Mycorrhizal fungi have an important role in the agroecosystem because of its ability to establish symbiotic interactions with roots of approximately 95% of plant species (Smith and Read, 1997). Mycorrhizal fungi contribute to plant development by means of enlarging absorbing surface root system via colonization of roots, increasing uptake of macro and micro nutrients initially phosphorus and providing liveliness of roots (Marschner and Dell, 1994). In addition, mycorrhizal fungi have an important role in protecting plant against pathogens via induction of some resistance mechanisms against them, particularly soil-borne pathogens such as Pythium sp., Phytophthora sp., Fusarium sp. and Verticillium sp. (Azcon-Aguilar and Barea, 1992; Trotta et al., 1996; Garmendia et al., 2004).
Some studies were conducted using disease reactions as well as microscopic
and biochemical studies related with the effectiveness of mycorrhizal fungi
against plant pathogens. Mycorrhizal fungi reduced disease severity of the disease
by forming a mechanical barrier as a result of root colonization and or enhanced
plant development (Dumas-Gaudot et al., 2000). Also, mycorrhizal fungi
induce biochemical resistance mechanisms depending on the root colonization.
In mycorrhized plants, protection against soil-borne fungal pathogens is due
to the activation of defense plant responses such as production of Pathogenesis
Related (PR) proteins (Lambais, 2000; Azcon-Aguilar et al., 2002). Dassi
et al. (1998) reported that the amount of PR proteins were increased
in G. mosseae-colonized tomato roots after inoculation with Phytophthora
parasitica. Symbiotic microorganisms caused an increase in PR proteins especially
chitinases, β-1,3-glucanases and peroxidases (Dumas-Gaudot et al.,
1996; Pozo et al., 1998, 1999; Lambais et al., 2003). Garmendia
et al. (2006) also reported that, the colonization of pepper roots by
G. deserticola induced isoforms of asidic chitinases and peroxidases
and mycorrhizal fungi plus inoculation with Verticillium dahliae inoculation
slightly induced the peroxidases. Devi and Reddy (2002) reported that the amount
of phenolic compounds were increased such as trans-p-coumaric
asit, trans-ferulic acid and vanillic acid in G. mosseae and/or
Rhizobium inoculated roots and upper part of peanut plants.
In this study, the amount of phenolic compounds and PR proteins were determined by mycorrhizal fungal inoculations against Phytophthora blight caused by P. capsici in pepper.
MATERIALS AND METHODS
Plant, pathogen and mycorrhizal fungi: Capcicum annum L. cv. Charliston Bagci was used as plant material. Phytophthora capsici (Pc5 isolate) was isolated from naturally infected plants at Adana.
Four different mycorrhizal fungi species, increased in Zea mays L. were used: G. mosseae (GM), G. etunicatum (GE), G. fasciculatus (GF) and Gi. margarita (GiM).
Studies were conducted at the Mycology and Molecular Biology Laboratories, Department of Plant Protection, University of Suleyman Demirel during 2005 to 2008.
Seedling production with mycorrhizal fungal inoculation and establishment of pot trial: Pepper seedlings were produced in plastic containers (30x40 cm). Growing medium consisted of soil:sand:pumice (1:1:1/v:v:v) autoclaved twice at 121°C, 1 kPa. GM, GE, GF and GiM inocula consisted of soil+root fragments+spores+mycelia were placed 2-3 cm below seeds before planting (Menge and Timmer, 1982). Mycorrhizal inoculum densities in soil were, 188 spores 10 g-1 soil, 2.1 kg for GM; 268 spores 10 g-1 soil, 1.3 kg for GE; 229 spores 10 g-1 soil, 1.7 kg for GF and 235 spores 10 g-1 soil, 1.6 kg for GiM. Seedlings were produced without mycorrhizal fungal inoculations representing control group. Containers were placed in growth room at 25±2°C and a 12 h photoperiod. Plants were watered with deionized water during seedling production until the 3-4 leaves stage.
Pot trials were established when seedlings reached the stage of 3-4 leaves with or without mycorrhizal inoculation. Mycorrhizal inocula were applied in the planting hole when seedlings were transplanted to 15 cm diameter pots at a ratio of 50 g for GM, 35 g for GE, 45 g for GF and 40 g for GiM depending on the spore number in 10 g soil, to reach a final concentration of ˜1000 spores.
Treatments were as follows: G. mosseae (GM), G. etunicatum (GE), G. fasciculatus (GF) and Gi. margarita (GiM), P. capsici (Pc), Control (C) and the combination of each of the four mycorrhizal fungi species with P. capsici. Each treatment was replicated five times.
For P. capsici inoculation, the oomycete was grown on oatmeal agar plates at 28°C for 7 days and placed under fluorescent light for sporulation. Culture plates were incubated in sterile distilled water for 40 min at 4°C and then during 30 min at 25°C room temperature. Zoospore released from sporangia of P. capsici were collected by filtering through two-layers of cheesecloth and zoospore concentration was adjusted to 1x106 zoospores mL-1 using haemocytometer (Sunwoo et al., 1996). A 10 mL spore suspension was applied to soil around the root of the plant in each pot after 15 days.
One week after inoculation, symptoms were evaluated according to a 0-5 scale: where 0 = no visible disease symptoms; 1 = leaves slightly wilted with brownish lesions beginning to appear on stems; 2 = 30-50% of entire plant diseased; 3 = 50-70% of entire plant diseased; 4 = 70-90% of entire plant diseased and 5 = plant dead (Sunwoo et al., 1996).
Disease severity values were expressed as a percentage of affected tissue according to Tawsend-Heuberger transformation (Gomez and Gomez, 1983).
Microscopic observations: Roots from mycorrhizal inoculated plants were
observed at three different stages of plant development:
||(15 days after mycorrhizal fungal inoculation) Determination
of hyphal development in or on roots
||(25 days after mycorrhizal fungal inoculation) Formation of arbuscul or
vesicle of mycorrhizal fungi
||(45 days after inoculation) Formation of arbuscule, vesicle or spores
The roots were cleared and stained as described by Koske and Gemma (1989) and
the percentage of root colonization was estimated by a gridline intersection
method (Giovannetti and Mosse, 1980).
Determination of phenolic compounds: For determination of phenolic compounds, root extractions were performed for each treatment. Roots were harvested for each treatment and extracted with ethyl alcohol according to Mahadevan et al. (1965). Amount of total phenolic compounds were determined according to Bray and Thorpe (1954) and Johnson and Scholl (1957).
Thin layer chromatography were performed on Whatman No. 1 chromatographic paper using concentrated root extracts with two dimension progression technique. Applied solvent systems were benzen-acetic acid-water (60:70:30, upper phase) and sodium formate -formic acid-water (10:1:200), respectively. Points on chromatographic paper were examined under UV light and/or by spraying reagents (Sulphanidic asit and p-nitraline) and ferric chloride with 1% according to Rf values.
According to Rf, regions without sprayed point were dissolved in ethyl alcohol (90%) and extract was evaporated until dryness. Extract was mixed with 3 mL distilled water and 0.5 mL of Folins reagent and shacked. After 3 min, 1 mL of diluted sodium carbonate was added and completed to 10 mL within 1 h. Measurements were performed using spectrophotometer at 725 nm after formation of blue color.
Preparation of stem extracts: Stem extractions were performed for
each sample from harvested plants according to Hwang et al. (1997). Stem
pieces were homogenized in 0.5 M sodium acetate buffer with 15 mM of mercaptoethanol.
Crude extracts were centrifuged at 4°C, 10.000 g for 60 min and then supernatant
was centrifuged at 4°C, 20.000 g for 60 min.
Protein in collected supernatant was determined using as standard known bovine serum albumin.
Proteins in crude extract were mixed with 4 part acetone and precipitated at 20°C overnight. Precipitation was centrifuged at 15.000 g for 15 min and washed with cold acetone twice and dried.
Residue was suspended in 30 mM of sodium acetate buffer (pH 5.2) for cleaning and then samples were kept at -70°C until use.
Measurement of β-1,3-glucanase activity: β-1,3-glucanase activity was performed using a calorimetric assay method as described by Kauffman et al. (1987). For enzyme activity detection, laminarin was used as a substrate to determine the decreased sugar amount arising from laminarin in extract.
Substrate buffer included 0.1 M of sodium acetate buffer (pH 5.2) contain laminarin (1 mg mL-1 buffer). Reaction mixture was prepared using 0.9 mL of subtrate buffer and 0.1 mL of enzyme solution (stem extract).
Reaction tubes were kept at 37°C for 1 h and the decrease in sugar amount was determined according to Nelson (1944). Accordingly, glucose was used as standard and 1 kat (katal) defined as enzyme activity equivalent 1 mol catalyzed glucose.
Measurement of chitinase activity: Chitinase activity in crude extract was determined using calorimetric assay (Hwang et al., 1997).
Reaction mixture was prepared as final volume of 0.5 mL of 0.1 M sodium acetate buffer (pH 5.2) including 0.5 mg of washed chitin and enzymatic solutions in different volume. Mixture was kept in water bath at 37°C for 1 h and incubated by shaking.
For determining the chitinase activity, 0.3 mL of supernatant was incubated at 37°C to hydrolyze chitin oligomers using 5 μL, 25% glucoronidase. To induce the formation of N-acetyl glucosamin after reaction, 0.6 M of 0.1 mL potassium tetraborat was added and kept in boiling water for 3 min and incubated with 1 mL diluted reagent and glacial acetic acid (1:2, v:v) at 37°C for 20 min after cooling. Reagent stock solution was prepared using glacial acetic acid and 11.5 M of HCl, 87.5 mL:12.5 mL (v/v) mixture contain 10% (w/v) 4 (dimethyl amine) benzaldehyde. Resulting Glc-Nac was determined according to Legrand et al. (1987).
Statistical analysis: The data were subjected to analysis of variance (F-test). Treatment means were compared using Fishers Least Significant Difference (LSD) test at p = 0.05 (Gomez and Gomez, 1983).
Microscopic observation: The colonization percent of mycorrhizal fungi,
vesicule, arbuscule and spores in and outside of the host cell were observed
in different stages of seedling development. Results were shown in Table
At seedling stage I, root colonization percentage after seedling emergence were 9.0, 5.0, 8.0 and 5.0% for GM, GE, GF and GiM, respectively. Structures of the mycorrhizal fungi were not observed at this stage.
At stage II, the colonization percentage of GM and GF found as 21.0 and 18.0%, respectively; whereas colonization percentage of GE and GiM were 15%. Mycorrhizal arbuscules were observed at this stage of seedling development.
The highest colonization rates were obtained from GM and GF treated plants
by 53.0 and 50.0%, respectively at stage III. Arbuscules and spores were observed
in all inspected roots at this final stage of seedling development.
||Root colonisation (%) and structures of G. mosseae,
G. etunicatum, G. fasciculatus and Gi. margarita
|*C: Colonisation (%); A: Arbuscule; V: Vesicule; S: Spore;
+,-: Colonisation ratio are means of values
||Amounts of total phenolic compounds in G. mosseae,
G. etunicatum, G. fasciculatus, Gi. margarita and/or
P. capsici inoculations
|aMeans fallowed by different letters are significantly
different (p = 0.05) according to Fishers LSD test
Analysis of phenolic compounds: The amounts of total phenolic compounds
(μg g-1 fresh weight) 15 days after inoculation are shown in
The amounts of total phenolic compounds were increased in treated compared as compared to control plants. The amount of the total phenolic compounds was 45.0 μg g-1 of fresh weight in control compared to 68.0 μg g-1 fresh weight in Pc. Mycorhizal fungal inoculations individually provide increased the amount of phenolics compounds in root and the highest amount was obtained from GM treatment (72 μg g-1 of fresh weight). However, phenolic compounds concentration was highest in the combined treatment of mycorrhizal fungi and P. capsici. The amounts of phenolic compounds in GM + Pc and GF+Pc were 115.0 and 110.0 μg g-1 of fresh weight, respectively.
Several phenolic compounds were identified in treated plants: Caffeic acid, trans-coumaryl, capsaicin, p-aminobenzaldehyde, aspartic acid, chlorogenic acid, glutamic acid, linoleic acid, cis-feruloyl acid, stearic acid, capsicoside and an undefined F1.
Diseases severity (%) was determined in Pc inoculated plants when at the same
time as phenolic compounds 15 days after inoculation. Disease symptoms consisted
in root and crown rot. Mycorrhizal fungi reduced disease severity compared to
that observed on non mycorrhized Pc infected plants (Table 3).
Enzyme analysis: Activities of β-1.3-glucanase and chitinase enzymes
in the different treatments are shown in Table 4 and 5.
In all treatments, the β-1.3-glucanase and chitinase activities were maximum
6 days after inoculation, decreasing at the initial and final assessments 3
and 9 days after inoculation, respectively. The levels of both, β-1.3-glucanase
and chitinase enzymes in control plants ranged 0-3 μ kat mg-1
||Phytophthora root rot severity (%) in different inoculation
of G. mosseae, G. etunicatum, G. fasciculatus, Gi.
margarita and P. capsici
||Activity of β-1.3-glucanase (μ kat mg-1
protein) in G. mosseae, G. etunicatum, G. fasciculatus,
Gi. margarita and/or P. capsici inoculations
|aMeans within each column fallowed by different
letters are significantly different (p = 0.05) according to Fishers
||Activity of chitinase (μ kat mg-1 protein)
in G. mosseae, G. etunicatum, G. fasciculatus, Gi.
margarita and/or P. capsici inoculations
aMeans within each column fallowed by different
letters are significantly different (p = 0.05) according to Fishers
The level of β-1.3- glucanase enzyme was higher in Pc infected plants
(19 μ kat mg-1 of protein) and those with combined inoculation
of mycorrhizal fungi and Pc treatments (19-25 μ kat mg-1 of
protein), as compared to that at the single mycorrhizal treatments (10-16 μ
kat mg-1 of protein) 6 days after inoculation (Table
4). Similarly, chitinase activity was higher in Pc infected plants (11 μ
kat mg-1 of protein) and those with combined inoculation of mycorrhizal
fungi and Pc treatments (9-14 μ kat mg-1 of protein), as compared
to that at the single mycorrhizal treatments (5-7 μ kat mg-1
of protein) 6 days after inoculation (Table 5).
In this study, the formation of root structures of mycorrhizal fungi (G. mosseae, G. etunicatum, G. fasciculatus and Gi. margarita) as well as the level of phenolic compounds and the pathogenesis related proteins β-1.3-glucanase and chitinase were determined.
The root colonization of the host plant by mycorrhizal fungi were increased with the stage of seedling. GM and GF showed the highest root colonization level. Mycorrhizal arbuscule formation was observed at the Stage II of host development, while both, arbuscule and spores were observed at stage III.
Severity of Phytophthora root rot symptoms were reduced on the combined mycorrhizal fungi and Pc treatments as compared to Pc inoculations alone. At the same time, the amount of total phenolic compounds were higher in all inoculated treatments than in the control treatment. However, the levels of phenolic compounds were much higher in the combined mycorrhizal fungi and pathogen inoculation treatments. Twelve different phenolic compounds were determined in the different treatments, except for one that could not be identified. Rabie (1998) reported that the amount of total phenolic compounds were increased by mycorrhizal fungus and nodule bacteria in fababean and Botrytis fabae pathosystem.
In present study, mycorrhizal fungi plus Pc inoculation resulted in an increase in the level of β-1.3-glucanase and chitinase compared to control treatment. The enzymes activities were the highest on the 6th day after inoculation in all treatments and decreased on the 3rd and 9th days after inoculation. Mycorrhizal fungal inoculations individually resulted in increasing levels of the both enzymes that were maximum for the combination of mycorrhizal fungi and Pc. Pozo et al. (1999) reported that G. mosseae and G. intraradices inoculation combined with Phtophthora parasitica inoculation of tomato resulted in an increase of β-1,3 glucanase activity 4 weeks after inoculation. Acibenzolar-S-methyl an abiotic inducer resulted in increasing level of enzymes activity as found by Suo and Leung (2002), the reported that ASM at 50 μM induced accumulation of extra cellular polysaccharides in rose against the rose scab pathogen Diplocarpon rosae. On the other hand, Garmendia et al. (2006) reported that in G. deserticola inoculated pepper plants, peroxidases activity slightly increased when plants were also inoculated with the Verticillium wilt pathogen.
A reduction of the severity of soil borne diseases by mycorrhizal fungi has
been demonstrated in some plant-pathogens systems (Caron et al., 1986;
Dar et al., 1997; Ozgonen et al., 2001). The known preventing
effects of mycorrhizal fungi against infection and/or colonization of plant
roots by soil borne pathogens as well as an increase in uptakes of plant nutrients
have been reported. However, physical protection against the plant pathogens
by enhancing some of the biochemical mechanisms have also been reported (Dumas-Gaudot
et al., 2000).
Dugassa et al. (1996) investigated the effects of G. intraradices against Fusarium oxysporum f.sp. lini in flax by physiological and biochemical methods and resulted in increased resistance to this pathogen due to the colonization of plant roots by mycorrhizal fungi. These effects are reported to be related with the highest root colonization and increased concentrations of phytohormones, as gibberellins and oxin in shoots and ethylene in roots.
Plant hydrolytic enzymes (chitinases, β-1,3-glucanases) are among those antifungal compounds which have potential roles in protection against plant pathogens (Dumas-Gaudot et al., 1996).
This findings showed that mycorrhizal symbiosis could work as signalling compounds in this system during the colonization process. It can be concluded that the amount of phenolic compounds and increased level of enzymes activity were related the specific mycorrhizal fungi and likely could be involved in protection of pepper plant against P. capsici.
We thank to Süleyman Demirel University Scientific Research Project Unit for supporting this project (Project number: BAP 1050-M-05).