RESEARCH ARTICLE IN VITRO ACTIVITY OF EXTRACTS OF FIVE MEDICINAL PLANT SPECIES ON PLANT PATHOGENIC FUNGI

and vegetables GRAPHICAL ABSTRACT ABSTRACT The antifungal effectiveness of extracts of five medicinal plant species was determined. The inhibitory activity of extracts of Eucalyptus tereticornis , Xanthium sibiricum , Artemisia argyi , Tupistra chinensis and Pyrola calliantha were evaluated against the mycelial growth of Aspergillus niger , Botrytis cinerea, Penicillium digitatum , P. expansum, P. italicum and Rhizopus stolonifer . All plant extracts were prepared at 60°C using solvents (either water, 50% ethanol ( v/v ), 95% ethanol ( v/v ), ethyl acetate or petroleum ether). Fungicidal effects of all plants tested were confirmed. Different extracts from the same plant species gave different degrees of inhibition. All aqueous extracts had weak or no activity on all fungi tested. Ethyl acetate and 95% ethanol extracts from T. chinensis rhizomes gave greater inhibition and a broader spectrum inhibition than the other extracts. T. chinensis may have potential as a new natural fungicide and may be used for the preservation of agricultural and forestry products such as fruits and vegetables.


INTRODUCTION
Many plant pathogens are fungi that cause considerable damage by postharvest decay of fruits and vegetables [1]. Common pathogenic fungi causing postharvest rots are in the genera Aspergillus, Botrytis, Colletotrichum, Penicillium and Rhizopus, with major species being Aspergillus niger, Botrytis cinerea, Colletotrichum musae, Penicillium digitatum, P. expansum, and Rhizopus stolonifer [2].
Postharvest fungal rots affect the quality and shorten the shelf life of fruit and vegetables, leading to considerable economic loss and the waste of important resources. In addition to causing disease in fruit and vegetables, many species of Alternaria, Aspergillus and Penicillium are also sources of mycotoxins that are of concern in animal and human health [3,4]. Aflatoxins produced by Aspergillus may cause DNA damage and liver cancer [5,6] and gliotoxin, citrinin and patulin affect interferon-γ production [7].
Synthetic fungicides have long been the most important method of protecting agricultural and forestry products against fungal damage. However, most synthetic fungicides on the market are toxic and have undesirable effects on non-target organisms in the environment [8]. Furthermore, some synthetic fungicides such as benzimidazole accumulate in plants, waters and soils and can affect humans by transfer via the food chain [9].

Preparation of plant extracts
Ethanol, ethyl acetate and petroleum ether were used to prepare extracts. Dried ground plant material (100 g) was soaked separately in 1 L of water, 50% ethanol (v/v), 95% ethanol (v/v), ethyl acetate, and petroleum ether in 1 L conical flasks sealed with plastic film. All conical flasks were placed in an ultrasonic cleaner for 30 min [18] and allowed to stand for 3 h at 60°C for the extraction process [19]. The supernatant from each flask was filtered through Whatman filter paper and vacuum evaporated at 45-55°C. The plant materials underwent three further extractions to obtain the maximum metabolite yield. Extracts were freeze-dried or vacuum dried, weighed separately, transferred into small vials, and the percentage yield from the extraction calculated. These samples were stored at 4°C before use.

Activation of strains and fungal cultures
Lyophilized fungal strains were revived in accordance with the supplier's instructions. PDA slopes were inoculated with the fungal strains and incubated at 25-28°C in the dark for 7 d then sub-cultured in the same way.
Spores were harvested by adding sterile water and glass spheres (6 mm) to the flask and shaking for 3-5 min. Spore concentration was determined using a hemacytometer [20] and was adjusted to a final concentration of 1 × 10 6 spores mL −1 . Spore suspensions (10 mL) containing 1 × 10 6 spores mL −1 were added to PDA plates and incubated at 25 ± 2°C in the dark for 3-5 d.

Mycelial growth inhibition
Three main steps were followed to assess the mycelial growth inhibition of the plant extracts [21]. First, extracts were dissolved separately into the extraction solvents water, 50% ethanol, 95% ethanol, ethyl acetate and petroleum ether. Carbendazim was dissolved in ethanol. All samples were then diluted and emulsified with 0.1% Tween 80 (cell culture level) to final concentrations of extracts of 10 g·L −1 . Secondly, PDA medium containing 1 g·L −1 solvent extract and a separate control medium with carbendazim (1 g·L −1 ) were prepared. Two types of negative control were also prepared, PDA medium and PDA medium containing the corresponding solvents used for extraction (including the 0.1% Tween 80) from the first step. The final concentrations of the organic solvents were < 2%. The broad-spectrum fungicide carbendazim was used to make comparisons with extracts. Thirdly, plates with extracts (1 g·L −1 ) were inoculated with the six fungal strains using a 6-mm-diameter agar disk (placed centrally) from an actively growing region of the fungus on PDA agar plates. The inoculated plates were incubated in the dark at 25 ± 2°C. Colony diameters were measured using a caliper every 24 h until control plates were filled with fungal growth. The diameter of mycelial growth was calculated using the equation D = D0 -0.6 (cm), where D0 represents the diameter of mycelial growth measured. Mean growth values were converted into the inhibition percentage of mycelial growth relative to the control treatment using the following equation: where Dc and Dt represent mycelial growth diameter in control and treated Petri dishes, respectively. Three replicate plates of each treatment were assessed.

Total phenol and flavonoid contents in the extracts
The total phenol and flavonoid contents in the extracts were assayed as described by Hossain et al. [22]. The absorbance of all samples was measured at fixed wavelengths of 760 nm (total phenol) and 510 nm (total flavonoids) using a UV-visible spectrophotometer. Gallic acid (total phenol) and rutin (total flavonoids) standards were used for the calibration curve.

Statistical analysis
All analysis was carried out on at least three replicates and results are expressed as the mean values ± SE. Analysis of variance (ANOVA) of data and a multiple comparison of mean values using Duncan's multiple range test at the 5% protection level were conducted using the IBM SPSS 20.0 statistical software package. Figure 1 shows that the yield of extraction was significantly affected by the solvent used. It is likely that the solvent polarity was the main factor determining the ability to extract soluble substances. The polar solvents water, 50% ethanol and 95% ethanol extracted greater proportions of plant constituents than the non-polar solvents ethyl acetate and petroleum ether.

Mycelial growth inhibition
The inhibitory effects of 1 g·L −1 crude extracts on mycelial growth were tested on P. expansum, P. italicum, A. niger, P. digitatum, B. cinerea, and R. stolonifer (Fig. 2). A. argyi extract inhibited all the microorganisms tested ( Fig. 2(a)). Except for the aqueous extracts of A. argyi, P. calliantha and X. sibiricum, ethyl acetate extracts of E. tereticornis, and 95% ethanol extracts of X. sibiricum, all extracts had in vitro inhibitory effects on A. niger. T. chinensis extracts using 95% ethanol and ethyl acetate had the greatest inhibitory effect on A. niger (˃ 60%).

A .a rg yi P .c al lia nt ha E .te re tic or ni s X .s ib ir ic um T. ch in en si s C ar be nd az im
Aqueous extracts of all species and all P. calliantha extracts had effectively no inhibitory effect on P. expansum ( Fig. 2(b)). T. chinensis extracts using 95% ethanol and ethyl acetate had stronger inhibitory effects on P. expansum than the other extracts.
The inhibitory effects of the plant extracts on P. digitatum are shown in Fig. 2(c). P. calliantha and E. tereticornis extracts had effectively no inhibitory effect on P. digitatum. Ethyl acetate and 95% ethanol extracts of T. chinensis had greater inhibitory effects on P. digitatum than the other extracts.
All ethyl acetate extracts, and 95% ethanol and petroleum ether extracts from T. chinensis significantly inhibited P. italicum (Fig. 2(d)). Water and 50% ethanol extracts gave less inhibition than the ethyl acetate extracts, 95% ethanol and petroleum ether extracts.
Most crude extracts (except aqueous extracts) significantly inhibited all the fungal pathogens (Fig. 2(e) and Fig. 3). Overall, the mycelial growth inhibition associated with 95% ethanol, ethyl acetate, and petroleum ether extracts of E. tereticornis, X. sibiricum and T. chinensis was greater than for the others tested after 24 h. However, most mycelial diameters of R. stolonifer were ˃ 8.4 cm after 3 d, except with 95% ethanol and ethyl acetate extracts of T. chinensis. Mycelial growth diameters with 95% ethanol and ethyl acetate extracts of T. chinensis were only 2.07 cm and 5.88 cm, respectively, after 5 d (Table 1).  The inhibitory effects of the extracts on B. cinerea are shown in Fig. 2(f) and Fig. 3. All extracts except aqueous extracts inhibited B. cinerea; 95% ethanol and ethyl acetate extracts gave the greatest inhibition of B. cinerea, followed by E. tereticornis extracts. The aqueous extracts gave the greatest mycelial growth.

Model of mycelial radial growth of Botrytis cinerea
The fitted models and equations for mycelial radial growth of B. cinerea with the addition of water, 50% ethanol, 95% ethanol, ethyl acetate and petroleum ether extracts are shown in Fig. 4. The mycelial growth diameter of B. cinerea became linear with time (during the first 6 d). The fitted equation indicates the growth rate and linear correlation coefficients (r).     With the exception of aqueous extract, E. tereticornis extracts had a stronger effect than A. argyi or P. calliantha extracts on growth; the slopes of the mycelial radial growth equation for B. cinerea with the addition of E. tereticornis extracts were ˂ 1.12. The growth inhibition with ethyl acetate and petroleum ether E. tereticornis extracts was greater than with 95% ethanol or 50% ethanol extracts.
The effect X. sibiricum extracts on B. cinerea growth is shown in Fig. 4(d). The inhibition with X. sibiricum extracts was slightly weaker than with extracts from A. argyi, E. tereticornis and P. calliantha; the slopes of the mycelial radial growth equation of B. cinerea with X. sibiricum extracts were ˃ 1.34. The growth inhibition with ethyl acetate and petroleum ether extracts was greater than with 95% ethanol and 50% ethanol extracts.
The effect of T. chinensis extracts on B. cinerea growth is shown in Fig. 4(e). The inhibition with 95% ethanol and ethyl acetate extracts was greater than with petroleum ether and 50% ethanol extracts, and the slopes of the growth equation were ˂ 0.72. Aqueous extract promoted the growth of B. cinerea compared to the control.
In summary, ethyl acetate extracts gave the greatest inhibition of B. cinerea mycelial radial growth of the extracts tested. Conversely, all water extracts gave the least negative effect on growth, or promoted growth. Ethyl acetate and 95% ethanol extracts of T. chinensis gave the greatest inhibition of B. cinerea growth.

Phenol and flavonoid contents of the plant extracts
The total phenol contents of the extracts are given in Fig. 5(a). Total phenol contents in water extracts, 50% ethanol extracts and 95% ethanol extracts from leaves of A. argyi and E. tereticornis were greater than in the other extracts. However, total phenol contents in extracts from T. chinensis were less than the other extracts, which did not parallel their antifungal activity, thus other compounds must be responsible for the antifungal activity of T. chinensis extracts.
Total flavonoid contents in different extracts are shown in Fig. 5(b). Total flavonoid contents in water extracts, 50% ethanol extracts and 95% ethanol extracts from A. argyi were also higher than other extracts. In general, the total flavonoid contents of extracts from T. chinensis were less than the other extracts, in the sequence ethyl acetate extracts > 95% ethanol extracts > 50% ethanol and petroleum ether extracts > water, which effectively parallels their antifungal activity. Flavonoids might be one of the antifungal constituents of extracts from T. chinensis.  [23].
In addition, different solvent extracts from the same plant showed different degrees of inhibitory effect on fungal growth. The ethyl acetate and 95% ethanol extracts of T. chinensis had the greatest antifungal effects, with lower effects of water, 50% ethanol and petroleum ether extracts. This observation agrees with Thembo et al. [24] who found that all extracts except aqueous extracts inhibited Fusarium spp. with the most active being methanol and hexane extracts of Vigna unguiculata and Amaranthus spinosus.
Similarly [25], alcoholic extracts of Melia azedarach had greater antimicrobial activity than methanol, petroleum ether or water extracts. Bakht et al. [26] found that the butanol extracts of Allium sativum gave the greatest inhibition of Bacillus cereus. However, petroleum ether, methanol and water did not inhibit the test microbes.
B. cinerea is a plant pathogen that attacks over 200 crop species worldwide, especially grapes, vegetables and berries [27,28]. B. cinerea is difficult to control because it has a wide range of hosts as inoculum sources and various modes of infection. It can also survive as conidia, mycelia and/or sclerotia for extended periods as in crop debris [29]. The present study indicates that all extracts other than water extracts can inhibit B. cinerea. However, carbendazim, a common fungicide, had almost no in vitro activity on B. cinerea. The 95% ethanol and ethyl acetate extracts of T. chinensis rhizomes and ethyl acetate and petroleum ether extracts of E. tereticornis leaves inhibited B. cinerea by ˃ 50% at 1 mg·mL −1 , indicating that these plant species are good prospects as potential sources of antifungal agents for control of B. cinerea.

CONCLUSIONS
In conclusion, in vitro tests were conducted with plant extracts against six plant pathogenic fungi. The 95% ethanol and ethyl acetate extracts of T. chinensis gave the greatest antifungal activity against these fungi. Based on in vitro tests, 95% ethanol and ethyl acetate extracts of T. chinensis rhizomes might provide good antifungal agents for controlling R. stolonifer and B. cinerea in postharvest fruits. T. chinensis has potential to be a new natural fungicide resource and may be used for the preservation of agricultural and forestry products such as fruits and vegetables. Clearly, further studies are justified on antifungal compounds from plant sources for extending fruit storage.