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The effects of laurel (Laurus nobilis L.) on development of two mycorrhizal fungi
International Biodeterioration & Biodegradation 65 (2011) 628e634
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The effects of laurel (Laurus nobilis L.) on development of two mycorrhizal fungi Christos N. Hassiotis*, Evanthia I. Dina Technical University of Larissa, Department Natural Environment and Forestry, 43100 Karditsa, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2011 Received in revised form 17 March 2011 Accepted 18 March 2011 Available online 12 April 2011
Hundreds of aromatic plant species are growing naturally around Mediterranean. Plant essential oils are incorporated in aromatic plant material and follow the litter fall. During litter degradation, the presence of essential oils can affect soil microorganisms. Mycorrhizal fungi have never been investigated so far under the presence of volatile oils. The aim of this study was to explore the effect of aromatic Laurus nobilis L. on development of two mycorrhizal species Glomus deserticola and Glomus intraradices. The response of fungi colonization and host growth were monitored under different concentrations of L. nobilis leaves and essential oil. The major compounds of L. nobilis essential oil were 1,8-cineole (49.6%), sabinene (7.8%), a-pinene (6.0%), eugenole (5.6%), a-terpinyl acetate (5.2%) and b-pinene (5.1%). Both mycorrhizal fungi colonized successfully the host plants whose growth was positively influenced by mycorrhizal fungi. G. deserticola presented higher infection level than G. intraradices. The addition of L. nobilis leaves in the soil resulted in mycorrhiza inhibition. The level of inhibition was positively correlated with the added amount of aromatic leaves in the soil. The essential oil presented a little higher inhibition than the leaves. The presence of this aromatic plant in many different ecosystems could contribute in mycorrhiza inhibition and it is suggested, when it’s possible, reduction of laurel litter before reforestation programs. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: AMF Mycorrhiza Aromatic plants Essential oil Laurus nobilis Allelopathy
1. Introduction Aromatic plants make a remarkable contribution to the flora and vegetation of the Mediterranean environment. Aromatic plants contain essential oils, have the capacity to synthesize, accumulate and emit volatiles that may act as aroma and flavour molecules due to interactions with living organisms. Aromatic plants produce volatile organic compounds (VOCs) diffusing into the atmosphere and the soil. Plant foliage is the source of at least two-thirds of global VOC emissions depending on land characteristics, species composition, foliar density, and other factors (Guenther, 1997). The fade of essential oil during degradation is significant since it is affect soil microorganisms. Plant essential oils are incorporated in plant material and follow the litter fall. Litter deposit depends primarily on the productivity of plant communities, affected by climate, soil fertility, soil water retention and species composition (Pausas, 1997). A growing interest in biologically active volatile chemicals within soil and their effect on microorganisms has been demonstrated (Joner et al., 2001; Prati and Bossdorf, 2004; Bainard et al., 2009). The majority of these chemicals when applied to soil at low concentrations (1 ml g1 of soil) can increase microbial numbers
* Corresponding author. Tel.: þ30 6946 501110; fax: þ30 23920 92221. E-mail address: [email protected] (C.N. Hassiotis). 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.03.006
and their respiration, while at higher concentrations they become inhibitory (Schlesinger and Hasey, 1981; Bowers and Locke, 2000; Kumbhar et al., 2001; Koide et al., 2005). Secondary compounds emanating have been reported to cause a shift in the microbial population structure upon contact with the soil, reducing the number of fungi and increasing the numbers of bacteria (Letessier et al., 2001; Fujii et al., 2005). The importance of mycorrhiza is well documented by several authors. It is the mutualistic symbiosis (non pathogenic association) between soil-born fungi and roots of higher plants (Frank, 1885) to describe the union of two different beings to form a single, morphological organ, in which the plant nourishes the fungus and the fungus the plant. The most common mycorrhiza association is the AM type, found in most plant families. Arbuscular mycorrhizal fungi (AMF) invade only the primary cortex and have not been found to be present in the vascular tissue, secondary cortex or thick fleshy roots of plants, although the outer cortex may be colonized by infection hyphae (Sutton, 1973). AM fungi may play an important role as transport paths for nutrients in nutrient cycling processes (Bowen, 1980; Jehne, 1980; Thangaswamy et al., 2004; Smith and Read, 2008). Investigations of Robinson (1972) in many mycorrhizal fungi and Rose et al. (1983) on ectomycorrhizal fungi stated the sensitivity of mycorrhizal fungi to phenolics. However, if mycorrhizal fungi are able to infect tree roots, they may render trees less
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vulnerable to the negative effects of phenolics (Hanson and Dixon, 1987; Lendzemoa and Kuyper, 2001). Recently Piotrowskia et al. (2008), testing cottonwood foliage and litter extracts on the colonization of AMF, found that all tested compounds significantly reduced AMF colonization but they did not affect colonization of non-AMF root-colonizing fungi. Fungi in soils are also strongly affected by water soluble substances coming from the litter of needles and leaves of trees and herbaceous plants (Persidsky et al., 1965; Dix, 1974; Boufalis and Pellissier, 1994). The water soluble substances produced or leached from humus can affect the metabolism of mycorrhizal fungi in the soil (Rice, 1984; Pellissier, 1993; Brucknera et al., 2003; Weir, 2007). Phenolics and water soluble substances are well documented but it is a question of interest how plant essential oils, even abundant, widespread and widely used have not been investigated against mycorrhizal fungi. Laurus nobilis is a perennial aromatic shrub, abundant in Mediterranean region, rich in essential oil which is reported as toxic to many microorganisms (Hassiotis, 1997; Simic et al., 2004; Erturk, 2006; Unver et al., 2008). Despite ongoing research the role of this aromatic species containing essential oils on mycorrhizal fungi has not been investigated so far. The aim of the present study is (1) to investigate whether and in which way the aromatic L. nobilis (leaf material and essential oil) can affect two species of AM fungi in mycorrhiza establishment, (2) to explore the level of mycorrhiza development according to different treatments with laurel, (3) to investigate if the antifungal activity of L. nobilis is because of its essential oil content and (4) to monitor the growth of host plants (leeks). 2. Materials and methods 2.1. Experimental Leek (Allium porrum L.) was used as host plant because of its fast growth; it’s relatively good response to mycorrhiza and its easy production of plants. Two arbuscular mycorrhizal fungi were used to establish mycorrhiza with leeks. These were Glomus deserticola and Glomus intraradices, both registered in the European Bank of Glomales as BEG 73 and BEG 72 respectively. These inoculants were chosen because of their ability to form abundant internal vesicles, considered by many authors as chlamydospores (Berch, 1988), because of their growth promoting effect on various plants (Planchette and Fortin, 1982; Furlan et al., 1983) and their protective influence against some root pathogens (Caron et al., 1985). The seeds of A. porrum L. were surface sterilized by soaking in 15% H2O2 for 30 min, carefully washed with sterilized water and then germinated in sterile soil (temperature 120 C, pressure 2 atm for 2 h). The seedlings remained for six weeks in the germination pots and then transplanted in new pots. 2.1.1. L. nobilis leaves The experimental potting mixture was made by 6 parts of soil, 9 parts of sand (sterilized as above) and 15 parts of vermiculite. Five different L. nobilis leaves concentrations were used in this experiment. Those treatments were C: 0 g L1, 1: 0.75 g L1, 2: 1.5 g L1, 3: 3 g L1 and 4: 6 g L1 (per litre of soil). The given amount of mature collected and chopped L. nobilis leaves were carefully mixed to the above substrate. The plant material prior was sterilized for 30 min in 15% H2O2. Four leek plants were planted in cylindrical pots (R: 10 cm, H: 15 cm) and placed under controlled conditions, in a very tidy and clean place avoiding any contamination of the host plants. Ten replications (pots) per each treatment were made. The experiment was established in the village of Kato Scholari (N: 40 260 44, E: 23 010 02) near Thessaloniki, Greece on the 1st of March, 2007.
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2.1.2. L. nobilis essential oil The following year the experiment was repeated in the same manner using essential oil instead of plant material from L. nobilis. The essential oil was applied in the soil as described by Hassiotis (2010). Cylindrical pots, as used before, of 4 L volume were used. In the center of the pot, a specific tube construction with tiny holes was placed, allowing the essential oil evaporation radially. The essential oil was extracted by water steam distillation from the same laurel trees used in the previous experiment. Five different L. nobilis essential oil concentrations were used in this experiment. Those treatments were C: 0 mg L1, 1: 6 mg L1, 2: 12 mg L1, 3: 24 mg L1 and 4: 48 mg L1 (per litre of soil). These oil concentrations were respective to the essential oil amount that was incorporated in laurel leaves that were used in the previous experiment. The essential oil was applied in an absorbent paper stick and then was placed into the tube construction. The paper sticks were replaced every two weeks. This experiment was initiated on the 1st March, 2008. 2.2. Mycorrhiza assay The root samples were extracted by using a cylindrical corer (10 mm). The soil was removed by soaking the roots in water and gently washing them, to ensure that all the thinner roots and tips remained intact. The staining procedure was made according to Vierheilig et al. (2005) modified to parameters of the present study. The roots were cut into small pieces and placed in a beaker (5% KOH) for 30 min in a water bath at 90 C. The roots were then rinsed with tap water and acidified with 5% lactic acid at room temperature for 12 h. Finally the roots were stained with a solution consisting of 875 ml of laboratory lactic acid, 63 ml of glycerin, 63 ml tap water and 0.1 g acid fuscin for 30 min at 70 C and then destained in laboratory lactic acid for 15 min. 10 root segments were mounted onto slides and examined at 100e400 magnification under a Nikon YS100 microscope. Beneath the glass slide an acetate film with 10 thin lines was adapted. At crossing points between roots and lines, each point that had an infection was recorded and the number of infections was expressed as percentage. 2.3. Inoculation, root infection and growth recording Six weeks after transplant, leeks were checked for absence of infection, and inoculated. The AM fungi used in this assay were G. intraradices (Schenck and Smith) and G. deserticola (Trappe, Bloss, and Menge). Sixteen grams of active inoculum were placed in every pot, divided in four inoculation points. The final 50 pots (10 pots 5 treatments with L. nobilis) used as control without inoculation with fungi. At inoculation, and every fifteen days afterwards, root colonization, stem diameter and height of leeks were measured and recorded. Four glass slides from every pot were prepared in order to evaluate root colonization. The host growth was evaluated as average of leek stem diameter and height of leek. 2.4. Essential oil analysis 400 g of fresh and chopped plant material was steam distilled in a 2 L water steam distillation unit for 90 min, at 100e105 C and a flow rate of approximately 8.5 mL min1 (Furnis et al., 1989). The amount of oil was expressed (w/w) as essential oil/fresh raw material. The essential oil was collected and its volatile constituents were established by GCeMS analysis. GCeMS analysis was performed on a Shimadzu GC-2010 e GCMS-QP2010 system operating in EI mode (70eV) equipped with a split/splitless injector (230 C), a split ratio 1/30, using a fused silica HP-5MS capillary column
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(30 m 0.25 mm (i.d.), film thickness: 0.25 mm). The temperature cycle was initiated at 50 C for 5 min and was programmed to 290 C with increments of 4 C per min. Helium was used as a carrier gas at a flow rate of 1.0 mL min1. Injection volume of each sample was 1 mL. Retention indices for all compounds were determined according to the Van den Dool and Kratz (1963) approach using n-alkanes as standards. The identification of the components was based on comparison to their mass spectra with those of NIST21 and NIST107 (Massada, 1976) and those described by Adams (2001) as well as by comparison of their retention indices with literature data (Davies, 1990; Bisio et al., 1998). Eight samples of 400 g of L. nobilis were distilled and analyzed in order to determine the chemical composition of the oil. 2.5. Statistical analysis Experimental data was performed with Statistical Package for Social Sciences (SPSS, version 12, Chicago, IL, USA). The significance of time post inoculation and fungi species on colonization was determined by one-way analysis of variance (ANOVA). Two-way ANOVA was employed to test for significant differences between time (weeks) and fungi species. Where the F values were significant, Fisher’s protected least significant difference (LSD) was used to test for differences among treatments (different concentrations of laurel leaves and laurel oil) and control. The Pearson correlation was used to explore the level of relationship between the different concentrations of laurel leaves and laurel oil and mycorrhiza colonization and plant growth. All analyses were evaluated at a ¼ 0.05 significance level. The percentage of fungi colonization presented in Table 1, Fig. 1 is mean values of 40 measurements (10 pots 4 glass slides) of each fungi species and control (non inoculated plants). The number of host growth presented in Table 1, Fig. 2 is mean values of 80 measurements (10 pots 4 plants 2 parameters (stem and height)) of each fungi species and control. The compound percentage presented in Table 2 is mean values of 16 measurements (8 distillations 2 GCeMS runnings).
3. Results and discussion 3.1. Mycorrhiza infection The leeks infection by mycorrhizal fungi was recorded periodically every fifteen days after inoculation with fungi. The leeks responded immediately to arbuscular mycorrhizal fungi and symbiosis was established. This happened successfully for both fungi G. deserticola and G. intraradices used for inoculation. Fifteen days after inocula introduction the infection level was 4.2% and 3.2% respectively (average for all treatments with laurel leaves) in the first experiment used different concentration of L. nobilis mature leaves. The colonization level was progressively increased during the experiment resulting in a final average infection percentage of 89.4% for G. deserticola and 69.0% for G. intraradices, twelve weeks after inoculation (Table 1). A high correlation was found between days of inoculation and fungi colonization (r ¼ 0.969 for G. deserticola and r ¼ 0.961 for G. intraradices, P < 0.05). It was also observed that G. deserticola presented higher colonization level than G. intraradices. Comparing the ability of the two AM fungi to colonize the leek roots, it was found that G. deserticola presented 16% higher colonization level than G. intraradices twelve weeks post inoculation. The differences in the colonization levels between the AMF (P < 0.01) and between the weeks after inoculation (P < 0.001) were statistically significant (Table 1). In a similar way, the host plants and fungi responded in the second year of the experiment, where L. nobilis essential oil was used instead of mature leaves. It is highlighted that the amount of essential oil used in every treatment was respective to the essential oil amount that was incorporated in laurel leaves and used in the previous experiment. Among other objectives this evaluation was targeting to investigate, if the antifungal activity of laurel leaves demonstrated in the previous year was due to essential oil presence. The leeks responded immediately to arbuscular mycorrhizal fungi and symbiosis was established. Even statistically not significant (P > 0.05) the colonization level between host and fungi in this
Table 1 Colonization level of two arbuscular mycorrhizal fungi and host growth treated with different concentrations of Laurus nobilis leaves and different concentrations of Laurus nobilis essential oil. Weeks after inoculation
Laurus nobilis leaves
Laurus nobilis essential oil
Fungi colonization (%)
Host growth (%)
Average
Min
Max
Glomus deserticola 0 2 4 6 8 10 12
0.0 4.2 11.6 45.0 69.0 85.0 89.4
0.0 3.0 9.0 30.0 45.0 60.0 62.0
0.0 5.0 15.0 60.0 95.0 100.0 100.0
Glomus intraradices 0 2 4 6 8 10 12
0.0 3.2 8.9 34.8 53.4 65.5 69.0
0.0 2.0 6.0 20.1 30.2 40.2 41.5
0.0 4.3 12.9 51.6 81.7 86.0 86.0
Significance Weeks AMF Weeks AMF
*** ** **
*** ** **
*P < 0.05, **P < 0.01, ***P < 0.001.
*** ** **
Fungi colonization (%)
Host growth (%)
Average
Min
Max
0.0 3.3 7.5 13.1 20.6 24.7 26.3
0.0 3.8 10.4 40.5 62.1 76.5 80.5
0.0 2.7 8.1 27.0 40.5 54.0 55.8
0.0 4.5 13.5 54.0 85.5 90.0 92.1
0.0 3.0 6.8 11.8 18.5 22.3 23.7
0.0 3.2 7.1 11.4 16.5 21.4 23.1
0.0 2.8 7.8 30.3 46.4 57.0 60.0
0.0 1.7 5.2 17.5 26.2 35.0 36.1
0.0 3.7 11.2 44.9 71.1 74.8 75.8
0.0 2.8 6.1 10.0 14.4 18.6 20.1
** * *
*** ** **
*** ** **
*** ** **
** * *
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631
120.0
colonization level (%)
100.0
a a
a a
a ab b a
bc
80.0
a a
a
a
a
c
b
60.0
d
bc c
40.0 d
20.0
0.0 C
1
2
3
4
C
1
2
3
4
Treatments with L. nobilis leaves and Fig. 1. Colonization level twelve weeks after inoculation with two arbuscular mycorrhizal fungi subjected to different concentrations of Laurus nobilis leaves (C: 0 g L1, 1: 0.75 g L1, 2: 1.5 g L1, 3: 3 g L1 and 4: 6 g L1) and essential oil (C: 0 mg L1, 1: 6 mg L1, 2: 12 mg L1, 3: 24 mg L1 and 4: 48 mg L1) per litre of soil (The amount of essential oil incorporated in L. nobilis leaves is the same with the essential oil amount that was applied pure in the respective concentration). Different letters within a parameter (AMF) denote significant differences (LSD, P ¼ 0.05) according to essential oil concentrations.
experiment was lower compared to the respective weeks of the previous experiment for both fungi species (Table 1). This observation led us to thought that the essential oil vapours from laurel were presented more effective against fungi than the total leaf with the same amount of oil. The storage of plant essential oil in glands in the leaves may trap the vapours of the oil, allowing gradual release and consequently the aromatic leaf is less effective than the essential oil itself. The final average colonization percentage was reached at 80.5% for G. deserticola and 60.0% for G. intraradices, twelve weeks after inoculation. A high correlation was found between days of inoculation and fungi colonization (r ¼ 0.957 for G. deserticola and r ¼ 0.941 for G. intraradices, P < 0.05). It was also observed that G. deserticola presented higher colonization level than G. intraradices. Fig. 1 summarizes the colonization levels for G. deserticola and G. intraradices according to 5 different concentrations of laurel leaves and the respective concentration of laurel essential oil twelve weeks after inoculation. G. deserticola presented higher infection level than G. intraradices between the treatments and
reached at 100% for G. deserticola in treatment 1 in the first experiment (0.75 g L1 leaves) and in the second (6 mg L1 essential oil). For the particular treatment 1, G. intraradices reached at 82.3% in the first and 83.0% in the second respectively. A remarkable point is that the higher colonization levels being always observed for both fungi in treatment 1 and not in control leading to the indication that a small amount of L. nobilis leaves (0.75 g L1) or essential oil (6 mg L1) can enhance fungi infection. The lower mean colonization level was recorded for either fungi in treatment 4 with higher concentration of laurel leaves (6 g L1) or essential oil (48 mg L1). Values with common letter within colonization levels are not significantly different according to Fisher’s LSD test (P ¼ 0.05). A reverse correlation was observed in progressively increased amount of laurel leaves and oil in the substrate and level of mycorrhiza colonization (r ¼ 0.919 for G. deserticola and r ¼ 0.967 for G. intraradices, P < 0.05). The differences between the highest and the lowest colonization values were 62.3% for G. deserticola and 83.3% for G. intraradices. It is accomplished that G. intraradices not only presented lower infection than G. deserticola
40.0 35.0
Host growth (%)
30.0 25.0 20.0 15.0 10.0 Linear (Without AMF) Poly. (G. intraradici)
5.0
Linear (Without AMF) Poly. (G. intraradici)
Poly. (G. deserticola)
Poly. (G. deserticola)
0.0 C
1
2
3
Treatments with L. nobilis leaves
4
C
1
2
3
4
Treatments with L. nobilis essential oil
Fig. 2. Host growth twelve weeks after inoculation with two arbuscular mycorrhizal fungi according to five different treatments with Laurus nobilis leaves and essential oil. Error bars are also presented.
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but it was also stronger influenced by the presence of L. nobilis leaves and essential oil. The differences in the infection levels, according to the treatments with L. nobilis were statistically significant (P < 0.01) for both experiments. The interaction between treatments with L. nobilis and fungi species resulted in statistically significant differences (P < 0.01) according to ANOVA test. 3.2. Leeks growth The average leek growths after inoculation with arbuscular mycorrhizal fungi for inoculated and non inoculated leeks according to the different treatments with laurel are presented in Fig. 2 for both experiments. There was a remarkable growth of leeks (P < 0.01) after inoculation with the AM fungi in contrast to none inoculated. The average host growth (for all treatments) twelve weeks post inoculation for those were inoculated with G. deserticola was 26.3%, for G. intraradices 23.1% and for non inoculated 15.4% in the first experiment that L. nobilis leaves were used; In the second experiment (with essential oil) the respective growth values were 23.7%, 20.1% and 12.3%. Non inoculated leeks in the first experiment with L. nobilis leaves presented better growth as the concentration of leaves gradually increased leading to the thought that nutrients incorporated in leaves may used as nutrient tank for leeks growth. This was not observed in the experiment with the essential oil. Even these differences were statistically non significant (P > 0.05) and this idea needs further investigation with different experimental design. For inoculated leeks it was observed a positive correlation between AM fungi infection level and leeks growth (r ¼ 0.975 for G. deserticola and r ¼ 0.908 for G. intraradices, P < 0.05). 3.3. Essential oil analysis The steam distillations yielded an average oil amount of 3.2 g/ 400 g fresh leaves of L. nobilis which is expressed as 0.8% (w/w) of the total plant material. The amount of essential oil was incorporated in the different treatments with L. nobilis was 24 mg pot1, 48 mg pot1, 96 mg pot1 and 192 mg pot1 for treatments 1, 2, 3 and 4 respectively. Nineteen compounds contributing more than 0.5% of the total oil were identified and evaluated by GCeMS analyses comprising a final percentage of 97.74 of the total oil (Table 2). The small differences observed in essential oil composition between replications and the years of the study are given as standard deviation. The major compound of L. nobilis essential oil was 1,8-cineole (49.60%), followed by sabinene (7.80%), a-pinene (5.96%), eugenol (5.60%), a-terpinyl acetate (5.25%) and b-pinene (5.12%). These six compounds comprise the 76.33% of the total oil. Our data is in agreement with many other authors working on L. nobilis essential oil in surrounding countries like Italy (Flamini et al., 2007), Turkey (Dadalioglu and Evrendilek, 2004; Kilic et al., 2004; Ozcan and Chalchat, 2005) and Croatia (Politeo et al., 2007). The toxicity or the repellent effect of L. nobilis against microorganisms is well established. In this study the antifungal activity of L. nobilis against two AMF was tested and was found high, especially in concentrations of 24 and 48 mg essential oil L1 soil. Recently Fawzi et al. (2009) working in vitro, testing five plant extracts against pathogenic fungi Alternaria alternata and Fusarium oxysporum found that L. nobilis can be successfully used for antifungal control. He has also reported that plant extracts can be used as natural fungicides to control pathogenic fungi, thus reducing the dependence on the synthetic fungicides. Unver et al. (2008) working with L. nobilis methanolewater extracts showed that these had very high antimycotic activity against all tested yeasts. Simic et al. (2004) examined the antifungal activity of some
Table 2 Chemical composition of Laurus nobilis essential oil. Compoundsa
RIb
Composition (%) SDc
a-Thujene a-Pinene
926 932 946 968 974 985 1024 1027 1029 1100 1146 1190 1292 1305 1307 1355 1472 1405 1421
0.91 5.96 1.21 7.80 5.12 1.62 1.52 2.32 49.60 1.50 2.12 1.90 0.60 0.91 5.25 5.60 1.10 2.10 0.60 97.74
Camphene Sabinene b-Pinene b-Myrcene p-Cymene Limonene 1,8-Cineole Linalool Terpinen-4-ol a-Terpineol Linalyl acetate Bornyl acetate a-Terpinyl acetate Eugenol b-Elemene Methyleugenol b-Caryophyllene
0.01 0.14 0.04 0.09 0.12 0.04 0.02 0.07 0.89 0.04 0.08 0.11 0.01 0.02 0.13 0.17 0.07 0.10 0.04
a In table are presented compounds contributing more than 0.5% of the total oil. Compounds listed in order of elution from an HP-5MS column. b Retention indices as determined on an HP-5MS column using a homologous series of n-alkanes. c Standard deviation.
essential oils from Lauraceae family against 17 micromycetes (among the tested fungal species were food poisoning, spoilage fungi, plant and animal pathogens) and found first Cinnamomum zeylanicum and second L. nobilis to be more toxic against them. Even though the essential oil of L. nobilis reported as toxic to microorganisms in the present study was found that small amounts of plant leaves or pure essential oil were beneficial for mycorrhiza development. This happened because of the volatile oil compounds which were contained in laurel material and were used as energy source for fungi. Kaplan and Hartenstein (1979) and Harder and Foß (1999) found that low concentrations of toluene in cultural media encouraged the growth of 19 species of bacteria and fungi which used toluene as their only source of carbon. In 1984, Vokou et al. (1984) working with Coridothymus and Satureja showed that addition of these volatile oils to the soil significantly activated soil respiration. These data were later confirmed with other volatile compounds (Vokou and Liotiri, 1999; Vokou et al., 2002). Our results support the opinion that small amounts of aromatic material or volatile compounds can be used as energy source by fungi. The antifungal activity of L. nobilis is strongly linked with the presence of essential oil and particularly by its main compounds. Halligan (1975) has reported that the two most toxic components against microbes were camphor and 1,8-cineole and these two compounds contributed the major toxicity in the field. Riaz et al. (1989) found that 1,8-cineole and eugenol were the main compounds of L. nobilis oil and the oil to be toxic against soil bacteria. In 1991, Shaaya et al. (1991) demonstrated that 1,8-cineole was the most effective compound against Rhyzopertha dominica and Oryzaepfilus surinamensis. Kivanc and Akgul (1986) working with cumin, laurel and oregano and Bounatirou et al. (2007) showed that these aromatics had the most toxic oil against Staphylococcus aureus and Proteus vulgaris which were more sensitive. Two other compounds could possibly created mycorrhiza inhibition c et al. (2009) working with were sabinene and a-pinene. Sokovi Daucus carota and its antibacterial and antifungal properties against eight bacterial and eight fungal strains found strong inhibition of the essential oil whose main constituents were a-pinene (7.05e51.23%) and sabinene (2.68e36.69%). Also Singh et al. (2008) working with essential oil of Elettaria cardamomum tested and
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found strong antifungal activity against the food-borne fungi Aspergillus terreus, Penicillium purpurogenum, Fusarium graminearum and Penicillium madriti. The major compound of E. cardamomum essential oil was a-terpinyl acetate with 44.3%. Unfortunately there is no relative work done so far on AM fungi inhibition related to essential oils to compare our data with, so this study is an attempt to investigate the influence of one aromatic plant species on mycorrhiza development. Neither data concerning the antifungal activity of individual compounds composing the laurel oil against AM fungi are available. So we tried to report the antifungal activity which is being presented by other essential oils whose the major compounds are common with the oil of L. nobilis. Moreover, our approach e to grow the host plants in flower pots and let the AMF species to contaminate the roots under different concentrations of L. nobilis leaves and essential oil e represented a more realistic test of allelopathy than bioassays. In order to quantify which of the compounds were responsible for allelopathy effect a new experiment is needed in order to evaluate the antifungal activity of laurel oil individual compounds. It is assumed from our data that the major compounds of laurel essential oil were responsible for mycorrhiza inhibition. It is beyond any doubt that the management of native AM fungi is an effective strategy in revegetation of semiarid areas with shrub species (Caravaca et al., 2005). Combined treatments with mycorrhizal inoculation and composted residue are effective for increasing the growth and the N and P contents in shoot tissues, and could facilitate the establishment and development of new plants in the surrounding area (Caravaca et al., 2003). The abundant presence of aromatic L. nobilis in many different ecosystems around Mediterranean, as resulted from the present study, can generate allelopathy effect against mycorrhizal fungi. It is suggested, when it is possible, to reduce the degraded litter of this species accumulated every year in these ecosystems, allowing the development of mycorrhiza and resulting in more successful new vegetation establishment. Acknowledgements We would like to thank Missis Elena Koutsilieri, Aristotle University of Thessaloniki, for helping in statistical analysis, and Dr. Elpida Bombi for English corrections and linguistic improvements in the manuscript. References Adams, R., 2001. Identification of essential oil components by gas chromatography/ mass spectroscopy. Allured Publishing Co, Carol Stream, Illinois. Bainard, L.D., Brown, P.D., Upadhyaya, M.K., 2009. Inhibitory effect of tall hedge mustard (Sisymbrium loeselii) allelochemicals on rangeland plants and arbuscular mycorrhizal fungi. Weed Science 57, 386e393. Berch, S., 1988. Compilation of the endogonaceae. Mycologue Publication, Waterloo, Canada, pp. 238. Bisio, A., Ciarallo, G., Romussi, G., Fontana, N., Mascolo, N., Capasso, R., Biscardi, D., 1998. Chemical composition of essential oils from some Salvia species. Phytotherapy Research 12, S117eS120. Boufalis, A., Pellissier, F., 1994. Allelopathic effects of phenolic mixtures on respiration of two spruce mycorrhizal fungi. Journal of Chemical Ecology 20, 2283e2289. Bounatirou, S., Smiti, S., Miguel, M.G., Faleiro, L., Rejeb, M.N., Neffati, M., Costa, M.M., Pedro, L.G., 2007. Chemical composition, antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link. Food Chemistry 105, 146e155. Bowen, G.D., 1980. Mycorrhizal roles in tropical plants and ecosystems. In: Mikola, P. (Ed.), Tropical mycorrhizal research. Clarendon Press, London, pp. 165e190. Bowers, J.H., Locke, J.C., 2000. Effect of botanical extracts on the population density of Fusarium oxysporum in soil and control of Fusarium wilt in the greenhouse. Plant Disease 84, 300e305. Brucknera, D.J., Lepossa, A., Herpai, Z., 2003. Inhibitory effect of ragweed (Ambrosia artemisiifolia L.) e Inflorescence extract on the germination of Amaranthus hypochodriacus L. and growth of two soil algae. Chemosphere 51, 515e519.
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Pausas, J.G., 1997. Litter fall and litter decomposition in Pinus sylvestris forests of the eastern Pyrenees. Journal of Vegetation Science 8, 643e650. Pellissier, F., 1993. Allelopathic effect of phenolic acids from humic solutions on two spruce mycorrhizal fungi: Cenococcum graniforme and Laccaria laccata. Journal of Chemical Ecology 19, 2105e2114. Persidsky, D.J., Loewenstein, H., Wild, S.A., 1965. Effects of extracts of prairie soils and prairie grass on the respiration of ectotrophic mycorhizae. Agronomical Journal 57, 311e312. Piotrowskia, J.S., Morfordb, C.L., Rilliga, M.C., 2008. Inhibition of colonization by a native arbuscular mycorrhizal fungal community via Populus trichocarpa litter, litter extract, and soluble phenolic compounds. Soil Biology and Biochemistry 40, 709e717. Planchette, V.F., Fortin, A.J., 1982. Effects of different endomycorrhizal fungi on five host plants grown on calciend clay. Journal of the American Society for Horticultural Science 107, 535e538. Politeo, O., Jukic, M., Milos, M., 2007. Chemical composition and antioxidant activity of free volatile aglycones from bay (Laurus nobilis L.) compared to its essential oil. Croatia Chemica Acta 80, 121e126. Prati, D., Bossdorf, O., 2004. Allelopathic inhibition of germination by Alliaria petiolata (Brassicaceae). American Journal of Botany 91, 285e288. Riaz, M., Asshraf, C., Chaudhary, F.M., 1989. Studies of the essential oil of the Pakistani Laurus nobilis Linn. in defferent seasons. Pakistan Journal of Scientific Research 32, 33e35. Rice, E.L., 1984. Allelopathy. Academic Press, Orlando, Florida, p. 422. Robinson, R.K., 1972. The production by roots of Calluna vulgaris of a factor inhibitory to growth of some mycorrhizal fungi. Journal of Ecology 60, 219e224. Rose, S.L., Perry, D.A., Pilz, D., Schoeneberger, M.M., 1983. Allelopathic effects of litter on the growth and colonization of mycorrhizal fungi. Journal of Chemical Ecology 9, 1153e1162. Schlesinger, W.H., Hasey, M.M., 1981. Decomposition of chaparral shrub foliage: Losses of organic and inorganic constituents from deciduous and evergreen leaves. Ecology 62, 762e774. Shaaya, E., Ravid, U., Paster, N., Juven, B., Zisman, U., Pissarev, V., 1991. Fumigant toxicity of essential oils against four major stored products insects. Journal of Chemical Ecology 17, 499e504.
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The effects of laurel (Laurus nobilis L.) on development of two mycorrhizal fungi Christos N. Hassiotis*, Evanthia I. Dina Technical University of Larissa, Department Natural Environment and Forestry, 43100 Karditsa, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2011 Received in revised form 17 March 2011 Accepted 18 March 2011 Available online 12 April 2011
Hundreds of aromatic plant species are growing naturally around Mediterranean. Plant essential oils are incorporated in aromatic plant material and follow the litter fall. During litter degradation, the presence of essential oils can affect soil microorganisms. Mycorrhizal fungi have never been investigated so far under the presence of volatile oils. The aim of this study was to explore the effect of aromatic Laurus nobilis L. on development of two mycorrhizal species Glomus deserticola and Glomus intraradices. The response of fungi colonization and host growth were monitored under different concentrations of L. nobilis leaves and essential oil. The major compounds of L. nobilis essential oil were 1,8-cineole (49.6%), sabinene (7.8%), a-pinene (6.0%), eugenole (5.6%), a-terpinyl acetate (5.2%) and b-pinene (5.1%). Both mycorrhizal fungi colonized successfully the host plants whose growth was positively influenced by mycorrhizal fungi. G. deserticola presented higher infection level than G. intraradices. The addition of L. nobilis leaves in the soil resulted in mycorrhiza inhibition. The level of inhibition was positively correlated with the added amount of aromatic leaves in the soil. The essential oil presented a little higher inhibition than the leaves. The presence of this aromatic plant in many different ecosystems could contribute in mycorrhiza inhibition and it is suggested, when it’s possible, reduction of laurel litter before reforestation programs. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: AMF Mycorrhiza Aromatic plants Essential oil Laurus nobilis Allelopathy
1. Introduction Aromatic plants make a remarkable contribution to the flora and vegetation of the Mediterranean environment. Aromatic plants contain essential oils, have the capacity to synthesize, accumulate and emit volatiles that may act as aroma and flavour molecules due to interactions with living organisms. Aromatic plants produce volatile organic compounds (VOCs) diffusing into the atmosphere and the soil. Plant foliage is the source of at least two-thirds of global VOC emissions depending on land characteristics, species composition, foliar density, and other factors (Guenther, 1997). The fade of essential oil during degradation is significant since it is affect soil microorganisms. Plant essential oils are incorporated in plant material and follow the litter fall. Litter deposit depends primarily on the productivity of plant communities, affected by climate, soil fertility, soil water retention and species composition (Pausas, 1997). A growing interest in biologically active volatile chemicals within soil and their effect on microorganisms has been demonstrated (Joner et al., 2001; Prati and Bossdorf, 2004; Bainard et al., 2009). The majority of these chemicals when applied to soil at low concentrations (1 ml g1 of soil) can increase microbial numbers
* Corresponding author. Tel.: þ30 6946 501110; fax: þ30 23920 92221. E-mail address: [email protected] (C.N. Hassiotis). 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.03.006
and their respiration, while at higher concentrations they become inhibitory (Schlesinger and Hasey, 1981; Bowers and Locke, 2000; Kumbhar et al., 2001; Koide et al., 2005). Secondary compounds emanating have been reported to cause a shift in the microbial population structure upon contact with the soil, reducing the number of fungi and increasing the numbers of bacteria (Letessier et al., 2001; Fujii et al., 2005). The importance of mycorrhiza is well documented by several authors. It is the mutualistic symbiosis (non pathogenic association) between soil-born fungi and roots of higher plants (Frank, 1885) to describe the union of two different beings to form a single, morphological organ, in which the plant nourishes the fungus and the fungus the plant. The most common mycorrhiza association is the AM type, found in most plant families. Arbuscular mycorrhizal fungi (AMF) invade only the primary cortex and have not been found to be present in the vascular tissue, secondary cortex or thick fleshy roots of plants, although the outer cortex may be colonized by infection hyphae (Sutton, 1973). AM fungi may play an important role as transport paths for nutrients in nutrient cycling processes (Bowen, 1980; Jehne, 1980; Thangaswamy et al., 2004; Smith and Read, 2008). Investigations of Robinson (1972) in many mycorrhizal fungi and Rose et al. (1983) on ectomycorrhizal fungi stated the sensitivity of mycorrhizal fungi to phenolics. However, if mycorrhizal fungi are able to infect tree roots, they may render trees less
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vulnerable to the negative effects of phenolics (Hanson and Dixon, 1987; Lendzemoa and Kuyper, 2001). Recently Piotrowskia et al. (2008), testing cottonwood foliage and litter extracts on the colonization of AMF, found that all tested compounds significantly reduced AMF colonization but they did not affect colonization of non-AMF root-colonizing fungi. Fungi in soils are also strongly affected by water soluble substances coming from the litter of needles and leaves of trees and herbaceous plants (Persidsky et al., 1965; Dix, 1974; Boufalis and Pellissier, 1994). The water soluble substances produced or leached from humus can affect the metabolism of mycorrhizal fungi in the soil (Rice, 1984; Pellissier, 1993; Brucknera et al., 2003; Weir, 2007). Phenolics and water soluble substances are well documented but it is a question of interest how plant essential oils, even abundant, widespread and widely used have not been investigated against mycorrhizal fungi. Laurus nobilis is a perennial aromatic shrub, abundant in Mediterranean region, rich in essential oil which is reported as toxic to many microorganisms (Hassiotis, 1997; Simic et al., 2004; Erturk, 2006; Unver et al., 2008). Despite ongoing research the role of this aromatic species containing essential oils on mycorrhizal fungi has not been investigated so far. The aim of the present study is (1) to investigate whether and in which way the aromatic L. nobilis (leaf material and essential oil) can affect two species of AM fungi in mycorrhiza establishment, (2) to explore the level of mycorrhiza development according to different treatments with laurel, (3) to investigate if the antifungal activity of L. nobilis is because of its essential oil content and (4) to monitor the growth of host plants (leeks). 2. Materials and methods 2.1. Experimental Leek (Allium porrum L.) was used as host plant because of its fast growth; it’s relatively good response to mycorrhiza and its easy production of plants. Two arbuscular mycorrhizal fungi were used to establish mycorrhiza with leeks. These were Glomus deserticola and Glomus intraradices, both registered in the European Bank of Glomales as BEG 73 and BEG 72 respectively. These inoculants were chosen because of their ability to form abundant internal vesicles, considered by many authors as chlamydospores (Berch, 1988), because of their growth promoting effect on various plants (Planchette and Fortin, 1982; Furlan et al., 1983) and their protective influence against some root pathogens (Caron et al., 1985). The seeds of A. porrum L. were surface sterilized by soaking in 15% H2O2 for 30 min, carefully washed with sterilized water and then germinated in sterile soil (temperature 120 C, pressure 2 atm for 2 h). The seedlings remained for six weeks in the germination pots and then transplanted in new pots. 2.1.1. L. nobilis leaves The experimental potting mixture was made by 6 parts of soil, 9 parts of sand (sterilized as above) and 15 parts of vermiculite. Five different L. nobilis leaves concentrations were used in this experiment. Those treatments were C: 0 g L1, 1: 0.75 g L1, 2: 1.5 g L1, 3: 3 g L1 and 4: 6 g L1 (per litre of soil). The given amount of mature collected and chopped L. nobilis leaves were carefully mixed to the above substrate. The plant material prior was sterilized for 30 min in 15% H2O2. Four leek plants were planted in cylindrical pots (R: 10 cm, H: 15 cm) and placed under controlled conditions, in a very tidy and clean place avoiding any contamination of the host plants. Ten replications (pots) per each treatment were made. The experiment was established in the village of Kato Scholari (N: 40 260 44, E: 23 010 02) near Thessaloniki, Greece on the 1st of March, 2007.
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2.1.2. L. nobilis essential oil The following year the experiment was repeated in the same manner using essential oil instead of plant material from L. nobilis. The essential oil was applied in the soil as described by Hassiotis (2010). Cylindrical pots, as used before, of 4 L volume were used. In the center of the pot, a specific tube construction with tiny holes was placed, allowing the essential oil evaporation radially. The essential oil was extracted by water steam distillation from the same laurel trees used in the previous experiment. Five different L. nobilis essential oil concentrations were used in this experiment. Those treatments were C: 0 mg L1, 1: 6 mg L1, 2: 12 mg L1, 3: 24 mg L1 and 4: 48 mg L1 (per litre of soil). These oil concentrations were respective to the essential oil amount that was incorporated in laurel leaves that were used in the previous experiment. The essential oil was applied in an absorbent paper stick and then was placed into the tube construction. The paper sticks were replaced every two weeks. This experiment was initiated on the 1st March, 2008. 2.2. Mycorrhiza assay The root samples were extracted by using a cylindrical corer (10 mm). The soil was removed by soaking the roots in water and gently washing them, to ensure that all the thinner roots and tips remained intact. The staining procedure was made according to Vierheilig et al. (2005) modified to parameters of the present study. The roots were cut into small pieces and placed in a beaker (5% KOH) for 30 min in a water bath at 90 C. The roots were then rinsed with tap water and acidified with 5% lactic acid at room temperature for 12 h. Finally the roots were stained with a solution consisting of 875 ml of laboratory lactic acid, 63 ml of glycerin, 63 ml tap water and 0.1 g acid fuscin for 30 min at 70 C and then destained in laboratory lactic acid for 15 min. 10 root segments were mounted onto slides and examined at 100e400 magnification under a Nikon YS100 microscope. Beneath the glass slide an acetate film with 10 thin lines was adapted. At crossing points between roots and lines, each point that had an infection was recorded and the number of infections was expressed as percentage. 2.3. Inoculation, root infection and growth recording Six weeks after transplant, leeks were checked for absence of infection, and inoculated. The AM fungi used in this assay were G. intraradices (Schenck and Smith) and G. deserticola (Trappe, Bloss, and Menge). Sixteen grams of active inoculum were placed in every pot, divided in four inoculation points. The final 50 pots (10 pots 5 treatments with L. nobilis) used as control without inoculation with fungi. At inoculation, and every fifteen days afterwards, root colonization, stem diameter and height of leeks were measured and recorded. Four glass slides from every pot were prepared in order to evaluate root colonization. The host growth was evaluated as average of leek stem diameter and height of leek. 2.4. Essential oil analysis 400 g of fresh and chopped plant material was steam distilled in a 2 L water steam distillation unit for 90 min, at 100e105 C and a flow rate of approximately 8.5 mL min1 (Furnis et al., 1989). The amount of oil was expressed (w/w) as essential oil/fresh raw material. The essential oil was collected and its volatile constituents were established by GCeMS analysis. GCeMS analysis was performed on a Shimadzu GC-2010 e GCMS-QP2010 system operating in EI mode (70eV) equipped with a split/splitless injector (230 C), a split ratio 1/30, using a fused silica HP-5MS capillary column
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(30 m 0.25 mm (i.d.), film thickness: 0.25 mm). The temperature cycle was initiated at 50 C for 5 min and was programmed to 290 C with increments of 4 C per min. Helium was used as a carrier gas at a flow rate of 1.0 mL min1. Injection volume of each sample was 1 mL. Retention indices for all compounds were determined according to the Van den Dool and Kratz (1963) approach using n-alkanes as standards. The identification of the components was based on comparison to their mass spectra with those of NIST21 and NIST107 (Massada, 1976) and those described by Adams (2001) as well as by comparison of their retention indices with literature data (Davies, 1990; Bisio et al., 1998). Eight samples of 400 g of L. nobilis were distilled and analyzed in order to determine the chemical composition of the oil. 2.5. Statistical analysis Experimental data was performed with Statistical Package for Social Sciences (SPSS, version 12, Chicago, IL, USA). The significance of time post inoculation and fungi species on colonization was determined by one-way analysis of variance (ANOVA). Two-way ANOVA was employed to test for significant differences between time (weeks) and fungi species. Where the F values were significant, Fisher’s protected least significant difference (LSD) was used to test for differences among treatments (different concentrations of laurel leaves and laurel oil) and control. The Pearson correlation was used to explore the level of relationship between the different concentrations of laurel leaves and laurel oil and mycorrhiza colonization and plant growth. All analyses were evaluated at a ¼ 0.05 significance level. The percentage of fungi colonization presented in Table 1, Fig. 1 is mean values of 40 measurements (10 pots 4 glass slides) of each fungi species and control (non inoculated plants). The number of host growth presented in Table 1, Fig. 2 is mean values of 80 measurements (10 pots 4 plants 2 parameters (stem and height)) of each fungi species and control. The compound percentage presented in Table 2 is mean values of 16 measurements (8 distillations 2 GCeMS runnings).
3. Results and discussion 3.1. Mycorrhiza infection The leeks infection by mycorrhizal fungi was recorded periodically every fifteen days after inoculation with fungi. The leeks responded immediately to arbuscular mycorrhizal fungi and symbiosis was established. This happened successfully for both fungi G. deserticola and G. intraradices used for inoculation. Fifteen days after inocula introduction the infection level was 4.2% and 3.2% respectively (average for all treatments with laurel leaves) in the first experiment used different concentration of L. nobilis mature leaves. The colonization level was progressively increased during the experiment resulting in a final average infection percentage of 89.4% for G. deserticola and 69.0% for G. intraradices, twelve weeks after inoculation (Table 1). A high correlation was found between days of inoculation and fungi colonization (r ¼ 0.969 for G. deserticola and r ¼ 0.961 for G. intraradices, P < 0.05). It was also observed that G. deserticola presented higher colonization level than G. intraradices. Comparing the ability of the two AM fungi to colonize the leek roots, it was found that G. deserticola presented 16% higher colonization level than G. intraradices twelve weeks post inoculation. The differences in the colonization levels between the AMF (P < 0.01) and between the weeks after inoculation (P < 0.001) were statistically significant (Table 1). In a similar way, the host plants and fungi responded in the second year of the experiment, where L. nobilis essential oil was used instead of mature leaves. It is highlighted that the amount of essential oil used in every treatment was respective to the essential oil amount that was incorporated in laurel leaves and used in the previous experiment. Among other objectives this evaluation was targeting to investigate, if the antifungal activity of laurel leaves demonstrated in the previous year was due to essential oil presence. The leeks responded immediately to arbuscular mycorrhizal fungi and symbiosis was established. Even statistically not significant (P > 0.05) the colonization level between host and fungi in this
Table 1 Colonization level of two arbuscular mycorrhizal fungi and host growth treated with different concentrations of Laurus nobilis leaves and different concentrations of Laurus nobilis essential oil. Weeks after inoculation
Laurus nobilis leaves
Laurus nobilis essential oil
Fungi colonization (%)
Host growth (%)
Average
Min
Max
Glomus deserticola 0 2 4 6 8 10 12
0.0 4.2 11.6 45.0 69.0 85.0 89.4
0.0 3.0 9.0 30.0 45.0 60.0 62.0
0.0 5.0 15.0 60.0 95.0 100.0 100.0
Glomus intraradices 0 2 4 6 8 10 12
0.0 3.2 8.9 34.8 53.4 65.5 69.0
0.0 2.0 6.0 20.1 30.2 40.2 41.5
0.0 4.3 12.9 51.6 81.7 86.0 86.0
Significance Weeks AMF Weeks AMF
*** ** **
*** ** **
*P < 0.05, **P < 0.01, ***P < 0.001.
*** ** **
Fungi colonization (%)
Host growth (%)
Average
Min
Max
0.0 3.3 7.5 13.1 20.6 24.7 26.3
0.0 3.8 10.4 40.5 62.1 76.5 80.5
0.0 2.7 8.1 27.0 40.5 54.0 55.8
0.0 4.5 13.5 54.0 85.5 90.0 92.1
0.0 3.0 6.8 11.8 18.5 22.3 23.7
0.0 3.2 7.1 11.4 16.5 21.4 23.1
0.0 2.8 7.8 30.3 46.4 57.0 60.0
0.0 1.7 5.2 17.5 26.2 35.0 36.1
0.0 3.7 11.2 44.9 71.1 74.8 75.8
0.0 2.8 6.1 10.0 14.4 18.6 20.1
** * *
*** ** **
*** ** **
*** ** **
** * *
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631
120.0
colonization level (%)
100.0
a a
a a
a ab b a
bc
80.0
a a
a
a
a
c
b
60.0
d
bc c
40.0 d
20.0
0.0 C
1
2
3
4
C
1
2
3
4
Treatments with L. nobilis leaves and Fig. 1. Colonization level twelve weeks after inoculation with two arbuscular mycorrhizal fungi subjected to different concentrations of Laurus nobilis leaves (C: 0 g L1, 1: 0.75 g L1, 2: 1.5 g L1, 3: 3 g L1 and 4: 6 g L1) and essential oil (C: 0 mg L1, 1: 6 mg L1, 2: 12 mg L1, 3: 24 mg L1 and 4: 48 mg L1) per litre of soil (The amount of essential oil incorporated in L. nobilis leaves is the same with the essential oil amount that was applied pure in the respective concentration). Different letters within a parameter (AMF) denote significant differences (LSD, P ¼ 0.05) according to essential oil concentrations.
experiment was lower compared to the respective weeks of the previous experiment for both fungi species (Table 1). This observation led us to thought that the essential oil vapours from laurel were presented more effective against fungi than the total leaf with the same amount of oil. The storage of plant essential oil in glands in the leaves may trap the vapours of the oil, allowing gradual release and consequently the aromatic leaf is less effective than the essential oil itself. The final average colonization percentage was reached at 80.5% for G. deserticola and 60.0% for G. intraradices, twelve weeks after inoculation. A high correlation was found between days of inoculation and fungi colonization (r ¼ 0.957 for G. deserticola and r ¼ 0.941 for G. intraradices, P < 0.05). It was also observed that G. deserticola presented higher colonization level than G. intraradices. Fig. 1 summarizes the colonization levels for G. deserticola and G. intraradices according to 5 different concentrations of laurel leaves and the respective concentration of laurel essential oil twelve weeks after inoculation. G. deserticola presented higher infection level than G. intraradices between the treatments and
reached at 100% for G. deserticola in treatment 1 in the first experiment (0.75 g L1 leaves) and in the second (6 mg L1 essential oil). For the particular treatment 1, G. intraradices reached at 82.3% in the first and 83.0% in the second respectively. A remarkable point is that the higher colonization levels being always observed for both fungi in treatment 1 and not in control leading to the indication that a small amount of L. nobilis leaves (0.75 g L1) or essential oil (6 mg L1) can enhance fungi infection. The lower mean colonization level was recorded for either fungi in treatment 4 with higher concentration of laurel leaves (6 g L1) or essential oil (48 mg L1). Values with common letter within colonization levels are not significantly different according to Fisher’s LSD test (P ¼ 0.05). A reverse correlation was observed in progressively increased amount of laurel leaves and oil in the substrate and level of mycorrhiza colonization (r ¼ 0.919 for G. deserticola and r ¼ 0.967 for G. intraradices, P < 0.05). The differences between the highest and the lowest colonization values were 62.3% for G. deserticola and 83.3% for G. intraradices. It is accomplished that G. intraradices not only presented lower infection than G. deserticola
40.0 35.0
Host growth (%)
30.0 25.0 20.0 15.0 10.0 Linear (Without AMF) Poly. (G. intraradici)
5.0
Linear (Without AMF) Poly. (G. intraradici)
Poly. (G. deserticola)
Poly. (G. deserticola)
0.0 C
1
2
3
Treatments with L. nobilis leaves
4
C
1
2
3
4
Treatments with L. nobilis essential oil
Fig. 2. Host growth twelve weeks after inoculation with two arbuscular mycorrhizal fungi according to five different treatments with Laurus nobilis leaves and essential oil. Error bars are also presented.
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but it was also stronger influenced by the presence of L. nobilis leaves and essential oil. The differences in the infection levels, according to the treatments with L. nobilis were statistically significant (P < 0.01) for both experiments. The interaction between treatments with L. nobilis and fungi species resulted in statistically significant differences (P < 0.01) according to ANOVA test. 3.2. Leeks growth The average leek growths after inoculation with arbuscular mycorrhizal fungi for inoculated and non inoculated leeks according to the different treatments with laurel are presented in Fig. 2 for both experiments. There was a remarkable growth of leeks (P < 0.01) after inoculation with the AM fungi in contrast to none inoculated. The average host growth (for all treatments) twelve weeks post inoculation for those were inoculated with G. deserticola was 26.3%, for G. intraradices 23.1% and for non inoculated 15.4% in the first experiment that L. nobilis leaves were used; In the second experiment (with essential oil) the respective growth values were 23.7%, 20.1% and 12.3%. Non inoculated leeks in the first experiment with L. nobilis leaves presented better growth as the concentration of leaves gradually increased leading to the thought that nutrients incorporated in leaves may used as nutrient tank for leeks growth. This was not observed in the experiment with the essential oil. Even these differences were statistically non significant (P > 0.05) and this idea needs further investigation with different experimental design. For inoculated leeks it was observed a positive correlation between AM fungi infection level and leeks growth (r ¼ 0.975 for G. deserticola and r ¼ 0.908 for G. intraradices, P < 0.05). 3.3. Essential oil analysis The steam distillations yielded an average oil amount of 3.2 g/ 400 g fresh leaves of L. nobilis which is expressed as 0.8% (w/w) of the total plant material. The amount of essential oil was incorporated in the different treatments with L. nobilis was 24 mg pot1, 48 mg pot1, 96 mg pot1 and 192 mg pot1 for treatments 1, 2, 3 and 4 respectively. Nineteen compounds contributing more than 0.5% of the total oil were identified and evaluated by GCeMS analyses comprising a final percentage of 97.74 of the total oil (Table 2). The small differences observed in essential oil composition between replications and the years of the study are given as standard deviation. The major compound of L. nobilis essential oil was 1,8-cineole (49.60%), followed by sabinene (7.80%), a-pinene (5.96%), eugenol (5.60%), a-terpinyl acetate (5.25%) and b-pinene (5.12%). These six compounds comprise the 76.33% of the total oil. Our data is in agreement with many other authors working on L. nobilis essential oil in surrounding countries like Italy (Flamini et al., 2007), Turkey (Dadalioglu and Evrendilek, 2004; Kilic et al., 2004; Ozcan and Chalchat, 2005) and Croatia (Politeo et al., 2007). The toxicity or the repellent effect of L. nobilis against microorganisms is well established. In this study the antifungal activity of L. nobilis against two AMF was tested and was found high, especially in concentrations of 24 and 48 mg essential oil L1 soil. Recently Fawzi et al. (2009) working in vitro, testing five plant extracts against pathogenic fungi Alternaria alternata and Fusarium oxysporum found that L. nobilis can be successfully used for antifungal control. He has also reported that plant extracts can be used as natural fungicides to control pathogenic fungi, thus reducing the dependence on the synthetic fungicides. Unver et al. (2008) working with L. nobilis methanolewater extracts showed that these had very high antimycotic activity against all tested yeasts. Simic et al. (2004) examined the antifungal activity of some
Table 2 Chemical composition of Laurus nobilis essential oil. Compoundsa
RIb
Composition (%) SDc
a-Thujene a-Pinene
926 932 946 968 974 985 1024 1027 1029 1100 1146 1190 1292 1305 1307 1355 1472 1405 1421
0.91 5.96 1.21 7.80 5.12 1.62 1.52 2.32 49.60 1.50 2.12 1.90 0.60 0.91 5.25 5.60 1.10 2.10 0.60 97.74
Camphene Sabinene b-Pinene b-Myrcene p-Cymene Limonene 1,8-Cineole Linalool Terpinen-4-ol a-Terpineol Linalyl acetate Bornyl acetate a-Terpinyl acetate Eugenol b-Elemene Methyleugenol b-Caryophyllene
0.01 0.14 0.04 0.09 0.12 0.04 0.02 0.07 0.89 0.04 0.08 0.11 0.01 0.02 0.13 0.17 0.07 0.10 0.04
a In table are presented compounds contributing more than 0.5% of the total oil. Compounds listed in order of elution from an HP-5MS column. b Retention indices as determined on an HP-5MS column using a homologous series of n-alkanes. c Standard deviation.
essential oils from Lauraceae family against 17 micromycetes (among the tested fungal species were food poisoning, spoilage fungi, plant and animal pathogens) and found first Cinnamomum zeylanicum and second L. nobilis to be more toxic against them. Even though the essential oil of L. nobilis reported as toxic to microorganisms in the present study was found that small amounts of plant leaves or pure essential oil were beneficial for mycorrhiza development. This happened because of the volatile oil compounds which were contained in laurel material and were used as energy source for fungi. Kaplan and Hartenstein (1979) and Harder and Foß (1999) found that low concentrations of toluene in cultural media encouraged the growth of 19 species of bacteria and fungi which used toluene as their only source of carbon. In 1984, Vokou et al. (1984) working with Coridothymus and Satureja showed that addition of these volatile oils to the soil significantly activated soil respiration. These data were later confirmed with other volatile compounds (Vokou and Liotiri, 1999; Vokou et al., 2002). Our results support the opinion that small amounts of aromatic material or volatile compounds can be used as energy source by fungi. The antifungal activity of L. nobilis is strongly linked with the presence of essential oil and particularly by its main compounds. Halligan (1975) has reported that the two most toxic components against microbes were camphor and 1,8-cineole and these two compounds contributed the major toxicity in the field. Riaz et al. (1989) found that 1,8-cineole and eugenol were the main compounds of L. nobilis oil and the oil to be toxic against soil bacteria. In 1991, Shaaya et al. (1991) demonstrated that 1,8-cineole was the most effective compound against Rhyzopertha dominica and Oryzaepfilus surinamensis. Kivanc and Akgul (1986) working with cumin, laurel and oregano and Bounatirou et al. (2007) showed that these aromatics had the most toxic oil against Staphylococcus aureus and Proteus vulgaris which were more sensitive. Two other compounds could possibly created mycorrhiza inhibition c et al. (2009) working with were sabinene and a-pinene. Sokovi Daucus carota and its antibacterial and antifungal properties against eight bacterial and eight fungal strains found strong inhibition of the essential oil whose main constituents were a-pinene (7.05e51.23%) and sabinene (2.68e36.69%). Also Singh et al. (2008) working with essential oil of Elettaria cardamomum tested and
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found strong antifungal activity against the food-borne fungi Aspergillus terreus, Penicillium purpurogenum, Fusarium graminearum and Penicillium madriti. The major compound of E. cardamomum essential oil was a-terpinyl acetate with 44.3%. Unfortunately there is no relative work done so far on AM fungi inhibition related to essential oils to compare our data with, so this study is an attempt to investigate the influence of one aromatic plant species on mycorrhiza development. Neither data concerning the antifungal activity of individual compounds composing the laurel oil against AM fungi are available. So we tried to report the antifungal activity which is being presented by other essential oils whose the major compounds are common with the oil of L. nobilis. Moreover, our approach e to grow the host plants in flower pots and let the AMF species to contaminate the roots under different concentrations of L. nobilis leaves and essential oil e represented a more realistic test of allelopathy than bioassays. In order to quantify which of the compounds were responsible for allelopathy effect a new experiment is needed in order to evaluate the antifungal activity of laurel oil individual compounds. It is assumed from our data that the major compounds of laurel essential oil were responsible for mycorrhiza inhibition. It is beyond any doubt that the management of native AM fungi is an effective strategy in revegetation of semiarid areas with shrub species (Caravaca et al., 2005). Combined treatments with mycorrhizal inoculation and composted residue are effective for increasing the growth and the N and P contents in shoot tissues, and could facilitate the establishment and development of new plants in the surrounding area (Caravaca et al., 2003). The abundant presence of aromatic L. nobilis in many different ecosystems around Mediterranean, as resulted from the present study, can generate allelopathy effect against mycorrhizal fungi. It is suggested, when it is possible, to reduce the degraded litter of this species accumulated every year in these ecosystems, allowing the development of mycorrhiza and resulting in more successful new vegetation establishment. Acknowledgements We would like to thank Missis Elena Koutsilieri, Aristotle University of Thessaloniki, for helping in statistical analysis, and Dr. Elpida Bombi for English corrections and linguistic improvements in the manuscript. References Adams, R., 2001. Identification of essential oil components by gas chromatography/ mass spectroscopy. Allured Publishing Co, Carol Stream, Illinois. Bainard, L.D., Brown, P.D., Upadhyaya, M.K., 2009. Inhibitory effect of tall hedge mustard (Sisymbrium loeselii) allelochemicals on rangeland plants and arbuscular mycorrhizal fungi. Weed Science 57, 386e393. Berch, S., 1988. Compilation of the endogonaceae. Mycologue Publication, Waterloo, Canada, pp. 238. Bisio, A., Ciarallo, G., Romussi, G., Fontana, N., Mascolo, N., Capasso, R., Biscardi, D., 1998. Chemical composition of essential oils from some Salvia species. Phytotherapy Research 12, S117eS120. Boufalis, A., Pellissier, F., 1994. Allelopathic effects of phenolic mixtures on respiration of two spruce mycorrhizal fungi. Journal of Chemical Ecology 20, 2283e2289. Bounatirou, S., Smiti, S., Miguel, M.G., Faleiro, L., Rejeb, M.N., Neffati, M., Costa, M.M., Pedro, L.G., 2007. Chemical composition, antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link. Food Chemistry 105, 146e155. Bowen, G.D., 1980. Mycorrhizal roles in tropical plants and ecosystems. In: Mikola, P. (Ed.), Tropical mycorrhizal research. Clarendon Press, London, pp. 165e190. Bowers, J.H., Locke, J.C., 2000. Effect of botanical extracts on the population density of Fusarium oxysporum in soil and control of Fusarium wilt in the greenhouse. Plant Disease 84, 300e305. Brucknera, D.J., Lepossa, A., Herpai, Z., 2003. Inhibitory effect of ragweed (Ambrosia artemisiifolia L.) e Inflorescence extract on the germination of Amaranthus hypochodriacus L. and growth of two soil algae. Chemosphere 51, 515e519.
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