EFFECT OF TEMPERATURE, pH AND ESSENTIAL OILS ON THE MYCELIAL GROWTH OF RHIZOCTONIA SOLANI Kühn (CANTHARELLALES: CERATOBASIDIACEAE) ISOLATES

This research aimed to study diversity among a collection of Rhizoctonia solani strains, isolated from many crops in Morocco, based on radial mycelial growth on potato dextrose agar (PDA) at different pH and temperature levels. The in vitro antifungal efficacy of essential oils (EOs) derived from aromatic and medicinal plants of Thymus vulgaris, Origanum compactum, Rosmarinus officinalis, Eucalyptus sp., Salvia sp., Cistus ladanifer and Lavandula stoechas was also evaluated by measuring the mycelial growth of this plant pathogenic fungus after placing active mycelial plugs of each strain on Petri dishes using a disc diffusion method of EOs. The Minimum Inhibitory Concentration (MIC) of each EO was also determined. The growth rates observed under different temperatures and pH levels varied among isolates and did not show similar responses for the same levels of the two growth parameters. However, the maximum mycelial growth of the majority of isolates was reached between 20 and 30°C as well as at pH 7. Among the EOs tested, the results revealed that EO of Thymus vulgaris and Origanum compactum were very effective on controlling R. solani strain RS1 with growth inhibition rate of 75,9% and 60,6%, respectively. The MIC of these EOs was at 0.03%. According to the gas chromatography analysis, Carvacrol (61.8%) and thymol (47.8%) are the major constituents of O. compactum and T. vulgaris EOs, respectively, suggesting that these natural compounds have important potential to control R. solani and could be useful for developing effective organic fungicides.


INTRODUCTION
Rhizoctonia solani Kuhn (Teleomorph: Thanatephorus cucumeris) is one of the most serious soil-borne fungal pathogens, causing disease on a large number of host plant species (Sneh et al., 1991). Isolates of this pathogen has a complex biology and diversity in terms of myclial colour, size of aerial mycelium, growth rate, zonation, type and number of sclerotia, saprophytic behaviour, enzyme production and pathogenicity (Hyakumachi et al., 1988;Salunkhe et al. 2008). In addition to the recognized methods to group isolates of R. solani taxonomically, diversity of growth character within different pH and temperatures levels has been noted by Chang (1985). The most favorable temperature level for R. solani development is 23-28°C, while lower and higher optima have been noted for different isolates (Abe, 1978). Another report showed the optimal temperature between 25 to 35°C with the most convenient media were potato-dextrose-agar, malt-extract and Sabouraudglucose (Csöndes et al., 2007). In Morocco, no detailed information is available on the variability in R. solani species, in particular the diversity of isolates and their behavior under different pH and temperature regimes. Chemical fungicides are often used to avoid the substantial yield losses caused by R. solani. However, the intensive use of chemical fungicides has not only created problems of fungicide resistance, but has also resulted in the increase of the soil contamination, high toxicity of microbial communities and a deterioration effect on the ozone layer (Huang et al., 2011). In addition, chemical control is not completely effective, and Rhizoctonia disease still damages diverse crops (Goudjal et al., 2014). To overcome this challenge, the use of plants extracts may be an alternative to conventional fungicides to control R. solani. The activity pathway of these biopesticides against fungi is unrevealed but may be interrelated to their general capability to soften or otherwise dislocate the reliability of cell membranes and cell walls (Isman, and Machial, 2006). The objectives of this study were to i) demonstrate the existing diversity among R. solani isolates based on growth at different pH and temperature regimes on culture media, ii) determine the efficacy of some plant-derived EOs against the growth of R. solani in laboratory assays, iii) identify the major constituents of these oils by gas chromatography method using appropriate detectors.

Rhizoctonia solani isolates
Naturally diseased plants of different crops (melon, water melon, sugar beet, bean, olive tree, strawberries, potato and tomato) showing typical symptoms of root rot disease were collected from several locations in Morocco (Tab1). For fungal isolation, the collected plant roots were washed carefully under running tap water followed by sterile water, then dried between two filter papers. Roots were cut into small pieces, that were transferred into ethanol 70° during 3 min for surface sterilization. Surface sterilized pieces were then washed several times with sterilized water to wash out the remaining disinfectant solution. The pieces were then dried on sterilized filter papers. Using sterilized forceps, plot dried pieces were then transferred into Petri dishes containing potato dextrose agar medium (PDA) (Rashad et al., 2012). Concerning potato tubers, sclerotia (black scurf) detached from tubers were disinfected in ethanol 70° for 1 min, then washed with sterile distilled water and dried on sterile filter paper. Disinfected sclerotia were divided in many pieces and left to culture on PDA medium (Djébali et al., 2014). The dishes were then incubated at 25±1°C, and the fungal growth were checked two days after incubation. In order to obtain pure cultures, purification of the isolates was done using the hyphal tip technique (Rashad et al., 2012). Identification of the pure isolates were done according to the cultural properties, in particular morphological and microscopical characteristics (Sneh et al., 1991). were evaluated in this trial. Fifteen milliliters of PDA media were added into each of the Petri dish (9 cm diameter) then an agar plug of fungal inoculums (4 mm diameter) was cut aseptically with a sterile scalpel from the margin of 2 days old culture of the sixteen R. solani isolates. One disc was placed in the center of individual Petri dish and three dishes were used for each isolate. After inoculation, the Petri dishes were incubated maintaining every time one of the eight temperature levels.
Radial mycelial growth was calculated by averaging the two diameters taken at right angle of each colony when mycelial growth of some isolates reached the edge of the Petri dish (Goswami et al., 2011).

Effect of pH on mycelial growth of R. solani isolates
Five different levels of pH "5; 6; 7; 8 and 8.5" were maintained to study the isolates mycelial growth variation at different pH levels adjusted by adding HCI or NaOH before solidifying the PDA media (Goswami et al., 2011). The method followed for inoculation of mycelial discs of R. solani isolates with five pH levels was the same as described before. After inoculation, the Petri plates were incubated at 25±1°C. Radial mycelial growth was calculated by averaging the two diameters taken at right angles for each colony when mycelial growth of some isolates reached the edge of the Petri dish.
In order to facilitate the conduct of the other tests, eight representatives isolates were selected from the sixteen isolates. This selection was based on the origin (plant) of isolates and the average growth rates (mm/d) of the sixteen isolates under different temperature and pH levels.

Antifungal activity evaluation
Petri dishes with 9 cm diameter and containing 20 ml of PDA media were used to evaluate the antifungal activity of EOs by the disc diffusion method (Duru et al., 2003). Sterile Whatman paper discs of 6 mm diameter were placed on the PDA media, equidistant and near the border, where the EO (5 µL/disc) was added separately. An agar plug of fungal inoculums (6 mm diameter) was removed from a young culture of the eight fungal strains studied, and placed near the other border of the Petri dishes. The plates were incubated at 25±1ºC for 5 days, until the growth in the control plates reached the edge of the plates. Concerning the control, 5 µL of sterilized water was added to each disc. For each treatment, plates were prepared in triplicate and the percentage of growth inhibition was calculated using the following formula (Topps and Wain 1957):

Determination of Minimum Inhibitory Concentrations (MICs)
The MIC values of R. solani isolates were detremined following the agar dilution method as defined previously by Gul et al. (2002). Appropriate volume of the EO was added aseptically to sterile molten PDA medium to produce the concentration range of 0,01% -0,5%. After vortexing, the resulting PDA solutions were immediately poured into Petri plates. An agar plug of fungal inoculums (6 mm diameter) was removed from a young culture of all the fungal isolate tested, and placed in the center of the Petri dishes. The plates were prepared in triplicate for each treatment and were incubated at 25±1ºC for 5 days, until the growth in the control plates (PDA not mixed with EOs) reached the edge of the plates. The MIC values were determined as the lowest concentrations of the EO where the absence of growth was recorded.

Characterization of essential oils by Gas Chromatography analysis
Determination of the EOs chemical composition was done using a Hewlett-Packard model HP6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a DB-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm; Agilent Technologies, USA) and coupled to an HP model 5973 mass selective detector. The column temperature is programmed from 50 to 300 °C with an increase of 7 °C/min. The injector temperature was 290 °C. Purified helium was used as a carrier gas with a flow rate of 1 mL/min, and the split ratio was 60:1. Mass spectra (MS) was obtained, in EI mode, at 70-eV ionization energy and the mass range was from 35 to 400 m/z. 10 μL of each EO was diluted in 990 μL of pure hexane, and for the analysis 1 μL was injected. The apparatus was piloted by an HP electronic system equipped with a ChemStation Software "G1701BA, version B.01.00", and the data were analysed out with the same software. After each compound analysis, the Kovats retention index (RI) was calculated comparing to a standard mix of n-alkanes between C8 and C26 (Sigma-Aldrich Co.) analyzed under same conditions. The components were identified by comparing the IR and MS spectra with those reported in the literature (Adams 2007, Habbadi et al., 2018) and by computer matching using standard reference databases (NIST98, Wiley275, and CNRS libraries).

Statistical analysis
Analysis of variance (ANOVA) was used to analyse results related to the effects of plant EOs on mycelial growth of R. solani strains and to check the existence of variability among R. Solani strains under different temperature and pH regimes on PDA media. Least Significant Difference (LSD) test was used at the 5% level of significance; Statistical analysis was performed using SAS statistic software.

Effect of temperature on mycelial growth of R. solani isolates
According to the ANOVA results, there is significant interaction between R. solani isolates and temperature (P<0.0001) and significant main effect of both temperature and isolates on growth rate (Tab 3). Comparison between the sixteen isolates showed that isolates don't develop under 5°C and 35°C except isolate RS7 under 35°C with the average growth rate (9.6 mm/d) (

Effect of pH on mycelial growth of R. solani isolates
Diagnostic of the ANOVA results (Tab 5) revealed significant interaction between R. solani isolates and pH level (P<0.0001) and significant main effect of both pH level and isolates on growth rate. Average growth rate showed significant difference among different pH levels for each isolate. All sixteen isolates of R. solani were able to develop on PDA medium at all levels of pH within the range of 5-8.5 with certain similarities between average growth rate under pH=7 and the control (Tab 6). The average growth rate also showed high value (20.0 mm/d) for the isolate RS1 under pH=7 and lower value (1.5mm/d) under pH=8.5 for the isolate RS10.

Antifungal activity
The ANOVA results (Tab 7) revealed significant interaction between isolates and EO (P<0.0001) and significant main effect of both EO and isolates on growth inhibition rate. The growth inhibition rates of eight R. solani isolates using seven EOs of Salvia sp., Cistus ladanifer L., Lavandula stoechas L., Thymus vulgaris L., Origanum compactum Benth., Rosmarinus officinalis L. and Eucalyptus sp. are shown in Figure1. The growth inhibition rate showed high value for the isolate RS1 (75.9%) using the EO of Thymus vulgaris L. and the lower value was for the isolate RS4 (1.9%) using the EO of Rosmarinus officinalis L. EO of Origanum compactum showed the higher level of growth inhibition rates followed by Thymus vulgaris L. EO on all R. solani isolates except the RS1 isolate, suggesting the great potential of these two EOs in controlling the development of all R. solani isolates tested in this study. However, EO of Cistus ladanifer L. showed the lowest level of growth inhibition rates on most of R. solani isolates (Figure 2).

Determination of Minimum Inhibitory Concentrations (MICs)
Results of this assay showed that the plant EOs have different MIC on R. solani isolates. However, the MIC of T. vulgaris and O. compactum EOs on R. solani isolates was similar with an average of 0.03% (Figure 3). Whereas, the others EOs showed a MIC higher than 0.15%. This difference could be due to the chemical compounds of each plant EO.

Essential oil composition
In this study, the GC-MS analysis revealed that the major compounds of the EOs examined were monoterpene hydro-carbons and phenolic monoterpenes.   Chang (1985) found that both mycelial growth and sclerotial developpement of R. solani were maximal at pH 7 which confirm the results of our study. Sharma and Chowdhury (1984) observed that R. solani has a low incidence in cauliflower at neutral pH comparing to pH 7.4 -8.5. Also, Marcelo and Vega (1988) found that the most favorable pH for R. solani development was in pH 6-6.5. Moreover, Kobayashi (1985) found that soil pH has a direct influence on the hyphal growth of R. solani.
In the last several years, many researches have been conducted for the development of safer antifungal agents such as EOs and plants extracts to control plant pathogens in agriculture ( In the present study, among the EOs that were selected for GC analysis, the carvacrol and thymol were identified as main constituents for O. compactum and T. vulgaris, respectively. It is well known that some plants contain compounds that are able to inhibit microbial growth (Naovi et al., 1991). Additionally, antagonistic or synergistic effects between some EOs constituents may also affect the observed antimicrobial activity of these EOs. The use of these EOs tested on R. solani fungus may be a good source of new alternative active ingredient for an effective and sustainable management of this disease. Also, EOs have two importants advantages; low toxicity for people and environment due to their natural characteristics and low risk for resistance development by pathogenic microorganisms. However, further formulation and field experiments are necessary to achieve this goal.