MN Anwar (2006). Fungitoxic and cytotoxic activity of a novel compound 1,2-benzenedicarboxylic acid, diisooctyl ester of Plumbago zeylanica Linn. Asian J Microbiol Biotechnol 8:461‒464

Shafique HA, V Sultana, S Ehteshamul-Haque, M Athar (2016). Management of soil-borne diseases of organic vegetables. J Plant Prot Res 56:221‒230

Tsuzuki JK, TIE Svidzinski, CS Shinobu, LFA Silva, E Rodrigues-Filho, DAG CortezI, ICP FerreiraI (2007). Antifungal activity of the extracts and saponins from Sapindus saponaria L. An Acad Bras Cienc 79:577‒583

Ullah N, KP Akhtar, MJ Asghar, G Abbas, (2019). First report of Macrophomina phaseolina causing dry root rot of lentil in Pakistan. J Plant Pathol 101:429‒429

Wang F, P Zhang, S Qiang, Y Zhu, L Xu (2008). Effect of epicuticular wax from Digitaria sanguinalis and Festuca arundinacea on infection by Curvularia eragrostidis. Australas Plant Pathol 37:43‒52

Woldemichael GM, M Wink (2001). Identification and Biological Activities of Triterpenoid Saponins from Chenopodium quinoa. J Agric Food Chem 49:2327‒2332