Antifungal activity and GC-MS detection of leaves and roots parts of Chenopodium album extract against some phytopathogenic fungi

Plant materials
 
Leaves and roots of C. album were collected from Wasit University during March 2018. It is identified by at Field Crop Department, College of Agriculture, Wasit University. Herbarium of C. album was prepared and preserved in the Crop field laboratory, Wasit University. The roots have been separated from the stem by a sharp blade and washed by rinsing in tap water. They allowed to dry and then dried by electric oven at 75°C. The plant materials were ground to a fine powder using a mechanical grinder.
 
Fungal pathogens
 
Five isolates of phytopathogens fungi A. alternata, F. solani, R. solani, P. aphanidermatum and S. sclerotium were obtained from the Field Crops Department, Agriculture College, Wasit University, Wasit, Iraq. These fungi have been grown in Potato dextrose agar (PDA) media with autoclaved at 120°C for 20 min.
 
Preparations of water extracts
 
Two hundred gram of C. album were soaked in 1 L of distilled water separately and incubated overnight. Then, filtered through a Whatmann No. 41 filter paper. The filtered extract has been evaporated by a rotary evaporator and oven dried at 45°C. Different concentrations 0, 5, 10 and 15 % of dried powder extraction were prepared from leaves and roots dry powder to 100 ml sterilized distilled water separately. After that, they are ready to use them in inhibition growth of pathogens fungi.
 
Antifungal activity
 
To determine the effects of the leaves and roots of C. album water extract on the mycelia length growth of pathogens in dual-culture techniques. Four concentration 0, 5, 10 and 15 % concentrations have been added as 3 ml for each concentration into 60 ml sterilized P.D.A media (40°C ± 2°C) separately. The media was mixed properly and poured into Petri dishes and then inoculated with 5-mm mycelia plugs of the pathogens placed in the centers of the plates separately. Control treatments consisted of PDA and disks of pathogens, without filtered extract. There were three repeated plates for each concentration per pathogen. The inoculated plates have been incubated at 27°C ± 1°C until the fungus grows completely in the control dish. Percent mycelia growth inhibition was calculated according to the following formula:
 

                Percent inhibition = C – T / C * 100

 
C = Control of radial growth of the pathogen,
T = Treatment of radial growth of the pathogen with leaves or roots of C. album water extract.
 
GC-MS analysis
 
GC-MS 7820A gas chromatography equipped with detector 5977E mass spectrometer (Agilent Technologies, USA). Helium was used as a carrier gas with 99.999% supplied by (BOC, gas, Sydney, AU). Inlet temperature of the instrument was 250°C. Oven was preset at 50°C for 5 min, raised up to 250°C. The total run time for equipment was 45 min. the flow rate was 0.7 ml/min, the split less was 20 ml/min at 1.5 min. The total run time for equipment was 45 min. The compounds were identified by comparison of the mass spectrum with the US National Institute of Standards and Technology (NIST, 2014) database.
 
The samples have been analyzed in biosecurity lab., Murdoch University, Australia. Limit of detection (LOD) has been identified in this study as well. Alkane standard C7-C30 (Supelco, Bellefonte, USA) has been used to identify limit of detection. One microliter of standard was used in injection into GC-MS with 250°C injection port.
 
Statistical analysis
 
In vitro experiments, three replicates were performed for each treatment according to the randomized design of the experimental experiments. The results data were analyzed for the variance using by Principal component analysis (PCA) and One-way Analysis of Variance (ANOVA) methods using online MetaboAnalyst 3.0 a comprehensive tool for metabolomics analysis and interpretation.

Antifungal activity
 
The results of Invitro screening showed that the ability of C. album water extraction to inhibition of radial growth of some fungi pathogens. The percent mycelial inhibition of Alternaria alternata, Fusarium solani, Rhizoctonia solani, Pythium aphanidermatum and Sclerotinia sclerotium reached at 5 % concentration of leaves water extract at 27.90, 8.55, 20.12, 10.73 and 3.52 % respectively, while reached 48.18, 37.62, 62.40, 74.26 and 40.52 % respectively at 10 % concentration and reached 100, 83.60, 100, 9.33 and 91.42% respectively at 15 % concentration. For roots water extract at 5 % concentration, it reached 32.18, 17.76, 21.60, 16.30 and 19.00% respectively, while reached 63.80%, 71.95, 78.10, 80.46 and 85.15.52 % respectively at 10 % concentration and reached 100 % for all fungi at 15 % concentration. Where reached at 15 % concentration of leaves water extract at 100%, 83.60%, 100%, 9.33% and 91.42% respectively, while reached 48.18, 37.62, 62.40, 74.26 and 40.52 % respectively at 10 % concentration. For roots water extract at 15 % concentration, it reached 100 % for all fungi growth. While reached 63.80%, 71.95, 78.10, 80.46 and 85.15.52 % respectively at 10 % concentration. However, mycelia inhibition of all fungi reached 0% at 5 and 0% concentration of both leaves and roots water extract (Table 1).
 

 
There are evidence from earlier studies that various weed species possess antifungal properties, Iqbal et al., (2001) found that the growth of mycelial growth of Phomopsis theae, Aspergillus niger and Rhizoctonia solani was reduce after treated with water extracts of  Phyllanthus debilis, Desmodium trifolium, Ageratum conyzoides, Vernonia cinerea and Oxalis corniculata weed species. Some extracts of C. album such as roots, leaves, stem and inflorescence were examined against F. oxysporum f. sp. cepae at different concentrations (0.5, 1.0,3.0%) extract suppressed pathogen growth by 2.5–100% (Rauf and Javaid, 2013).C. album has been found to have cinnamic acid amide, saponin, phenolic amide, apocortinoid, alkaloid chinoalbicin, lignans, phenols and xyloside as active phytoconstituents (Agrawal et al., 2014). Kamurthy et al., (2016) reported that some compounds extracted from weeds including, phenolic compounds, organic acids, malic acid, steroids, tannins, glycosides, triterpenoids, tartaric acid, alkaloids, flavonoids and citric acid may have the important role on inhibition of C. albicans growth.
 
GC-MS detection
 
GC-MS has been detected seven main compounds belonging to the aromatic dicarboxylic ester, fatty acid methyl esters and ketone groups which water leaves and roots extract from C. album (Table 2). The peak areas for these compounds were 6-methylenebicyclohept- 3-en-2-one (159.984); 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (377.746); 23h-furanone, dihydro-4,4-dimethyl (288.391); hexadecanoic acid, methyl ester (211.644); 9-octadecenoic acid (Z)-, methyl ester (153.649); 9,12-octadecadienoic acid (Z,Z)-, methyl ester (58.281) and farnesene (86.406) (Table 2). The names of compounds are shown alongside comparison with molecular formulas, molecular weights and time retention of the NIST database and peak areas in Table 2. Some of these compounds belong to the methyl ester, methyl ethylethyl, methyl ethyl group, linoleic, Esther and fatty acids (Agoramoorthy et al., 2007., Chandrasekharan et al., 2008., Lima et al., 2011., Hussein et al., 2016).
 

 
Agoramoorthy et al., (2007) have been described that the methyl esters and fatty acid were known to possess antifungal properties. Khurshid et al., (2018) have been evaluated the antimicrobial activity of methanolic leaf, stem and inflorescence extract of Cenchrus pennisetiformis against Fusarium oxysporum f. sp. lycopersici causing wilt disease in tomato, they found that the grass significantly reduced fungal biomass and GC-MS analysis showed the presence of 10 compounds 1-eicosene, 1-propanol2-2-hydroxypropxy, benzofuran 2,3-dihydro, hexadecanoic acid, ethyl-ester, Phenol, 2,4-bis, 1,1-dimethlethyl, Heptanal, 2-[phenylmethylene]-, E-15-heptadecenal, Ethanone, 1-[2,4,5-tri ethoxypenyl]- and Benzene, 1-[1,1-dimethylethyl]-3,5-dimethyl-2,4,6-trinitro. Fatty acid of seeds of Annona cornifolia, have been examined by Lima et al., (2011), palmitic acid methyl ester (16.9%), linoleic acid methyl ester (19.1%) and mainly oleic acid methyl ester (51.5%). Heat map and ANOVA analyses in these experiments have been described the differences of the compounds with their peak areas. The PCA visualizes both the covariance and correlation between the metabolites and the modeled class designation. Thereby the PCA-plot helps to identify statistically significant and potentially biochemically significant metabolites based both on contributions to the model and their reliability. An extension of the PCA, the heat map, was applied to compare the outcome of multiple classification models compared the peak area. The used example is a GC coupled MS-based metabolomics study in extracted samples were extracted from the weed seeds (Fig1). In the current study, the method explain inhibited the growth of five phytopathogenic fungi Alternaria alternata, Fusarium solani, Rhizoctonia solani, Pythium aphanidermatum and Sclerotinia sclerotiurm. Based on Agoramoorthy et al., (2007) showed the ability of fatty acid methyl ester extract of E. agallocha to suppressed growth of four species of Candida including C. parapsilosis, C. tropicalis, C. albicans and C. krusei because it is rich in palmitic acid methyl ester (56.02%). The 6-methylenebicyclo (3.2.0) hept-3-en-2-one has been used to show normalization of the compounds that are significantly influenced 1,2-benzene dicarboxylic 5acid and for mono (2-ethylhexyl) ester has been used successfully from marine metabolites of a cyanobacterium Oscillatoria terebriformis, Alternaria sp. and Streptomyces sp.


 
This compound has been used for a large number of biological studies, including cytotoxicity, anti-inflammatory, antioxidant, antimicrobial and antiviral activity (Mukund et al., 2014., Krishnan et al., 2014; Govindappa et al., 2014). It was a major compound, which has been found in the current study (377.746). Therefore, there was a possibility that it may have had a role in the antifungal activity of water leave and roots extract of C. album in this study.

As a result of the spread of this weed in a wide area of Iraq, it can be used as an environment-friendly fungicide. It can also be managed many important diseases that affect the fields of wheat and barley, which are grown primarily in Iraq instead of using the pesticides. This study concluded that the five phytopathogenic fungi A. alternata, F. solani, R. solani, P. aphanidermatum and S. sclerotium could be managed and control by the water leaves and roots extract of C. album. Antifungal activity of these extracts can be used to control a wide range of phytopathgens.

The authors would like to acknowledge Wasit University and Murdoch University to their support. We would like to thankful Dr. Nabel from Wasit University, Field Crop Department for identifing the weed and for the help in statistical analysis. Many thanks for everyone who helped in this study.