Experimental & AppliedAcarology, 19 (1995) 581-591 581

Chlorophyll fluorescence and leaf chlorophyll content of bean leaves

injured by spider mites (Acari: Tetranychidae)

G. Iatrou, C.M. Cook a, G. Stamou and T. Lanaras*. a Department of Ecology, Aristotle University, PO Box 119,

GR-540 06 Thessaloniki, Greece aDepartment of Botany, Aristotle University, PO Box 109,

GR-540 06 Thessaloniki, Greece

ABSTRACT

The use of chlorophyll fluorescence as a method for detecting and monitoring plant stress arising from Tetranychus urticae (Koch) feeding injury was investigated. The effect of mite density (1-32 mites per 1.5 cm z of leaf) and the duration of the feeding period (1-5 days) on the chlorophyll fluorescence parameters of bean (Phaseolus oulgaris) leaves were examined. Changes in chlorophyll fluorescence parameters were dependent both on mite density and duration of feeding. Decreases in Fo, the initial fluorescence and Fro, the maximum fluorescence led to a decrease in the ratio of variable to maximum fluorescence, Fv/F m. The decrease in F J F m is typical of the response of many plants to a wide range of environmental stresses and indicates a reduced efficiency of photosystem II (PSII) pho- tochemistry. 7"1/2, which is proportional to the pool size of electron acceptors on the reducing side of PSII, was also reduced in response to mite-feeding injury. The leaf chlorophyll content decreased with increasing mite density and duration of feeding but did not appear to contribute to the decrease in F v / F m. Chlorophyll fluorescence is an effective method for detecting and monitoring stress in T. urticae-injured bean leaves.

Key words: Tetranychus urticae, feeding damage, Phaseolus vulgaris, plant stress

INTRODUCTION

Severe spider-mite infestations cause major reductions in plant growth rates, flower formation and yield (Sances et al., 1981, 1982; Tomczyk and Kropezyfiska, 1985). Penetrat ion of cells by mite stylets and the injection of saliva cause both mechanical damage and changes in the cell cytology and physiological and biochemical processes of non-punctured adjacent cells (Tomczyk and Kropezyfiska, 1985). In leaves damaged by mite feed-

*To whom correspondence should be addressed.

0168-8162 © 1995 Chapman & Hall

582 G. IATROU ETAL.

ing a number of processes affect plant productivity: degeneration of chloroplast structure (Tanigoshi and Davis, 1978; Mothes and Seitz, 1982), reductions in chlorophyll concentration (De Angelis et al., 1983; Rilling and Diiring, 1990), partial inhibition of photosynthesis and stomatal con- ductance (De Angelis et al., 1983; Rilling and Diiring, 1990; Candolfi et al.. 1992). r educ t ions in stornatai (dayt ime or nho tone r iod ) t ran~nirat ion ~ - -7 . . . . ~ 7 . . . . . . . . . . . . . . . . . . . . . . ~----d . . . . . . f . . . . f . . . . . i . . . . . . f . . . . . . . .

(De Angelis et al., 1982; Rilling and Diiring, 1990; Candolfi et al., 1992) and increases in cuticular (nighttime or scotoperiod) transpiration (De Angelis et al., 1982). Mite-induced water stress brought on by damage to the leaf epidermis and cuticle causes stomatal closure. Leaf gas exchange is thereby reduced and as a consequence photosynthesis is inhibited.

The degree of severity of infestation and the effect on physiological and biochemical processes are variable depending on the mite species and plant host due to, for example, differences in leaf anatomy, mite feeding behaviour and stylet length (Youngman et al., 1986). Therefore, it is necessary to determine in each case the population density which causes plant stress. However, effects on plant processes such as photosynthesis may take weeks to become evident. Recently, chlorophyll fluorescence has been demonstrated to be an early indicator of plant stress caused by aphid feeding (Blanco et al., 1992). Chlorophyll fluorescence is a rapid, non- destructive method capable of evaluating the effect of a wide range of environmental factors on photosynthetic capacity in the absence of visible leaf symptoms (Bolhhr-Nordenkampf eta!. , 1989). Part of the light energy absorbed by the chlorophyll antenna of the photosynthetic apparatus of a leaf is re-emitted as fluorescence (Bolh~r-Nordenkampf et al., 1989; Kranse and Weis, 1991). Fluorescence emission can be related to pho- tosynthetic rate and efficiency. The sudden illumination of a dark-adapted leaf causes an immediate rise in fluorescence to an initial level termed Fo, representing the fluorescence when the majority of the photosystem II (PSII) reaction centres are open, i.e. the primary electron acceptor is oxidized. Further illumination causes fluorescence to increase from F o to a maximum Fro, as the PSII reaction centres become reduced and the rate of photochemistry declines. The ratio of the variable (F v) to maximum fluorescence ( ( F m - F o ) / F m) is proportional to the quantum yield of photochemistry. Half of the time (t l /2) taken for the fluorescence to rise from F o to F m is proportional to the pool size of electron acceptors (plastoquinones) on the reducing side of PSII (Krause and Weis, 1991). The size of the electron acceptor pool and, thus, t , /2, can be reduced by some stress treatments. The use of chlorophyll fluorescence in the assess- ment of plant stress caused by spider mite feeding has been investigated in this study.

CONTENT OF BEAN LEAVES INJURED BY SPIDER MITES 583

MATERIAL AND METHODS

Spider mite culture Mites (Tetranychus urticae Koch; Acari: Tetranychidae) were obtained from greenhouse roses in Thessaloniki and were maintained in detached leaf culture on leaves of Phaseolus mlgaris (Striven and McMurtry, 1971). Mature, newly-moulted virgin females were used in the experiments. The mites were confined in cages located on the lower surface of the leaves. Cages were a modification of the 'clip-on sandwich cage' descn'bed by Lewis (1973) and consisted of a lightweight metal spring clip to which two rings of foam were attached and between which the leaf was placed. The end of the cage was closed with a circle of wire gauze and transparent plastic sheeting. The leaf area enclosed by the cage was 1.5 cm 2. Experi- mentation was limited to 5 days to avoid the hatching of eggs and an increase in the number of mites.

Plant culture Beans (P. mlgaris L. var. Zargana Kavala) were germinated in the dark for 7 days in perlite saturated with water. Germinated beans were planted individually in pots (0.5 1) containing perlite and were watered with full strength Hoagland's solution (Hoagland and Arnon, 1950) every 2 days and the day prior to measurements. Plants were maintained under a L16 (24-2WC):D8 (19-20°C) regime. Plants were illuminated with cool-white fluorescence tubes giving an approximate light intensity of 140-200 #mol m - 2 s - 1 at the level of the leaves. The first pair of fully expanded leaves of 7 day old plants were used in the experiments. One cage was placed close to the petiole between the midrib and the edge of the leaf. The mean leaf area of the first 12 day old, P. mlgaris leaves was 87.09 + 5.80 em 2 (n = 10, ev, 21%).

Chlorophyll fluorescence measurements The chlorophyll fluorescence parameters, initial fluorescence (Fo) , maxi- mum fluorescence (Fm), variable fluorescence (Fv, where F v = F m - F o) and the half-time (tl/2) for the rise from F o to F m were measured using a portable fluorimeter (Plant Stress Meter, Biomonitor, SCI AB, Ume~, Sweden) as described by Oqulst and Wass (1988). Measurements were carried out on the leaf area enclosed by the cages of both mite-infested and control leaves after dark adaptation of the leaves for 45 rain, using a light intensity of 400 /~mol m -2 s -1. Measurements were carried out in situ on intact plants and at room temperature.

584 G. 1ATROU ETAL,

Chlorophyll determination The leaf area enclosed by each cage (1.5 ClTI 2) Was excised and extracted in dimethyl sulphoxide (DMSO) at 60°C for 1 h. The chlorophyll concentra- tion was calculated using the extinction coefficients given by Barnes et al. (1992).

Effect of mite density Individuals of T. urticae were placed on the lower surface of one leaf of each of the pairs of first fully expanded, 7 day old bean leaves at densities of 0 (control), 1, 2, 4, 8, 16 and 32 mites per cage and allowed to feed for 5 days. Five plants were used at each mite density. Measurements of chlorophyll fluorescence (on the upper and lower leaf surfaces) and chlorophyll concentration were made on the infested and control areas. Intraplant controls (uninjured leaves) were areas enclosed by cages with- out mites on the opposite leaf of the pair.

Temporal changes at foced mite density Individuals of T. urticae were placed on the lower surface of both of the first fully expanded, 7 day old bean leaves at a density of 16 mites per cage. The chlorophyll fluorescence and chlorophyll concentration were measured daily for 5 days. Four plants (eight measurements) were used each day. Control measurements were carried out daily on two plants (four measurements).

RESULTS

Chlorophyll fluorescence parameters (Fo, Fv, Fro, Fv//Fm and tl/2) changed with the density and duration of mite infestation (Table 1 and Figs 1 and 2). The responses were similar whether measured on the upper or lower leaf surface with the exception of F o (Table 1). Responses were more pronounced on the lower leaf surface for a given mite density and duration of feeding, where immediate mite damage was experienced (Table 1). Values of F o, F m and Fv measured on the lower surface of control leaves were statistically greater than those of the upper leaf surface (Table 1).

At an infestation level of 32 mites per cage F o, F m and F v, measured on the lower leaf surface, decreased to 50, 85 and 67% of the control (absence of mites), respectively. For the upper leaf surface F m and Fv also decreased but to a lesser extent (25 and 33%, respectively) while F o showed a 12% increase. The ratio Fv/F m decreased with increasing

CONTENT OF BEAN LEAVES INJURED BY SPIDER MITES 585

TABLE 1

Overall mean values + sE (n = 5) of chlorophyll fluorescence parameters for upper and lower surfaces of control and mite-infested leaves of P. vulgaris for all Z urticae infestation levels (1, 2, 4, 8, 16 and 32 mites per cage) after 5 days' exposure.

Control leaves Infested leaves

Lower leaf surface Upper leaf surface Lower leaf surface Upper leaf surface

F o 0.459 (0.002) 0.341 (0.004) 0.428 (0.013) 0.376 (0.008) F~, 2.503 (0.017) 2.045 (0.016) 1.711 (0.185) 1.817 (0.107) F v 2.040 (0.018) 1.707 (0.012) 1.284 (0.173) 1.442 (0.103) Fv/F m 0.816 (0.002) 0.834 (0.001) 0.718 (0.031) 0.785 (0.016) tl/2 39.490 (0.940) 65.250 (1.260) 27.350 (1.200) 34.510 (3.170)

infestation level (Fig. la) with 23 and 11% decreases for the lower and upper surfaces, respectively, at the highest infestation level. The lower leaf surface tl/2 value decreased by approximately 30% at an infestation level of one mite per cage and thereafter remained more or less constant with increasing infestation level (Fig. lb). When measured on the upper leaf surface it decreased with increasing infestation level to 50% at 16 mites per cage but showed no further change at 32 mites per cage. Overall mean values of tl/2 measured on the lower leaf surface of control leaves were almost half of those of the upper leaf surface (Table 1).

Changes in chlorophyll fluorescence parameters on the lower leaf surface were followed over a duration of 5 days at a fixed infestation level (16 mites per cage). These conditions were chosen since more pronounced changes in chlorophyll fluorescence were observed on the lower leaf surface and since no greater effect at higher mite-densities was observed. F o and F m decreased daily with total decreases of 59 and 25% on day 5, respectively, while they remained unchanged for control plants (Fig. 2a and b). The ratio Fv/F m decreased with increasing duration of mite infestation with major reductions occurring after day 3, while it remained unchanged for control leaves (Fig. 2c). The value of tl/2 decreased daily for both control and mite-infested leaves but decreases were significantly greater for infested leaves (Fig. 2d).

The chlorophyll concentration decreased significantly with increasing infestation level, falling to approximately 50% of initial values at an infestation level of 32 mites per cage and after exposure for 5 days (Fig. 3a). A daily decrease in chlorophyll was observed over a 5 day period for both control and infested leaves, but was more pronounced for infested leaves (Fig. 3b).

586 G. IATROU ETAL

o.s -ll

d 0.7 ~ ~ N ~ I

l 0o5 . . . . . , . . . . . . . . . .

a

40

'? 35

e ~ v 3~o

25

20 1

o i I i I - i . i . i . I . ,

4 8 12 16 20 24 28 32

Infestation level (mites per cage)

Fig. 1. The relationship between (a) the ratio of the variable to maximum fluorescence, F v / F m, and (b) the half-time for the rise from F o to F m, tl/2, in relation to, T. urticae infestation level (number of mites per cage). Chlorophyll fluorescence was measured on the lower surface of leaves in the areas exposed to mites for 5 days. Bars represent SE; n = 5 .

DISCUSSION

Changes in chlorophyll fluorescence parameters measured on the lower leaf surface were more sensitive indicators of damage caused by mite feeding than those on the upper leaf surface. Most mite species feed predominantly on the lower leaf surface (Tomezyk and Kropezyfiska, 1985) and the depth at which injury occurs is related to the length of the stylet, duration of feeding and population density (Mothes and Seitz, 1982). Although mite feeding on the lower leaf surface causes damage primarily in the spongy mesophyll tissue, damage to the palisade parenchyma, which contains more chloroplasts, may also occur (Tomezyk and Kropczyfiska, 1985). Injury to both the spongy and palisade parenehyma of bean leaves has been reported as a result of feeding by T. urticae (Mothes and Seitz, 1982).

CONTENT OF BEAN LEAVES INJURED BY SPIDER MITES 587

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o~

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Fig. 2. Temporal changes in chlorophyll fluorescence parameters over 5 days: (a) initial fluorescence, /7o; (b) maximum fluorescence, Fm; (c) the ratio of maximum to variable fluorescence, Fv/Fm; and (d) the half-time for the rise from F o to Fro, t l /2, measured on the lower surface of control ( • ) and T. urticae-infested (o) P. vulgaris leaves. Infested leaves (lower side) had a density of 16 mites per cage. Control leaves had empty cages. Values are the means of eight infested and two control measurements on four and two separate plants daily. Bars represent SE.

A comparison of the fluorescence characteristics of the upper and lower leaf surfaces of healthy leaves of several plant species indicated that both F o and F~ emissions (692 rim) were consistently higher on the lower than on the upper surface of the same leaf, with mean ratios of 1.52 and 1.58 for F o and Fro, respectively (Bj6rkman and Demmig, 1987). Likewise, F o and F m were consistently higher on the lower surface of the control bean leaves. F o and F m both decreased with respect to increasing mite density and duration of feeding, resulting in a decrease in F v / F m. This ratio is

5 8 8 G. IATROU E T A L .

E o

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O 4 8 12 16 20 24 28 32

Infestation level (mites per cage)

!

I , I i I , I i I , I

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Day

Fig. 3. The effect of T. urticae on P. vulgaris leaf chlorophyll O O l l l J ~ l l t l i : l . U O i l . $ , ~ L ] l l l l ~ l ~ l ~ l . t l O l l ~ i l l p

between chlorophyll concentration and infestation level. Chlorophyll was measured on leaves exposed to mites for 5 days. Bars represent SE; n = 6; (b) Temporal changes in chlorophyll concentrat ion measured daily over 5 days on control ( • ) and infested (o) leaves. Infested leaves had a density of 16 mites per cage. Control leaves had empty cages. Bars represent sE; n ffi4 for control and nffi8 for infested leaves. Chlorophyll was determined in all cases on leaf discs cut from the area enclosed by the cages.

remarkably consistent for a diverse range of healthy, non-stressed plant species, ranging from 0.778 to 0.860 (Bj6rkman and Demmig, 1987). The decrease in F v / F m for bean leaves in response to mite injury is typical of the response of many plants to a wide range of environmental stresses and indicates a reduced efficiency of PSII photochemistry (BjiSrkman and Demmig, 1987; Bolhbx-Nordenkampf et al., 1989; Krause and Weis, 1991). The relative decrease in F v / F m has also been used in the rapid assess- ment of plant susceptibility or resistance to aphids and of aphid phytotoxi- city (Blanco et al., 1992). The value tl/2, which is proportional to the pool size of electron acceptors on the reducing side of PSII, is also diminished by varying stress treatments (Bolhhr-Nordenkampf et al., 1989; Krause and Weis, 1991). The time-dependent decrease in tl/2 (Fig. 2d) indicates that the plastoqulnone pool on the reducing side of PSII is reduced in response to mite feeding injury and to a lesser extent by the presence of cages on the leaves.

The decrease in extractable chlorophyll in mite-injured leaf areas was dependent on the infestation level (population density) and the duration of the feeding period. A similar decrease in chlorophyll was observed in T. urticae-injured peppermint leaves, which was proportional to the injury

CONTENT OF BEAN LEAVES INJURED BY SPIDER MITES 589

sustained (De Angelis et al., 1983). Chlorophyll loss was proposed to result from cell disruption and the removal of chloroplasts rather than the enhanced metabolic degradation or decreased synthesis of chlorophyll; however, the possibility that mite-induced water stress influenced chloro- phyll metabolism was not discounted (De Angelis et al., 1983). The extent of the decrease in ehloronhvll content in mite-injured leaves ~enerallv . . . . . . . . . . . . . . . . . . . . . . . . r - - . I . . . . . . . . . . . . . . . . . . . . - . / . . . . . . . . . ~ . . . . . . . . J

depends on the amount of damage sustained by the palisade parenchymal cells which contain more chloroplasts (Tomczyk and Kropczyfiska, 1985). Damage is related to mite species, in terms of stylet length, salival composition and feeding behaviour and host-plant leaf morphology (Youngman et aL, 1986). The structural degeneration of chloroplasts in cells adjacent to those punctured (Tanigoshi and Davis, 1978; Mothes and Seitz, 1982) can also contribute to the decrease in leaf chlorophyll content (Tomczyk and Kropczyfiska, 1985). In T. urticae-injured bean leaves, cells in the lower layers of the palisade parenchyma are damaged (Mothes and Seitz, 1982) and the conversion of chlorophyll to phaeophytine has been reported (Van de Vrie et al., 1972).

Although chlorophyll content influences PSII fluorescence emission (Bj6rkman and Demmig, 1987; Morales et al., 1991; Jefferies, 1992) F v / F m

is generally unaffected in the range of chlorophyll concentrations found in fully developed green leaves of vascular plants (Bj6rkman and Demmig, 1987; Morales et al., 1991). F v / F m decreases only when drastic decreases in chlorophyll content occur (Bj6rkman and Demmig, 1987; Morales et al., 1991). The reduction in F v / F m observed for mite-infested bean leaves did not appear to be associated with a reduction in leaf chlorophyll concentra- tion. F v / F m remained constant in control leaves (Fig. 2c) despite a 14% decrease in chlorophyll (Fig. 3b). In addition, up to day 3, only a slight change in F v / F m (Fig. 2C) was observed in mite-injured leaves, while a marked decrease in chlorophyll concentration occurred (Fig. 3b). Further, after day 3 when a marked decrease in F v / F m occurred, only a slight change in chlorophyll concentration was observed (Figs 2c and 3b).

Plant stress in T. urticae-injured bean leaves may result from leaf tissue damage or saliva having direct effects on the photosynthetic apparatus or indirect effects on other physiological processes, which consequently feed back to photosynthesis. For example, tissue damage can lead to a lower leaf water potential and a loss in the functional integrity of the leaf cellular layers. Stomatal closure and increased mesophyll resistance can result causing a decrease in leaf conductance to CO 2. Assimilation of CO 2 is subsequently inhibited and this may contribute to the observed decrease in F v / F m (Sances et al., 1979; De Angelis et al., 1982, 1983; Candolfi et al.,

590 G. IATROU E T A L .

1992; Jefferies, 1992). Mite-induced water stress may also cause other metabolic changes which affect chlorophyll fluorescence. In aphid-infested barley leaves, observed decreases in chlorophyll content, CO z assimila- tion, total soluble carbohydrates and increases in proline content were due in part to the lower leaf water potential caused by aphid feeding (Cabrera et al., 1994). Chlorophyll fluorescence gives a rapid, overall picture of the photosynthetic capacity of intact plants in situ. The method has the potential to be standardized for particular plant-mite interactions to determine when intervention is necessary to control mite populations detrimental to plant growth and productivity.

ACKNOWLEDGEMENTS

The authors thank Dr J.P. Gutierrez (ENSA-M/INRA/URSTOM, Mont- pellier, France) for the identitieation of the spider mites. G. Iatrou and G. Stamou acknowledge the support of the MEDALUS II (Mediterranean Desertification and land Use) collaborative research project. MEDALUS II was funded by the EC under its Environment Programme, contract number EV5V 0128. The authors thank Mr A. Polyzos for technical assistance.

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