CHLOROPHYLL α FLUORESCENCE AS A DIAGNOSTIC TOOL FOR ASSESSMENT OF APPLE RESISTANCE AGAINST TWO-SPOTTED SPIDER MITE (TETRANYCHUS URTICAE KOCH.)

Wojciech Warabieda, Bożena Borkowska

ABSTRACT

The chlorophyll fluorescence measurements were used as a method of assessing the influence of Tetranychus urticae feeding on photosynthetic apparatus of ‘Lobo’ and ‘Jester’ apple cultivars. The photochemical efficiency measured with such parameters like: the initial fluorescence (F₀), the maximum fluorescence (F_m), the ratio of variable to maximum fluorescence (F_v/F_m), and the area under the fluorescence curve (S_c) decreases continuously with increasing infestation by mites. The only parameter not influenced by mites feeding was the half-rise time from F₀ to F_m (T_fm). Antennas of both ‘Lobo’ and ‘Jester’ cvs. were similarly injured by mites (comparable decrease of F₀) but photochemical reactions of the harvested energy of ‘Jester’ leaves were less influenced by this pest (lower decrease for F_v/F_m). It suggests a higher tolerance of the photosynthetic apparatus of ‘

Key words: apple, biotic stress, mite, resistance, chlorophyll fluorescence.

INTRODUCTION

Spider mites are common pests on many woody and herbaceous plants. Some species of that group, e.g. Two-spotted spider mite (Tetranychus urticae Koch.), Often building a huge population on different crops, including fruit trees. Chemical control of this species is expensive, highly controversial from ecological point of view and often far from full success, caused mainly by high fecundity and its capability to develop acaricide-resistant races. Growing apple resistant cultivars in addition to augmentation and introduction of natural enemies can form a base of pro-ecological approach in controlling T. Utricae in the apple commercial orchards. Differences in the level of infestation of the particular apple cultivars by mites were already observed in some previous studies indicating different levels of natural resistance to the mites [1, 3, 4, 11]. However a breading programme aimed to release new cultivars with higher level of resistance to the two-spotted spider mite would be r ather different task. One of the main reason is that the resistance of the plants against this mite species is probably of oligo or polygenic nature [5, 6].

Among the mechanisms of plant resistance to mites, the tolerance seems to be particularly important from practical point of view. Planting more tolerant cultivars may leads to avoid of needless pesticide applications and would be profitable for farmers and environment.

Screening techniques and methods have been developed, in order to detect and characterise the degree of plant resistance against pathogens or pests including spider mites [5]. Studies of biotic stresses using the chlorophyll α (Ch) fluorescence method revealed presence of heterogeneous photosynthetic activities in leaves that had been exposed to a pathogen. Using this method, an effect of biotic stress can be detected before any symptoms of it can be observed visually. This method allows also to evaluate the plant’s natural defence mechanism against infection [9].

The paper presents interactions between the two-spotted spider mite and the photosynthetic apparatus, using chlorophyll fluorescence analysis and checks the possibility of utilising this technique to assessment of apple tree tolerance to mite feeding.

MATERIALS AND METHODS

Plant material and infestation with mites. The experiment was conducted on one-year apple trees of ‘Lobo’ and ‘Jester’ cultivars grafted on M.9. These two apple cultivars characterize different level of resistance to T. urticae. ‘Lobo’ cv. represents the apple being more heavily infested by mites compared to ‘Jester’ cv. [12]. The trees were planted into 20 l pots filled with a mixture of peat and light soil in ratio 2:1. The experiment was carried out under greenhouse conditions.

Thirty trees of both cultivars were infested with mites at different level, on 25^th May, 1998. Every 10-14 days, the pest population density was measured by counting all the mobile mites on three leaves of each apple tree. The level of mites infestation of each tree was calculated as the cumulative mite days (CMD), according to the formula:

CMD = å 0.5 (P_a + P_b) D_{a – b}

where P_a and P_b are number of mites per leaf at time a and b, respectively, and D_{a – b} is the number of days between date a and b [13].

The area all of the checked leaves was measured using a Portable Leaf Area Meter CI 202 (ADC, England) device and the level of mite infestation was recalculated to 1 cm² of the leaf area.

Chlorophyll α fluorescence measurements. The chlorophyll fluorescence measurements were used as a method of assessing the influence of two-spotted spider mite feeding on photosynthetic apparatus of two selected apple cultivars. Ch fluorescence was measured on the abaxial side of the leaves by the fast direct fluorometer PEA (Hansatech, England) on 9^th August, 1998. The leaves were placed into clip, darkened for 20 min and then illuminated for 5 s with red light diodes (peak 650 nm, maximum PPFD at leaf surface was 3000 µmol m^-2s^-1). The samples were characterised by three basic parameters: the initial fluorescence (F₀), the maximum fluorescence (F_m), the half-rise time from F₀ to F_m (T_fm). The fast fluorescence rise starts from F₀ and reaches a maximum value F_m. The full kinetics of the fluorescence rise is given by value T_fm. Out of these parameters, se veral expressions were calculated. The basis of the calculated expressions is the ratio F_v/F_m. The value of this parameter is proportional to the quantum yield of photochemical reactions, where F_v (variable fluorescence) = F_m – F₀. Additionally, the parameter S_c was sampled. This parameter is a sum of non-reduced electron transfers between PSII and PSI [10].

Statistical analyses. The levels of infestation with mites of ‘Lobo’ and ‘Jester’ cultivars were compared using the Student’s t-test. Linear regression analysis was applied to determine relationship between the level of mite infestation of apple trees and particular parameters of chlorophyll fluorescence. Outliers (atypical observations), were determined using Cook's distance. Significance of differences between regression coefficients across both apple cultivars were evaluated using the method of comparison of few regression lines (“STAT” computer program). Other statistical analyses were conducted using “Statistica for Windows ver. 4.0” software package.

RESULTS

Experimental trees of both apple cultivars infested with various initial mite population have shown from 0.2 to 22 T. urticae specimen per cm² of leaf area, at the peak of the population growth on the June, 20. The mean level of leaf infestation with mites measured by CMD was similar for ‘Lobo’ and ‘Jester’ apple trees (fig. 1) allowing compare both cultivars with respect to the mites influence on the chlorophyll fluorescence parameters.

It was shown that the photochemical efficiency has decreased continuously with the increasing infestation by mites on both apple cultivars. It was expressed by the negative correlation between the value of most Ch fluorescence parameters and the level of mite infestation of leaves (fig. 2a, b, c, d). The only parameter that was not correlated with mite infestation was T_fm (fig. 2e).

In the regression graph, the intercept constitutes the predicted value of the analyzed parameter when the level of mite infestation measured with CMD is equal to zero. In this case the values of predicted basic parameters: F₀, F_m were similar for both compared cultivars (tab. 1). These findings indicate well functioning antennas and lack of non-radiative energy lost (heat dissipation). The value of the parameter F_v/F_m above 0.8, expresses a high potential activity of PSII in both apple cultivars (tab. 1). On the contrary, the lower S_c value for ‘Lobo’ cv. (p < 0.1) may indicate the handicapped electron transport between PSII and PSI (tab. 1). Comparison of T_fm for both apple cultivars using Student’s t-test revealed the significantly higher value of this parameter in ‘Jester’ cv. (p < 0.05).

Using the slope coefficients obtained in regression analysis seemed to be accurate in evaluation of the tolerance of both apple cultivars to mite infestation. Significant differences between slope coefficients of both apple cultivars were observed for the parameter F_v/F_m (tab. 1). The slope of the regression line for F_v/F_m was greater for ‘Lobo’ than for ‘Jester’ cv. (fig. 2c). Similar trend was found for the value of F_m, but unlike F_v/F_m the difference between slopes of the compared regression lines was not statistically significant (tab. 1, fig. 2b). On the contrary, the slopes of the regression lines for F₀ and S_c decreased faster in ‘Jester’ cv. as compared to ‘Lobo’, but no statistical differences were observed (tab. 1, fig. 2a, d).

DISCUSSION

Tolerance is the ability of a plant to yield and growth without any reduction, despite pest, pathogen or other stress presence. The plant response to biotic stresses have been poorly understood, mainly because its physiological effects are highly variable. An increasing number of reports suggest that the photosynthetic activity (especially light phase) is reduced when plants are exposed to environmental stresses, including pathogen or pest attack [2, 8]. Study of stress induced by T. urticae conducted on bean plants revealed a drop in F₀, F_m, F_v/F_m and T_fm parameter values on leaves that had been exposed to the pest. [8]. This confirms the results of our experiments. However, we have not noticed any influence of mites on T_fm. This unconformity is probably caused by different conditions of both experiments. In the experiment conducted on bean plants, the levels of mite density were fixed up from 1 to 32 mite specimen pe r 1.5 cm² of leaf area, and fluorescence measure was made after 1-5 days of mite feeding. In our study the infestation of a particular apple trees with mites was differentiated and fluorescence measurements were made about 10 weeks after mite introduction.

The comparison of both apple cultivars with regard to the T_fm parameter indicates the better kinetic of electron transport for ‘Jester’ cv. Furthermore, the higher value of S_c intercept parameter for ‘Jester’ means that the photosynthetic apparatus of the cultivar is highly effective in electron transfer between PSII and PSI. These findings suggest a better efficiency of light reaction in ‘Jester’ cv. as compared to ‘Lobo’. Antennas of both ‘Lobo’ and ‘Jester’ cvs. were injured by mites to a similar degree (comparable decrease of the slope for F₀) but photochemical reactions of harvested energy of ‘Jester’ leaves were less influenced by mites (the lower slope for F_v/F_m). It suggests a higher tolerance of photosynthetic apparatus of ‘Jester’ cv. to T. urticae feeding, as compared to ‘Lobo’ cv.

In our former publication we stated that differences in the level of tolerance of leaves to mite damage are accompanied by some anatomical characters of apple leaves [12]. After short period of feeding, intensive injuries of spongy mesophyll and epidermis of the lower side of the leaf were observed in cultivars Close and ‘Lobo’. Leaves of these cultivars are characterised by a loose structure of the spongy mesophyll and by a thin layer of cuticle covered epidermis. On the contrary, leaves of the cultivars ‘Jester’, ‘Marvit’ and ‘Piros’, with a tight cell arrangement of the mesophyll and a thick layer of cuticle were less damaged by this pest. We suggest that these anatomical differences create not only a physical barrier against the pest but also play an important role in the efficiency of the photosyntetic apparatus. The fluorescence measurement seems to be a useful tool for studies on plant – pest interactions, but must be used carefully and in combination with other techniques [7].

CONCLUSIONS

Results of the presented studies concerning the apple physiological reaction to infestation with two-spotted spider mite leading to the following conclusions:

1. The photochemical efficiency measured with Ch fluorescence method decreases continuously with increasing infestation by mites.

2. The negative correlation between the value of particular parameters of Ch fluorescence and the level of mite infestation was higher for ‘Lobo’ than ‘Jester’ cv.

3. The slope of the regression line for F_v/F_m decreased faster for ‘Lobo’ than for ‘Jester’ cv.

4. Results of the experiment suggest a higher tolerance of the photosynthetic apparatus of ‘Jester’ cv. to T. urticae feeding , as compared to ‘Lobo’ cv.

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