تاثیر اسید سالیسیلیک بر روی رشد و بیماریزایی قارچ Zymoseptoria tritici

Introduction

Septoria tritici blotch (STB) is one of the most damaging fungal diseases of wheat which is caused by a fungus, Zymoseptoria tritici (Desm.), Quaedvlieg et al. (1) previously identified as Mycosphaerella graminicola.The common method in the management of this disease is mainly based on the use of fungicides (1). There are many reports about disease control failure in agricultural systems causing fungicide resistance (2-4). However, this method is not appropriate because of the high cost and appearance of the new virulence patterns. Moreover, the adverse environmental effects of using fungicides are notable and alternatives including biological agents are used more and more (5-8). In studies related to hemibiotrophic pathogenic pathosystem, Z. tritici is a model pathogen which according to the results of conducted surveys, ranked among the ten most important phytopathogenic fungi in the world (9).

Salicylic acid (SA) is one of the secondary metabolites which is produced in a broad range of both prokaryotic and eukaryotic organisms such as plants (10). This compound as a phenolic phytohormone has different roles in physiological processes including growth, development, photosynthesis, transpiration, ion uptake and transport (11). In plant immunity system, the most documented role of SA is mounting immune responses (12). As an endogenous signal molecule, SA is primarily involved in triggering immunity responses against biotrophic and hemibiotrophic pathogens, and also in the establishment of Systemic Acquired Resistance (SAR). Establishment of SAR by inducing the expression of pathogenesis-related (PR) proteins led to protection against a broad range of pathogenic organisms (13).

Although, the detailed mechanism of SA-mediated plant resistance is not known, the key role of SA in plant defence is generally accepted (14-16). Studies have shown that the exogenous usage of SA or its functional analogues, like benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 2,6-dichloroisonicotonic acid can induce SAR (17-19). Inversely, plants with NahG gene, which converts SA to catechol, are more sensitive to various pathogens (20). Primary knowledge on the role of SA in plant defence arises from the recognition of an Arabidopsis thaliana mutant (sid2-2) that is disabled in SA biosynthesis (21).

The detailed role of SA in the Z. tritici-wheat pathosystem is still not well determined. However, results of some studies have shown that the exogenous application of SA has produced resistance in wheat (22). Qi et al. (23) studied the effects of SA on head blight fungus, Fusarium graminearum in wheat. Their finding showed that SA has significant effects on the efficiency of germination and mycelium growth of the fungi, and also, based on their results, 400µM SA was sufficient to prevent diseases in susceptible and moderate cultivars.

Since the use of resistant varieties is one of the best approaches in the management of STB, the effect of SA and SA-mediated plant resistance in this pathogen was studied.

In the present study, for the first time, the direct effect of SA on Z. tritici conidia germination and mycelium growth was investigated. Finally, the positive effects of proper concentrations of SA were evaluated in wheat defence responses. The results of this study should be considered as promising new leads for the development of a new strategy of this pathogen management.

Materials and methods

Strain and growth conditions: Z. tritici virulent strain IPO323 was used for the experiments. IPO323 isolate was cultured on Yeast Malt Dextrose Agar (YMDA) and maintained at 18°C for five days. Conidia obtained from these plates were spread into 50 ml Falcon tubes containing 30 ml (YMDB). The inoculated YMDB were placed at 18°Cat 135 rpm for five days. In order to prevent bacterial contamination, 50 mg/L kanamycin was added to all media. The experiment was conducted in both solid and liquid media.

The direct effect of SA on the germination of conidia and growth of mycelium was tested in liquid YMDB medium. To do this, spore concentration was adjusted to 200 spores per ml using a haemocytometer. Then, 500 μl of the prepared spore suspension was transferred into 50 ml YMDB media which amended with 0.3, 0.5, 0.8, 1, 2, 4, 5, 10 and 20 mM SA (Sigma). The inoculated YMDB media were maintained in shaker-incubator at 18°C at 135 rpm. At 0, 0.5, 2, 4, 8, 12 and 24 hours after the interaction, 500 μl was sampled from each flask and then inoculated on YMDA in three replications. Different concentrations of SA were provided from 1M stock solution in methanol. Plates with 1μl ml^-1 of methanol were used as control treatments. Five days after inoculation, the images of colonies on the plates were captured by a digital camera. All inoculations and photographs were made by the same operator during a routine working day.

In the other method to study the direct effect of SA on the germination of conidia and growth of mycelium, fungi were inoculated on YMDA containing 0, 0.3, 0.5, 0.8, 1, 2, 4, 5, 10 and 20 mM of SA. All concentrations with three replicate were inoculated by 500 μl conidia suspension and placed at 18°Cfor five days.

The size of colonies was measured with the powerful Java-based image processing tool (ImageJ 1.48v). Each Colony's growth was determined by calculating the relative size of 50-70 colonies on each plate and then the average values were used for analysis (24-26). In the analysis, only colonies obviously grown from single spore were considered, and fused colonies which were developed from two or more spores were discarded.

Pathogenicity assay: The experiment was performed in a randomized complete block factorial design with three replications. Susceptible bread wheat (Triticum aestivum) Chamran cultivar was grown in 8-cm diameter pots. The IPO323 isolate was grown for 5-7 days on YMDB; spore concentration obtained from the media was adjusted to 10⁷ spores/ml and supplemented with 0.15% Tween 20. Twenty-four hours before inoculation, seedlings were treated with 2 and 4 mM concentrations of SA. Ten-day-old wheat seedlings were inoculated using hand sprayer. After inoculation, plants were transferred into plastic bags to keep high humidity and maintained at 20-22°C under the dark conditions for 48 hours. Subsequently, they were brought out from the plastic bags and kept in the same greenhouse with high humidity (>75% RH). The first symptoms were visible between 14 and 16 days of post-inoculation. Finally, scoring was performed on first leaves at 21th day of post-inoculation by visual estimation of the percentage of leaf area with necrotic lesions bearing pycnidia (27).

Data analysis: To evaluate the data, analysis of variance (ANOVA) was performed with SPSS software version 20. Comparisons among means were conducted by Duncan at P= 0.01.

Results

In this study, the inhibitory effects of SA on Z. tritici at different concentrations in in vitro conditions, and also, the efficacy of its exogenous application in the control of STB in wheat under the greenhouse condition were investigated. In liquid media interactions based on the results of data analysis of variance, significant differences (p≤0.01) were observed among SA concentrations and time points. Also, the interaction between concentration and time point was also significant (p≤0.01) (data not shown). In our survey, low levels of SA had little inhibitory effect on conidia germination, but at high levels, a significant and proper impact was observed. At the first time points, conidia had more capacity of germination which, by increasing the duration of interaction, the reduction trend was observed in germination rate (Fig.1). On YMDB, by increasing the time period of interaction, conidia germination in lower concentration (up to 1mM) was affected negligibly. In 2 and 4 mM, the number of germinated conidia was decreased remarkably. However, the germination was observed at all time points. At 5mM, only a few numbers of zero time point spores were able to germinate and by increasing the time period of interaction, no increase in germination was observed. At 10 mM concentration only in time point of zero, one of the replications of plates contained three germinated spores. No colony growth and germination was observed in 20 mM concentration at all of the time points. Also, no significant changes were observed in colonies diameter on plates related to liquid medium interactions (Fig. 1).

In modified YMDA, based on the results, the germination ratio was significantly (p≤0.01) affected by different SA concentrations. The germination ratio of conidia was not significantly affected (p≤0.01) up to 0.8 mM (Fig. 2) and reduction in germination rate started from 1 mM concentration. However, germination was extremely prevented at 2 mM SA and halted by 4 mM. At 4 mM and higher concentrations, conidia were not able to germinate and in microscopic observation had wrinkled and shrunken appearance. Briefly, it was observed that there is a negative correlation between SA concentration and germination rate of conidia, i.e. as the concentration increases the germination rate decreases.

Fig. 1- The average values of germinated conidia on modified liquid YMDB media with 1, 2, 4, 5, 10 and 20 mM concentrations of SA at different time points.

Fig. 2- The average values of germinated conidia on YMDA media with different concentrations of SA. met: Methanol in the same concentration was used as solvent. Values followed by the same letter(s) are not significantly different at p≤0.01.

Inhibitory effect of YMDA plates with different concentrations of SA on mycelium growth was not significant up to 0.5 mM. In spite of that the single colonies, diameter was reduced significantly at concentrations above 0.8 mM (especially at 1 and 2 mM). At 4 mM and higher concentrations, no colonies were grown (Fig. 3 and 4).

To further clarify the relationship between SA and Z. tritici growth and pathogenesis, we considered the foliar application of this component. As the results of the research confirmed the in vitro inhibitory effects of 2 and 4 mM of SA, for further investigation, their impact on wheat defence responses stimulation was evaluated in greenhouse examinations. Based on the results of analysis of variance, there were significant differences (p≤0.01) in pycnidium coverage percentage in interaction with different SA concentrations. The results showed that 2 and 4 mM SA were effective to reduce disease symptom in the susceptible cv. Chamran, but none of them completely prevents the symptoms development in infected wheat. With 2 mM SA, inoculated leaves were partially protected but the application of SA at 4 mM indicated lower values of diseases symptoms on wheat leaves (Fig. 5).

Fig. 3- The average values of grown colonies on YMDA media containing different SA concentrations. Values followed by the same letter(s) are not significantly different at p≤0.01.

Fig. 4- The number and size of Z. tritici grown colonies in the presence of different concentrations of SA. A: 2 mM SA SA. B: 0.8 mM