Abstract: Fusarium oxysporum f. sp. gladioli represent one of the problems of greater importance in the culture of gladiolus. The resistance of this fungus to the fungicides has stimulated the search of new alternatives control measures. The natural plant extracts used in the study were safer to the environment and effective in the control of the plant pathogen tested. The present paper describes the in vitro fungicidal or fungistatic effect of powders (20 mg mL-1) and of aqueou, methanol and hexane extracts (5%) of 15 plant species on the development of F. oxysporum f. sp. gladioli on artificial growth media and volatile compound identification. Twelve plant species showed antifungal activity. The hexane extract of Chenopodium ambrosioides (by its fungicidal activity), the methanol extract with Spondias purpurea and Psidium guajava, as well as the aqueous extract of L. esculenta and Guazuma ulmifolia inhibited the mycelia growth with a percentage superior to 50%. Also, the powders of Byrsonima crassifolia diminished the percentage germination and sporulation of the pathogen. All the species presented antifungal activity in form of methanol extract. The 80% of the plant powders increased the rate of mycelial growth of the fungus. By chromatography of gases and spectrometry of masses, 90 volatile compounds in the powders and extracts were identified that showed activity on the fungus. The majority of the compounds were fatty acids (18.8%), monoterpenes (4.2%), sesquiterpenes (23.6%) and phenolic compound (6.3%). The high chemical diversity of the analyzed plant species, differentially affected the development of the fungus, either for the individual compounds or for synergism of some of them.
INTRODUCTION
Gladiolus is an easy-to-grow flower, especially valued for use in floral arrangements. A number of shades and colors have been developed in Gladiolus flower out of which white, pink and yellow are commonly grown on commercial scale. In Mexico Gladiolus flowers have, become one of the most important of Morelos growers cut flower industry. Fusarium corm rot, or yellows, is caused by the soilborne fungus Fusarium oxysporum f. sp. gladioli. This disease, generates serious economic losses and increases production costs. In México, this fungus is controlled by intensive fungicide applications that could have result in the development of resistant populations of the pathogens to fungicides, detriment of the human health and contamination of the environment. Therefore, use of plant natural products could be an attractive alternative for the management of this disease. In México, the use of plants extracts with fungicidal potential activity on postharvest fungal disease is scarce (Bautista-Banos et al., 2000 a, b). Most of plant species have not been investigated for their properties as fungicides for its use in the control of postharvest diseases. A chart of these Mexican plants has been prepared based on the fungicidal or bactericidal background previously reported in the literature. In vitro studies carried out by Shaukat et al. (2002) reported antifungal activity of Argemone mexicana L. against F. solani and Rhizoctonia solani. Caceres et al. (1993) reported that Byrsonima crassifolia L. inhibited the growth of Aspergillus flavus, Epidermophyton floccosum, Microsporum gypseum and Trichophyton rubrum. The essential oils of Ocimum basilicum L. inhibited Colletotrichum musae and F. proliferatum (Anthony et al., 2004). The aqueous extract of Persea americana Miller and Psidium guajava L. inhibited germination of Rhizopus stolonifer (Bautista-Banos et al., 2000a) and the development of C. gloeosporioides (Banos et al., 2002). The specific objectives of the present work were to determine the in vitro antifungal activity of 15, Mexican plant species on mycelial growth, conidial germination and sporulation of F. oxysporum f.sp. gladioli and to identify the volatile compounds.
MATERIALS AND METHODS
Plant material: This study was carried out in the Biotic Products Development Center in Yautepec, state of Morelos, México in January-December of 2008. Fifteen different plant species corresponding to various plant families and including some species reported with medicinal properties were collected in four different natural habitats within the state of Morelos (Table 1). In this four sampling sites the climate is tempered, wet warm and wet tropical with annual precipitancy of 754.6 to 1187 mm. Once harvested, leaves were cut, discarding damaged or diseased material. Plant material was dipped in 1% sodium hypochlorite, rinsed with distilled water, air-dried, macerated with the aid of a blender and a grinder and stored in amber bottles until further use.
Test microorganism: Fusarium oxysporum f.sp. gladioli were isolated from gladiolus corm rots at Cuautla, Morelos, México and the isolates were maintained on Potato Dextrose-Agar (PDA) in petri plates at temperature 26°C. To maintain biological properties of the fungus, periodic inoculations and reisolations from infected corms were carried out.
Preparation of powders and extracts: To evaluate botanic powders 20 mg mL-1 (w/v) of the macerated material was added to plates containing PDA. Leaves powders (50 g) were extracted with hexane, methanol and water (500 mL) for 24 h in each solvent system at room temperature according to Reyes-Chilpa et al. (1998). After each extraction step, the leaves extracts were filtered and concentrated in a rotary evaporator (Buchi R-114, Labortechnik Flawil, Switzerland) and then stored at 4°C in amber bottles until use. Plant powder and aqueous extracts were added to PDA, autoclaved (15 lb cm-2, 15 min) and poured into Petri plates (100x15 mm) (Bautista-Banos et al., 2000b). The hexane and methanol extracts were added to PDA after sterilization of the media and poured into Petri plates (60x15 mm). A 5 mm agar disc of a 9 days old colony of the pathogen was placed at the center of each plate and incubated at 25°C for 8 to 14 days in the dark. The rate of growth of the fungal colony was calculated as follows: y = bx + a, in which b is the slope of the regression line, is the intercept and x is the time of incubation. The percentage of inhibition was % I= [(C-T) /C] x 100 where, C represents the growth in the non amended control and T in the treatment. There were six replications for each treatment. Control Petri plates contained only PDA and solvent with PDA. Control of PDA was used for comparisons with treated plates. The colony diameter was recorded for each treatment until fungal colonies in the control treatment reached the edge of the plate.
For in vitro studies sporulation and germination of spores were measured as previously described by Bautista-Banos et al. (2000b). Petri dishes from each treatment were rinsed with 10 mL sterile distilled water, the surface scrapped with a glass rod and the filtrate passed through cotton wool. To test spore viability, a 0.5 mL aliquot of spore suspension was placed on a 20 mm diameter agar disk and after 8 h at room temperature (26°C) stained with lactophenol acid fuchsine. Both, sporulation and germination were determined at 400x magnification. The experiment was repeated twice.
| Table 1: | Botanical and common namet radiional usase and natural habitat in Morelos, Mexico |
| Traditional usage: Bactericidal (Ba), parasicidal (Pa) | |
Gas chromatography and spectrometry of masses (CG-MS): In Center of Chemical Investigations of the Autonomous University of the State of Morelos (C.I.Q. U.A.E.M) GC-MS analyses were performed using an Agilent 6890 series GC systems (Agilent Technologies, Santa Clara, CA, USA) coupled to a quadrupole mass spectrometer (model 5973) equipped with an HP5-MS with stationary phase of (5 % phenyl methyl siloxane) capillary column (25 m x 0.20 mm x 0.33 um film thickness). For GC-MS detection electron ionisation with ionisation energy of 70 Ev was used a scan range of 30-550 atomic mass units. Helium was the carrier gas, at a flow rate of 1 mL min-1 with a program of temperature of 40°C during 2 min with an increase of 10°C min-1 until reaching a temp of 260°C, staying thus during 20 min, work with a way splitters 10:1 and the line of transference of masses MSD to 280°C. For the case of the extracts, a solution of 5 mg in 0.5 mL of reliable the respective one was prepared. In the case of plant powders, before injecting it in the chromatograph, they were entered to the Headspace with a temperature of 100°C, after to the Loop to 120°C and the line of transference registred a temperature of 130°C. The components were identified by matching their recorded mass spectra with the data bank mass spectra (NIST-MS Version 1.7a) and by comparing their relative retention indices with those available in the literature.
Statistical analysis: Powders and plant extracts treatments were arranged in a completely randomized design with six replication. Standard analysis of variance (ANOVA) were used to determine the effects of plant extracts on mycelial growth and spore germination. Treatment means were compared using the Least Sgnificant Difference LSD multiple range test (p = 0.05).
Simple linear regression was carried out with data of daily mycelial growth. Sporulation data was analysed using a Kruskal-Wallis (this date did not present normal distribution) test previously to the ANOVA analysis. All the analyses were made in Stat Sigma version 3.5 (Systat Softwere Erkrath, Germany).
RESULTS
There were significant differences in mycelial growth among treatments (p = 0.001) (Table 2), the 80% of plant powders increased the growth rate of the fungus (7.9-8.3). The powders of B. crassifolia, P. guajava, S. purpurea presented the lowest growth rate (5.6-6.9). The aqueous extract of C. ambrosioides showed significantly greater growth rate compared to the control. The aqueous extracts of A. mexicana, B. crassifolia, G. ulmifolia, L. esculenta and O. basilicum showed the lower growth rates (2.4-4.6) with small differences among them. Methanol extracts of all the plant species presented a reduced growth rate compared to the control and ranged 1.7-6.6. Overall, 32 treatments of different powders and extracts diminished significantly (p≤0.001) the mycelial growth, with a percentage of mycelial inhibition that ranged 8 to 100% and 12 of them showed a mycelial inhibition to at least 40%, particularly methanol extracts, followed of the aqueous and hexane extracts. Among them, the hexane extract of C. ambrosioides were the only plant extract that completely inhibited mycelia growth of F. oxysporum f. sp. gladioli. Also the methanol extracts of S. purpurea, P. guajava and aqueous extract L. esculenta had a percentage of inhibition of 57.2-67 %, respectively (Table 3). Similarly to mycelia growth, there were important differences among treatments on the sporulation of the pathogen (p = 0.001) (Table 4), the treatments that produced the lower number of spores mL-1 as compared to the control were powders of B. crassifolia and aqueous extracts of P. guajava.
| Table 2: | Effect of powders and extracts of mexican plant on mycelial growth (mm) of Fusarium oxysporum f.sp. gladioli |
| Means followed by different letter(s) in each column are significantly different by LSD test at p = 0.05; Values in parenthesis indicate growth rate in mm day-1*** p≤0.001,**p≤0.05; nsNot significantl; Means without letter were not compared with the control and the rest of the treatments because was same that control | |
| Table 3: | Effect of powders and extracts of mexican plant on mycelial inhibition (%) of Fusarium oxysporum f.sp. gladioli |
| Values in parenthesis indicate SD of the mean | |
| Table 4: | Effect of powders and extracts of mexican plant on sporulation (spores mL-1) of Fusarium oxysporum f.sp. gladioli |
| Means followed by different letter(s) in each column are significantly different by Dunn`s test at p = 0.05 | |
Aqueous extract of A. diversifolia, C. ambrosioides, I. murucoides, and P.americana, produced a significantly higher (p = 0.05) number of spores mL-1 as compared to the control. The sporulation of F. oxysporum f. sp. gladioli was reduced significantly (p = 0.05) by eight methanol extracts (A. mexicana, B. crassifolia, G. ulmifolia, L. esculenta, O. basilicum, P. guajava, S. purpurea and S. humilis) and ranged 3.23x105 to 5.61x106 spores mL-1. Methanol extracts in where the higher number of spores mL-1 was obtained were those of A. diversifolia, C. pulcherrima, C. ambrosioides, E. americana, I. murucoides, P. americana and P. dulce (Table 4).
The hexanic extracts of B. crassifolia, C. ambrosioides, G. ulmifolia, I. murucoides, P. americana, P. guajava and S. purpurea, had a lower number of spores mL-1 with respect to control treatments (0.0 to 5.81x106 spores mL-1). On the opposite, hexanic extract of A. mexicana, A . diversifolia, C. pulcherrima, E. americana, P. dulce and S. humilis had a higher number of spores mL-1 that the control treatment (5.99x106 spores mL-1). Hexanic extract of C. ambrosioides inhibited the sporulation completely.
Percentage of F. oxysporum f. sp. gladioli conidial germination differed significantly among experimental treatments (p≤0.001). Powders of B. crassifolia (27.8%) and S. humilis (32.2%) had the lower percentage of spore germination. Powders of I. murucoides, A. mexicana, P. dulce, P. americana and C. pulcherrima was significantly lower than that on the control and ranged from 6.33 to 72.9%.
The percentage of conidial germination of F. oxysporum f. sp. gladioli was 52.0, 59.6, 60.1 and 60.6% with aqueous extracts of S. humilis, C. pulcherrima, P. dulce and S. purpurea, respectively. The treatment of S. humilis presented the lower average of germinated conidia (p = 0.05) in the experiment. In addition, treatments with lower percentage of germination that on the control (p = 0.05) were the methanol extract of G. ulmifolia, S. humilis, B. crassifolia, A. mexicana, S. purpurea and C. pulcherima, with percentage ranging 62.4 to 86%. Seven of those treatments of the methanol extracts, had a higher percentage of germination than the control. Concerning hexanic extracts, six treatments reduced (p = 0.05) germination level on the control, these treatments included extracts of C. pulcherrima, I. murucoides, S. purpurea, L. esculenta, A. diversifolia and P. dulce, with percentage of germination from a 57.8 to 75.7%. Treatments with O. basilicum (96.9%) and B. crassifolia (94.8%) reached the highest number of germinated conidia. On the opposite, treatment G. ulmifolia reached only 34.4 % of germination, and nil conidia germinated with C. ambrosioides (Table 5).
In agreement with the effect obtained on the mycelial growth of F. oxysporum f. sp. gladioli, 12 methanol extracts, four hexane extracts, five aqueous extracts and the powders of three plant species were selected to chromatographic analysis. In powders and extracts 90 compounds were identified, which, belong to 17 families of compounds which include, sesquiterpenes (23.6%), fatty acids (18.8%), hydrocarbons (5.8%), phenolic compounds (6.3%), monoterpenes (4.2%) and diterpenes (17.8%).
| Table 5: | Effect of powders and extracts of mexican plants on conidial germination (%) of F. oxysporum f.sp. gladioli after 8 h incubation at 25°C |
| Means followed by different letter(s) in each column are significantly different by LSD test at p = 0.05; *** p≤0.001;* p≤0.05 nsNot significantly | |
| Fig. 1: | Volatile compounds in the powders and extracts identified by chromatography of gases and spectrometry of masses |
Most of identified compounds were soluble in methanol and hexane (Fig. 1); the detected volatil compounds had molecular weights of between 126 to 500 MW. The diterpenes, triterpenes and fatty acids were present in most of the species. On the contrary, the sesquiterpenes were detected only in O. basilicum, P. american, P. guajaba, S. humilis and S. purpurea. The sesquiterpenes were not detected in A. mexicana, B. crassifolia, C. ambrosioides, C. pulcherrima, G. ulmifolia and L. esculenta.
DISCUSSION
The effect of different powders and evaluated extracts, was different according to the stage of development of F. oxysporum f. sp. gladioli, being the most sensitive the mycelial growth. This detrimental effect on mycelial growth must be caused by fatty acids and terpenes presents in the extracts, break of the cellular membrane caused by lipophilic compounds (Cowan, 1999), as most of the species that showed activity against the fungus contained these type of compounds. The methanol extracts of all the plant species tested showed a higher percentage of mycelial inhibition compared with aqueous extracts. This agrees with the report of Alanis et al. (2005) that consider the methanol extracts presented greater activity than the aqueous. This activity must be related to the type of compounds and not to the dissolvent used in the extraction process (Askun et al., 2009) but also the strong capacity of extraction of methanol compared to water (Bhattacharjee et al., 2006). With respect to the effect of the extracts on the conidial germination of F. oxysporum f. sp. gladioli, was the opposite to mycelia growth, the aqueous extracts that have a greater detrimental effect in comparison to the methanol extracts, indicating that the effect of some plant species varies with the fungal structure or stage of development of the pathogen. Montes- Belmont and Prados-Ligero (2006) observed that some extracts affected the mycelial growth of S. cepivorum, whereas others plant species, affected the formation of sclerotia. In the case of the hexane extracts, Qin et al. (2009) one nonpolar dissolvent extract lipophilic substances, obtaining mainly fatty acids. In our study, the hexane extract of C. ambrosioides had a fungicidal effect (complete inhibition of fungal growth) on F. oxysporum f. sp. gladioli. Other works reported a reduction of the rate of mycelial growth when applying the essential oils of this plant on F. oxysporum f. sp. gladioli (Barrera- Necha et al., 2009) and F. oxysporum (Kumar et al., 2007). Reported that the aqueous extracts of this species had a fungistatic effect on A. flavus and an inhibiting effect on its germination. In the present work, the aqueous extracts inhibited the germination of F. oxysporum f. sp. gladioli. Another important compound is the Ascaridole presented in the methanol and hexanic extract of C. ambrosioides in a percentage of 8,92% unlike the methanol extract with only 2,52%. The presence of this compound in the nonpolar extract and the extract of average polarity as is methanol, agrees with MacDonald et al. (2004), who mention that this compounds are extracted mainly with hexane. Rimada et al. (2007) reports that ascaridole is the major active principle of this specie. The hexane extracts of C. ambrosioides had greater number of monoterpenes compound (Pulegone, Ascaridole and Geraniol tiglato) in comparison to the methanol (Table 6), which could increase the activity of ascaridole. The β-sitosterol (10.06%) and δ-sitosterol (24.49 %) were identified only in methanol extracts, previous research reported antifungal activity of these compounds (Kiprono et al., 2000; Ghazala et al., 2004). The relation between composition and antifungal activity of these plant extract may be attributable both to their main more abundant components but also to that presents in small amount. It is possible that they may act synergistically to contribute to the toxicity of the totality of the tested plant extract. Powders, methanol and aqueous extracts of B. crassifolia had effect on the growth, sporulation and germination of the fungus. The major compounds in the methanol extracts was linoleic acid, a fatty acid that has antimicrobial properties (Santoyo et al., 2006). Powders, methanol and hexanic extract of P. guajava inhibited the mycelial growth and sporulation. The methanol extracts included mainly terpenes and fatty acids, groups considered with antimi crobial activity (Cowan, 1999). Methanol extract and powders of S. purpurea inhibited mycelial growth of F. oxysporum f. sp. gladioli and hexanic extract the sporulation. These extracts are characterized by the presence of several terpenes, fatty acids and phenolic compounds which has antimicrobial activity (Deans et al., 1995). With extracts obtained from leafs of this specie Bautista-Banos et al. (2000a) reported inhibition in the germination of R. stolonifer. Methanol extract of P. americana inhibited the mycelial growth of Fusarium sp. in 43.3%, this plant specie, completely inhibited to C. gloesosporoides (Banos et al., 2002), and also showed antibacterial activity (Pasewu et al., 2008; Castillo-Juarez et al., 2009). The presence of sesquiterpenes in P. guajava, S. purpurea and P. americana would be responsible for its antifungal activity.
| Table 6: | Percentage composition of volatile compounds of methanol and hexanic extracts of Chenopodium ambrosioides |
This hypothesis is consistent with Chang et al. (2008) who mentioned that antifungal activity of sesquiterpenes constituents were superior to monoterpenes constituents against different fungi. Reported that the oil of Curcuma longa which consists mainly of sesquiterpenes exhibited a complete mycelia growth inhibition against F. oxysporum.
Aqueous extract of L. esculenta inhibited mycelial growth in 57%, but antifungal or antimicrobial activity have not been reports previously. S. humilis, G. ulmifolia and A. mexicana (aqueous) inhibited mycelia growth and germination of F. oxysporum f. sp. gladioli. Previous studies have demonstrated the antibacterial activity of G. ulmifolia (Argueta, 1994; Camporese et al., 2003) and antifungal activity of A. mexicana (Shaukat et al., 2002; Masood and Ranja 1991). Methanol extracts of P. dulce inhibited the mycelial growth of F. oxysporum f. sp. gladioli in 47,7% and the germination in a 40% (aqueous extract). Barrera-Necha and Bautista-Banos (2002) observed that powders of seeds of P. dulce inhibited mycelial growth of F. oxysporum only 4,9%, and at 10 mg mL-1 stimulates the growth of the fungus. Different studies exist on their activity against pathogenic fungi, as Uromyces appendiculatus (Montes et al., 1990), Botrytis sp., Rhizopus stolonifer and Penicillium digitatum, in form of powders and etanolic extracts (Necha et al., 2002; Banos et al., 2002, 2003) and against C. gloeosporioides (Peraza-Sanchez et al., 2005). Results of this research demonstrated the antifungal potential activity of a range of plant species that could be used as potential antifungal agents for the control of fungal diseases in ornamental plants. Future research will include the isolation of the active compounds of those extracts with fungicidal properties.
CONCLUSION
Present data support the possible use of extracts of the tested plant species, in particular C. ambrosioides, against one important pathogenic fungus as F. oxysporum f. sp. gladioli. Such products of plants would be biodegradable, renewable in nature and safe to human health.