In vitro antifungal activity of polyphenols-rich plant extracts against Phytophthora cinnamomi Rands

1 Department of Agricultural Parasitology, Universidad Autónoma Agraria Antonio Narro, Buenavista, 25315, Saltillo, Coahuila, México. 2 Department of Food Research, School of Chemistry, Universidad Autónoma de Coahuila, 25000, Saltillo, Coahuila, Mexico. 3 Saltillo Experimental Station, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Buenavista, 25315, Saltillo, Coahuila, México.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License to others species as Eucalyptus and Pinus species (Linde et al., 1997), and pineapple (Allen et al., 1980).Their virulence is associated with temperature between 21 to 30°C, poorly drained soils and excessive moisture.This pathogen is diploid and heterothallic with two groups, A1 and A2 (Linde et al., 1997).
Its control is based on cultural practices, including management of soil moisture and improving ventilation by increasing drainage, and mineral nutrition care.The application of chemicals among which are the fungicides metalaxyl and fosetyl-aluminum to the soil, leaves or trunk injection (Whiley et al., 1986) and biological control agents including bacteria and fungi in soil, as Pseudomonas spp., Streptomyces spp.and Trichoderma spp., Myrothecium roridum, Aspergillus spp., or Paecilomyces spp., are other techniques useful to inhibit P. cinnamomi (Reeves, 1975;Gees and Coffey, 1989;Mass and Kotzé, 1990;Casale, 1990;Stirling et al., 1992;Duvenhage and Kotzé, 1993).
However, these management disease techniques present challenges and constraints in control of the disease, loss in efficiency, increased resistance to active ingredients and environmental hazards, so it is necessary to find new strategies for control, one of these strategies can be use of plant extracts as an alternative (Lira et al., 2007).
Several studies showed that secondary metabolites produced by plants have an effect on inhibiting the development of the mycelium of several pathogenic fungi (Hossein and Maldonado, 1982).Among the synthesis of secondary metabolites or phytochemicals are polyphenols which are a heterogeneous group of molecules having a structure of benzene substituted by various groups with hydroxyl functions, allowing them to be highly soluble is substances such as water.
These compounds are present in extracts of leaves, bark, wood, fruits and galls of certain ferns, gymnosperms and angiosperms (Swain, 1979).Polyphenols are important in plant physiology because they contribute to resistance to microorganisms, insects and herbivorous animals (Haslam, 1996).
Besides, these compounds help to preserve plant integrity during continuous exposure to environmental stressors, including ultraviolet radiation, high temperatures and dehydration (Lira et al., 2007).Polyphenol antioxidants are active in biological systems and probably the capacity or biological value explains its abundance in plant tissues (Meckes et al., 2004).Some plant species like Larrea tridentata, Turnera diffusa, Flourensia cernua, Jatropha diocesan among others are widely distributed in the Mexican Northern States, occupying an area of approximately 100 million hectares (González, 1975).These native plants have a high content of polyphenolic compounds (Lira et al., 2007).Plant extracts obtained with different solvents as methanol, acetone, chloroform, hexane, etc. have been reported with antimicrobial properties.
However, little attention has been given to obtaining polyphenols-rich extracts with unconventional solvents which have potential use in disease management of organic farming.The detected significant differences on the antifungal activity can be due to total polyphenols presents in the plant extracts.This is the first study on use polyphenols-rich plant extracts against P. cinnamomi, because there are some reports where plant extracts are used but to inhibit other Phytophthora species such as: Phytophthora infestans (Gamboa et al., 2003a, b), Phytophthora capsici (Galván, 2005) and Phytophthora palmivora (Mendoza et al., 2007) in vitro.
In addition, Nielsen et al. (2006), reported the effect of natural product derives from Quillaja saponaria which showed activity against root rot until 100% in disease control, this plant is native of desert regions and have high titers of saponins.Saponins have been reported to reduce surface tension in the nutrient solution of hydroponic systems in greenhouses and cause disintegration of the membrane of Phytophthora zoospores.
In this context this paper aims were to determine the in vitro antifungal activity of semi-desert plants extracts on inhibiting mycelial growth of P. cinnamomi and their MIC 50 .[Cactaceae], Lippia graveolens Kunth (Verbenaceae), Carya illinoensis K. Koch (Juglandaceae) and Yucca filifera Chabaud (Agavaceae)) were collected in the Southern region of Coahuila, (semi-desert region) during August and September, 2008.The collected plant material was transferred to the Microbiology Laboratory of The Food Research Department, School of Chemistry, Universidad Autonoma de Coahuila, for dehydration and milling.Dehydration was carried out at room temperature for 10 days and when required in an oven for two days to have moisture content between 5 to 10%, the milling process was carried out in a miller (Thomas Wiley) 1 mm mesh.The obtained fine powder was stored in amber bottles at room temperature until extraction of polyphenolic compounds was done.

Seven
The phytochemical compounds extraction was performed by a solid-liquid procedure, using four solvents (water, ethanol, lanolin and cocoa butter).For hydrophilic solvents group Soxhlet method was used and hydrophobic solvents group infusion method was used.In firth group distilled water and ethanol (70%) were used and second group mineral oil emulsions with 10% lanolin and cocoa butter were used.Each fine powder sample was mixed in a 1:4 (w /v) ratio with the corresponding extracting agent.Soxhlet method was performed in a rotary evaporator at 60°C for 7 h while infusion method was carried out heating the solvent at 60°C, once reached this temperature; the fine powder was added and remained under these conditions during 7 h.After this, extracts were filtered and stored at 5°C in container in ramber bottles until the extracted phytochemical compounds were identified and quantified.
In this case, only condensed and hydrolysable tannins were determined which belong to polyphenols group.Concentration of hydrolysable tannins (HT) was determined by the Folin-Ciocalteu method (Makkar, 1999).Condensed tannins (CT) were spetrophotometrically determined using the method reported by Swain and Hillis (1959).For condensed tannins determination, an aliquot of 0.5 ml of plant extract was placed in a tube, with 3 ml of HCl/butanol (1:9) and 0.1 ml of ferric reagent.
On the other hand, it was added to a tube assay series catechin (standard) in distilled water at different concentrations (0, 200, 400, 600, 800 and 1000 ppm) to determine the reference curve.Tubes were plugged tightly and were heated for 1 h in water bath at 90°C.After that, tubes were leaved to cool and absorbance was read at 460 nm.For hydrolysable tannins determination, a reference curve was done by placing 400 µl of gallic acid at different concentrations (0, 200, 400, 600, 800, and 1000 ppm) in assay tubes.Gallic acid concentrations were prepared using distilled water.Each one of the plant extract were diluted in a test tube respectively, immediately to each tube were added 400 µl of commercial Folin-Ciocalteu reagent, samples were vortexed and held for 5 min.Then 400 µl of NaCO3 (0.01 M) and 2.5 ml of distilled water were added.
Finally, absorbance was read at 725 nm in UV / visible spectrophotometer.Determination of polyphenolic compounds antifungal activity from 28 plant extracts on inhibition of mycelia growth was performed through the poisoned medium technique using different concentrations (ppm) of total polyphenols (hydrolysable plus condensed tannins).The response in inhibition mycelia growth was based in Minimum Inhibitory Concentration (MIC50) defined as: the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism in 50% of radial growth in contrast to control (Kumar et al., 2011).Potato dextrose agar (PDA -Bioxon) as culture medium was used, in this case, volume of each extract according to the final concentration was determined and quantified; this volume was added to a flask with the water volume and PDA and sterilized at 120°C for 15 min, then flasks were left to cool and poured in Petri dishes.Subsequently, 0.5 cm plug P. cinnamomi mycelia 7 days old was add and incubated at 25 ± 2°C, until the untreated control (PDA only) completely covered the Petri dish.The response variable was radial growth (cm).
This data was transformed to percent of mycelia growth inhibition by the following equation P = (CT) / C x 100, where P is inhibition percentage, C is colony diameter of the control treatment and T is the colony diameter of a specific treatment.Treatments were established under a completely randomized design with four replications.
In addition, Probit analysis by maximum likelihood method (Finney, 1971) to determine the minimum inhibitory concentrations at 50% (MIC50) of each extract was used.Data were analyzed using SAS V8.1 software.The MIC90 and MIC50 values were calculated as the 90th and 50th percentile of the minimum inhibitory concentration values and their fiducials limits respective.

RESULTS
The variance analysis detected significant differences on the antifungal activity by effect of polyphenols derives Mexican plants.We observed differences in percentage of mycelia growth inhibition of P. cinnamomi.These percentages in mycelia growth inhibition varied from 0% (control treatment) to 100% in the highest concentration treatment where plant extracts were used.In Figure 1, is shown as totals polyphenol concentration is increase, the algae mycelia growth inhibition also increases.
The antifungal effects of plant extracts on P. cinnamomi were variable.Figure 1a shows that the L. tridentata extracts promoted the high mycelium inhibition until 100% when those was obtained with ethanol and 80% when lanolin was used, while the lowest antifungal effect was observed with cocoa butter and water solvents.It also shows that as total polyphenols concentration increase P. cinnamomi mycelia growth inhibition also increases.
Results obtained with Flourensia cernua indicated that the highest fungal inhibition effect are reached using ethanol and water as solvents, while the lowest fungal inhibition effect was observed when lanolin was used during the extraction (Figure 1b).Although the fungal inhibition effect were equal (100%) with extracts obtained using water and ethanol, the concentrations required in the later case are lower (Figure 1b).
Pecan (C.illinoensis) nut husk extracts showed little or no effects on P. cinnamomi mycelium growth inhibition, the highest inhibition effect (16%) was observed when cocoa butter extracts was used as solvent (Figure 1f).In this case, it was so that the highest (3000 ppm) concentration inhibited only 50% the mycelium growth, extractions where ethanol, water and lanolin were used as solvents no inhibitory effect were observed.This is the first study reporting the use Opuntia ficusindica, Agave lechuguilla, Lippia graveolens and Yucca filifera extracts against P. cinnamomi.The highest fungal inhibition effects (100%) using Opuntia extracts were observed using water as solvents and a polyphenols concentration of 4000 ppm.While little or no mycelia growth inhibition was found with the other solvents (Figure 1c).
Agave extracts showed the best fungal inhibition effect (60%) observed when water was used during extraction at polyphenol concentration of 3000 ppm (Figure 1d).Not mycelium growth inhibition effects were observed with cocoa butter and lanolin emulsions, while inhibition effect (40%) was observed when ethanol was used in the extraction in polyphenols at 40 ppm concentration.The highest mycelium growth inhibition (80%) on P. cinnamomi by Lippia extracts was observed in lanolin at 40 ppm, while no fungal inhibitory effects were observed with aqueous extracts.In general it was observed less than 50% inhibition using ethanol and cocoa butter as solvents (Figure 1e).Yucca extracts showed little or no effect on P. cinnamomi mycelium growth inhibition at the evaluated concentration (Figure 1g).
The results obtained in the present study showed that the plant species has an effect on the level of P. cinnamomi mycelium growth inhibition.Figure 2 shows that the highest (75.3 and 68.1%) mycelium growth inhibition was reached when Flourensia cernua and L. tridentata were used as sources of extracts.On the other hand, all other plant species showed a maximum average effect on fungal inhibition of 30%.In general, it was observed plant and solvent interaction effects on mycelium    1).The extracts that did not inhibit P. cinnamomi mycelia growth are: Y. filifera using both lanolin and cocoa butter as solvents, O. ficus-indica with lanolin, cocoa butter and ethanol as solvents, A. lechuguilla with lanolin and cocoa butter as solvent, L. graveolens with cocoa butter and C. illinoensis with water, lanolin and ethanol as solvents (Table 1).

DISCUSSION
The solvents used permitting the extraction of polyphenols from plants in this study.It was demonstrated the solvents chemical structure interaction in specific manner with the polyphenols type extracted from vegetal tissue.Because it was used two groups of solvents, one highly hydrophilic (water and ethanol) and other hydrophobic (lanolin and cocoa butter) where polyphenols quantity differences obtained can be due to plant genera and solvent in this study (Figure 2).In addition, the polyphenols content in tissue is affected by season of plant tissue recollection, vegetative part, and plant growing conditions (Gamboa et al., 2003a;Hyder et al., 2005).
The differences shows on mycelia growth inhibition by polyphenols can be due to the chemical constitution of the polyphenols extracted associated with solvents (lanolin, cocoa butter, ethanol and water) may be due to the association formed between the hydrophobic region present in their structures, and the lipophilic region of the polyphenolic ester group, in comparison to the hydrophilic region of the water molecule.Lanolin is a complex mixture of esters of sterols, triterpene alcohols, esters of aliphatic alcohols and monohydroxyesters of sterols and triterpenes and aliphatic alcohols (Schlossman and McCarthy, 1978), while cocoa butter is composed by glycerides, mainly oleo-palmitostearin, oleo-distearin, oleodipalmitin, stearo-diolein, palmitodioleintrisaturatedtriolein and triunsaturedtriolein (Beckett, 1994).
On the other hand, results of this study suggest that emulsions obtained with lanolin and ethanol inhibit better this pathogen than extracts using water or cocoa butter as solvents at low concentrations.
The antifungal effect of all extracts on P. cinnamomi inhibition contrast with studies shown by other authors, because research works using different plant species.Gamboa et al. (2003) reported the use L. tridentata extracts against P. infestans and shown an antifungal activity of 100% at concentrations of 4000 ppm.Our results indicated that L. tridentata ethanol-extracts has potential on P. cinnamomi control because it was observed 100% fungal growth inhibition with concentrations as low as 20 ppm (Table 1).Galván (2005) reported 100% inhibition effects on P. capsici using ethanolic resin at concentrations of 500 ppm derives from F. cernua and Gamboa et al. (2003) found mycelium growth inhibition of 67.28% to 20,000 ppm using methanolic extracts against P.infestans.Osorio et al. (2010) mentioned effects in inhibition (100%) on Pythium sp. using C. illinoensis.
In general, it was observed that the polyphenols obtained from plant extracts using different solvents have effects on mycelium growth inhibition of P. cinnamomi.Results obtained with these plant species are similar to those obtained by Gamboa et al. (2003a, b) against Phytophthora spp.and confirm the antifungal activity of polyphenols derives from F. cernua and L. tridentata.
From this study results, it can be inferred that solvent selection play important role on metabolites extraction.The present study showed that aqueous solvents present major antifungal response to Oomycetes (Figure 3).Ethanolic extracts is 20 times better than water and 5 times better than lanolin extracts for have higher effect on P. cinnamomi growth inhibition (Figure 2).Also, lanolin can be the alternative solvent because it showed an interesting effect on polyphenol extraction from L. graveolens and excellent effect on antifungal response.
The MIC 50 obtained with more effective plant extracts on mycelia growth inhibition was such as 20 ppm and derives from L. tridentata and F. cernua.These doses are lower than those needed to in vitro inhibit 100% of P. cinnamomi mycelia growth using a commercial fungicide (Metalaxyl at 750 ppm) (Gamboa et al., 2003b).

Conclusions
It was possible in vitro mycelia growth total inhibition of P. cinnamomi using L. tridentata and F. cernua extracts obtained using ethanol and water, L. graveolens extracts obtained using lanolin.The best concentrations were lower than 20 ppm of total polyphenols.

Figure 2 .
Figure 2. In vitro average effect on inhibition of P. cinnamomi with different plant extracts.

Figure 3 .
Figure 3.In vitro average effect on P. cinnamomi mycelia growth inhibition using different solvents.

Table 1 .
Totals polyphenols minimum inhibitory concentrations (ppm) for inhibit mycelia of P. cinnamomi.Opuntia aqueous extract at 13039.00,Y. filifera ethanol extracts with 5378.00, for C. illinoensis extracts using cocoa butter as solvent with 2887 and L. graveolens ethanolic extracts with 2032 (Table