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a CSIRO Plant Industry and Australian Cotton CRC, Locked Bag 59, Narrabri NSW 2390, Australia
b CSIRO Land and Water, Waite Campus, Urrbrae SA 5064, Australia
c The University of New England, Armidale NSW 2351, Australia
* Corresponding author (lewis.wilson{at}csiro.au).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: afm, adult female mites (Tetranychus urticae Koch) • ai, active ingredient • Bt, Bacillus thuringiensis • Ci, intercellular CO2 concentration • Cry 1Ac, crystal protein 1Ac • DAS, days after sowing • gs, stomatal conductance to water vapor • LPR, leaf penetration resistance • +M, mite treatment (plants artificially infested with mites) • –M, control treatment (no mites) • PAR, photosynthetically active radiation • Pn, net photosynthesis • RUE, radiation use efficiency • S1, Season 1 • S2, Season 2 • T, transpiration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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At the leaf level, the effects of mites on photosynthesis have been studied in a range of crops, including cotton. Brito et al. (1986) reported that mite infestation increased leaf resistance to CO2 uptake and decreased photosynthetic rate in glasshouse-grown cotton plants. Bondada et al. (1995) studied T. urticae damage to cotton grown in the field and found alterations to the stomatal apparatus and internal damage to the mesophyll cells, which resulted in declining photosynthesis in parallel with declining stomatal conductance and transpiration. However, studies of the relationship between the density of mites on leaves and effects on the photosynthetic rate of cotton leaves in the field are limited. Many other studies on the impact of spider mites on host plants such as peach [Prunus persicae (L.) Batsch], almond (Prunus dulcis Mill.), apple (Malus domestic Borkh), tomato (Lycopersicon esculentum Mill.), strawberry (Fragaria ananassa Duch.), and peppermint (Mentha piperita L.) have recorded both reduced leaf photosynthesis and transpiration rates (Hall and Ferree, 1975; Poskuta et al., 1975; DeAngelis et al., 1983; Youngman et al., 1986; Youngman and Barnes, 1986; Hare and Youngman, 1987; Royalty and Perring, 1989; Mobley and Marini, 1990; Nihoul et al., 1992). Reductions in photosynthesis have been shown to result from decreased stomatal opening and increased mesophyll resistance (Welter, 1989). However, the order in which these changes occur and how this varies with different mite densities and intensity of damage has not been clarified previously.
A further limitation of the above studies was that none considered the possibility of compensation for reductions in leaf photosynthesis because of mite feeding damage. For instance, it is possible that the photosynthetic rate of an undamaged portion of a mite-damaged leaf could increase relative to a leaf in the same position in an uninfested plant, thereby maintaining the total level of photosynthate produced per leaf (Nowak and Caldwell, 1984; Welter, 1989). In cotton, the possibility of compensation in response to mite damage was raised in crop level, rather than leaf level, studies by Sadras and Wilson (1997a). These authors derived estimates of crop RUE from measures of dry matter accumulation and light interception. They found that until mite populations exceeded about 20 adult female mites per leaf, the RUE of mite-infested cotton was maintained at similar levels to those occurring in uninfested cotton. This implied that the photosynthetic rate of leaves infested at this mite density was also being maintained, though portions of leaves were quite heavily damaged. Hence, the possibility of within-leaf compensation was invoked but was not tested because the photosynthetic rate of damaged and undamaged potions of leaves was not measured.
This study had two aims. The first aim was to investigate the relationship between the intensity of T. urticae infestation, characterized as the number of adult female mites per leaf (Wilson and Morton, 1993), and leaf physiological responses, including photosynthesis, transpiration, stomatal opening and conductance, transpiration efficiency, intercellular CO2 concentration, and leaf chlorophyll content. The second aim was to evaluate the possibility of within-leaf compensation for mite damage by assessing photosynthesis on damaged and undamaged portions of leaves.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Crop Management
The cotton crops were sown into cracking gray clay soil (Vertisol, Typic Pellustert; Ug5.25) (Northcote, 1979; Constable et al., 1992), in rows 1 m apart, on 9 Oct. 1996 in Season 1 and on 16 Oct. 1997 in Season 2. The plant density was 13 plants per meter in Season 1 and 10 plants per meter in Season 2 because of differences in establishment between years. The cultivar, Deltapine NuCotn 37, was used in both seasons and produces Bacillus thuringiensis subsp. kurstaki insecticidal proteins (Cry IAc) that provide control of Helicoverpa spp. (Lepidoptera, Noctuidae), the main insect pest of cotton in Australia. All insect pests were monitored twice weekly and controlled according to thresholds published in Shaw (1996) by insecticides that had little or no effect on mites directly. Crops were fertilized with anhydrous ammonia at a rate of 100 kg N ha–1 2 mo before sowing and furrow irrigated before sowing. Soil water content was measured weekly with a neutron probe as described in Sadras et al. (1998). Crops were furrow irrigated whenever a soil water deficit of 50 to 60 mm was reached. Weeds were controlled with herbicides and interrow cultivation after crop emergence.
Treatments and Experimental Design
Two mite treatments, (i) –M, control and (ii) +M, plants artificially infested with mites, were included in a randomized block design which was replicated four times within a season. Plots were 8 rows by 18 m in Season 1 and 8 rows by 15 m in Season 2. The crops were mite-free before they were artificially infested with mites 83 d after sowing (DAS) in Season 1 and 91 DAS in Season 2, using the procedures developed by Wilson (1993)( 1994a). These procedures included (i) spraying of crops with a broad-spectrum insecticide [thiodicarb (3,7,9,13-tetramethyl-5,11-dioxa-2,8,14-trithia-4,7,9,12-tetra-azapentadeca-3,12-diene-6,10-dione) at 750 g ai ha–1] 2 d before mite infestation to eliminate mite predators and encourage mite establishment, (ii) infestation of the central rows of +M subplots with mite-infested cotton seedlings grown in a glasshouse, (iii) use of 4-m intervening bare soil gaps between plots to reduce the risk of cross infestation between +M and –M plots, and (iv) control of mites in –M plots with acaricides whenever mites exceeded an average of one adult female mite per leaf.
Measurements
Weekly measurements of photosynthetic rate, stomatal conductance, transpiration, intercellular CO2 concentration, transpiration efficiency, chlorophyll content, leaf damage, and mite abundance were made, beginning 1 wk before the introduction of mite infestations. To avoid possible effects of miticides (see above) on photosynthesis, measurements were not taken within 48 h of an application. Measurements were made on the top most fully expanded mainstem leaf, which was usually four nodes below the terminal; this is also the node most likely to contain the highest mite density within the cotton plant (Wilson and Morton, 1993). This means that the position of the leaf measured remained constant relative to the plant terminal but that it was at a progressively higher node position in relation to the cotyledons as the season progressed. Because the leaf that occupied this position changes from week to week the fate of particular leaves over time was not studied. Each week, two measurements were taken on one leaf on a previously tagged plant in the center row of each plot, one measurement at the basal and one at the distal portion (see below). A new plant was tagged in each plot each week (i.e., four leaves were measured per mite treatment per week, four new leaves the following week, and so on).
To assess within-leaf compensation, all plant measurements were taken from the basal portion of each leaf, where mites initially colonize leaves, and the distal portion of the leaves, which are colonized by mites later as mite populations build and spread (Wilson, 1994b) (i.e., two measurements per leaf, Fig. 1) . Mite abundance and leaf area damaged were recorded for the whole leaf.
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Leaf chlorophyll content was measured with a SPAD 501 chlorophyll meter (Minolta, Osaka, Japan), which has been tested in a number of plant species, including cotton (Wood et al., 1992). The relationship between actual chlorophyll content and output from the SPAD is nonlinear and was calibrated for analysis by the exponential decay equation (Markwell et al., 1995):
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The number of adult female mites and feeding damage were recorded for each tagged leaf in both the +M and –M plots. Feeding damage was scored to compare the leaf damage per mite between years and leaf portions and to test the relationship with photosynthetic rate and related components. In Season 1, damage was scored by estimating the percentage of the abaxial leaf area visibly damaged by mites, irrespective of damage intensity, as described in Wilson and Morton (1993). In Season 2, the damage intensity was similarly estimated but classified further as light damage, where the leaf showed the pale yellow mottling typical of a short period of mite feeding or heavy damage, where the leaf showed the dark red-brown scarred areas typical of prolonged mite feeding.
In Season 1, stomatal imprints were taken to (i) relate stomatal conductance to the proportion of open stomata and (ii) assess if mite feeding alters this relationship. Stomatal imprints were taken at solar noon on three dates, 21 Jan. (104 DAS), 10 Feb. (124 DAS), and 19 Feb. (133 DAS), 1997. Imprints were made by painting a thin layer of clear fingernail polish over a 1-cm2 area of the abaxial surface of the leaf. This was allowed to dry for 1 min. Adhesive tape was then placed over the nail polish and peeled off, removing with it the nail polish with stomatal imprints. The tape with nail polish was then stuck to a 35-mm glass microscope slide. The number of open and closed stomata was counted in a 1-cm by 1-mm area (about 500 stomata) of each imprint with a compound microscope (Nikon, model L-Ke, Nippon Kogaku Inc., Garden City, NY, USA). Measurements were taken at four sites on each leaf, a visibly damaged and an undamaged sample from both the basal and distal portions.
Data for leaf penetration resistance were obtained from Sadras et al. (1998) for Season 1 and unpublished data of Sadras and Wilson in Season 2. Briefly, penetration resistance was measured on attached leaves with a dial tension gauge (Sadras et al., 1998). Three leaves per plot and three positions per leaf near the insertion of the petiole, where mites prefer to feed (Wilson, 1994b), were measured and averaged at approximately weekly intervals.
Calculations and Statistical Analyses
Days after sowing were converted to cumulative degree days after sowing with a base temperature of 12°C, which is close to the threshold for development of both cotton and T. urticae (Wilson, 1993).
Linear and nonlinear curves were used to characterize the relationship between leaf response variables, i.e., photosynthesis, stomatal conductance, transpiration, intercellular CO2 concentration, transpiration efficiency and chlorophyll content, and the number of adult female mites per leaf (SigmaPlot 2000, SPSS Science, Chicago, IL, USA). The nonlinear curves fitted included exponential decay, which allows for a rapid drop in the dependent variable as the independent variable increases, and a negative logistic growth curve, which allows for a delay in the decline of the dependent variable as the independent variable increases. To account for ontogenetic and environmental effects, net photosynthesis and other components for the +M treatments were normalized with respect to controls. This was done by expressing the +M as a percentage of –M for each sample occasion. An analysis of variance including terms for block, mite treatment, leaf portion, and damage status was used when comparing the percentage of stomata open of +M and –M mite treatments (Genstat Version 5, Lawes Agricultural Trust, IACR, Rothamsted, UK). Mite numbers were loge transformed and damaged leaf percentages arcsine transformed before analysis to stabilize variances (Wilson, 1993), but untransformed values are shown in figures for easier interpretation. Significant differences were expressed at 95% (P < 0.05) confidence unless otherwise stated.
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RESULTS |
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Photosynthetic Compensation
There was no significant evidence for within-leaf photosynthetic compensation for mite damage in either season. At no time was there a significant increase in Pn in either basal or distal leaf sections of the +M leaves relative to the –M leaves (Fig. 4a,b)
. In contrast, Pn declined rapidly as mite numbers increased in the damaged basal areas and declined even in the undamaged distal portions of damaged leaves.
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Relative Sensitivity of Pn, gs, T, and TE to Mite Damage
In the basal portions, initial reductions of 10% in Pn, gs, T and TE, in +M compared with –M treatments, all occurred at less than 7 afm leaf–1 in both seasons (Table 2). Larger reductions of 50%, in +M compared to –M treatments, followed a more discernable sequence in both seasons with gs affected first at 8 to 11 afm leaf–1, followed by Pn at 8 to 14 afm leaf–1, followed by TE at 11 to 21 afm leaf–1, then T at 20 to 36 afm leaf–1. The more rapid decline in Pn compared with T probably explains why TE declined at a lower afm leaf–1 than T.
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Relationship between Ci and Mite Density
Ci showed a linear increase with increasing mite density in both seasons, with a greater increase per afm in the basal portions than the distal portions (Fig. 4i,j, Table 1). It is important to note that Ci is calculated from photosynthesis, transpiration, and vapor pressure deficit, thus Ci and Pn are correlated to a degree.
Stomatal Imprints
Stomatal imprints were taken in Season 1 after significant reductions in Pn, gs, and T had occurred in both the basal and distal leaf positions of the +M leaves (Fig. 5)
. Reductions in the percentage of open stomata were generally found on damaged and undamaged areas in both distal and basal portions of mite infested leaves compared with uninfested control leaves (Fig. 5, 6)
. On uninfested leaves, the proportion of open stomata ranged 50 to 70%. On damaged leaf areas of mite infested leaves, it ranged between 5 to 30% while on undamaged portions it was higher, ranging between about 20 to 55% but still less than the uninfested leaves. On the final measurement date, there was a similar percentage of open stomata on the undamaged portions of mite-infested leaves and control leaves.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The rate of increase in the mite populations of the +M plots in Seasons 1 and 2 was similar and the relationship between the mite abundance and damage was the same in both seasons (Fig. 3). However, the mites were added to plots about 385 day degrees earlier in Season 1, in terms of cotton development, which may have been a major cause for the difference in response between seasons. This suggestion is supported by Wilson (1993) and Sadras and Wilson (1996) who found that the earlier that mite infestations occur, the greater the potential reductions in cotton yield, fiber quality, seed viability and oil content.
Leaf penetration resistance was higher in Season 2 than in Season 1. The higher penetration resistance in Season 2 probably reflected differences in growing conditions. Jiang and Ridsdill-Smith (1996) found that increased leaf toughness in subterranean clover cotyledons (Trifolium subterraneum subsp. subterraneum L.), measured with a similar penetrometer to that used in this study, was negatively correlated with damage scores from redlegged earth mite [Halotydeus destructor (Tucker)]. Sadras et al. (1998) found that mites feeding on harder-leaved water-stressed cotton caused less marked symptoms of mite damage than those on softer-leaved cotton receiving optimal water despite similar levels of infestation. Given the differences in leaf hardness between Season 1 and 2, it is possible that the intensity of damage, in terms of the number of puncture holes in the leaf, was lower in Season 2, even though the proportion of leaf area damaged per afm was similar. Further, Sadras et al. (1998) showed that penetration resistance was positively correlated with specific leaf weight; that is, leaves with higher penetration resistance were thicker. If leaves were thicker in Season 2, it is possible that a higher proportion of the photosynthetic apparatus was undamaged, compared with Season 1, because it was beyond the reach of mite stylets (discussed below). Combined with differences in the timing of mite infestation and differences in growing conditions, a difference in leaf hardness could therefore contribute to differences in the severity of damage to cotton leaves and therefore the differences in the response of photosynthesis to mites between seasons.
Leaf Responses to Mite Damage
Both stomatal and nonstomatal components of photosynthesis, chlorophyll for example, were reduced by mite feeding damage and corresponded to reductions in photosynthetic rate. Where the mites caused the most severe damage, particularly the basal leaf portions, the greatest effects on leaf physiology occurred (Fig. 4). In the basal portions of leaves, transpiration, stomatal conductance, photosynthesis, chlorophyll content (Fig. 7), and transpiration efficiency were reduced quickly at low mite densities, while in the distal areas effects occurred at higher mite densities (Table 2).
Leaf transpiration efficiency and photosynthetic rate were reduced by 10% at a similar rate but photosynthetic rate was reduced by 50% more rapidly than leaf transpiration (Table 2). Anisohydric species, such as cotton and sunflower (Helianthus annuus L.), tend to keep stomata open and tolerate severe drops in leaf water potential in response to soil water deficit (Tardieu and Simonneau, 1998). Stomatal closure induced by mites (Fig. 5, 6) and consequent heating of the leaf and canopy (Sadras and Wilson, 1997a) therefore contrasts with the trend to maintain high conductance in water-stressed cotton.
The rapid closure of stomata in damaged areas, and resulting decline in gs may be related to the nature of feeding of mites. Their piercing mouthparts (stylets) are about 132 ± 27 µm long. Studies with strawberries as host plants found mites penetrated leaves to a depth of about 117.5 ± 24.9 µm (Sances et al., 1979). Cotton leaves are typically in the order of 255 µm thick (Pettigrew et al., 1993). Assuming mites penetrate the abaxial surface of cotton leaves to a similar depth to strawberry leaves then damage should occur mostly to the spongy mesophyll, and this has been shown by Bondada et al. (1995), though some damage to palisade mesophyll was also observed. Damage to spongy mesophyll has been associated with dehydration and a lack of turgidity of stomatal guard cells, resulting in stomatal closure (Bondada et al., 1995; Sances et al., 1979), which is likely to be one of the first components of photosynthesis to be affected.
There was a strong relationship between chlorophyll content and photosynthetic rate, particularly in the heavily mite damaged areas in basal leaf portions (Fig. 8). However, photosynthesis declined more rapidly than chlorophyll content, suggesting that the initial rapid decline in photosynthesis observed in the basal areas was probably driven primarily by rapid stomatal closure, thereby limiting gas exchange, rather than by loss of chlorophyll. After mite feeding, there are many penetration holes in damaged areas of the leaf surface, which presumably allow water loss independent of the stomata, hence transpiration may appear to be maintained. That gs and photosynthetic rate were reduced by 50% more rapidly than transpiration provides some support for this suggestion (Table 2).
In relatively undamaged areas (distal portions), photosynthesis was reduced and cannot be attributed to loss of chlorophyll, at least in the initial stages. In these areas, reductions in stomatal conductance occurred even though there was no direct feeding damage. We determined a sequence of events as a result of mite damage in distal areas for Season 2, where first stomatal conductance was reduced, then transpiration, then photosynthetic rate and finally transpiration efficiency (Fig. 9) . In Season 1, reductions in physiological processes were too rapid in the distal areas so a sequence of events could not be determined.
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Interestingly, intercellular CO2 concentration increased with increasing mite density in both basal and distal portions over the two seasons (Fig. 4). In contrast, Wong et al. (1979) showed that under many conditions where photosynthetic capacity and stomatal conductance declined, the ratio of intercellular and ambient CO2 concentration often remained constant. In order for plants to do this and therefore maximize their efficiency of water use, plants synchronize stomatal opening with the CO2 requirement of the assimilatory tissue (Farquhar et al., 1978, 2001). In our study, an increase in Ci was found when photosynthesis decreased as a result of mite feeding damage rather than a constant intercellular CO2 concentration as would be expected. This may be due to puncture holes in the plant (discussed above) caused by mite feeding damage or even possibly damage to some stomata leading to leakage of H2O, therefore preventing Ci from being maintained.
Within-Leaf Compensation
Two seasons of fieldwork did not show evidence of within-leaf compensation for mite damage. Sadras and Wilson (1997a) in studies of mite-cotton interactions at the crop level found that mite damage caused significant reductions in crop radiation use efficiency (RUE), which is essentially a crop level reflection of effects on photosynthesis. They also reported an initial tolerance of RUE to increasing mite density, indicating possible compensatory photosynthesis. In the study presented here, photosynthesis declined rapidly in response to increasing mite density in basal areas. The only evidence for such a lag in response was for the distal portion in Season 2; however, there was no evidence of elevated rates of photosynthesis that would suggest compensation. Instead, we report for the first time that damage to basal leaf areas also results in reductions in photosynthesis, stomatal conductance, transpiration and transpiration efficiency in the distal, undamaged portions of leaves. This response is delayed compared with the responses in the basal areas, particularly in Season 2, and indicates that damage to basal areas eventually has an effect on undamaged leaf portions.
Herbivores often induce biphasic responses in plants, where at low levels of herbivory an increase in production potential can occur, whereas extreme herbivory causes extreme reduction in productivity (Dyer et al., 1993). No evidence of such a response was found in this study. Furthermore, Welter (1989) noted that photosynthetic compensation has not been recorded as a result of mesophyll or selective tissue feeders such as spider mites. The results from this study agree with Welter (1989) as it would appear that selective tissue feeding of the mesophyll cells by mites has negatively affected the surrounding tissue.
This study does not explain the initial lag in decline of RUE reported by Sadras and Wilson (1997a). It is possible, however, that there could be within plant compensation, i.e., undamaged leaves on mite infested plants could have higher rates of photosynthesis than similar aged leaves on undamaged plants. This possibility is reinforced by the within plant distribution of mites which is initially skewed toward younger leaves, leaving the bulk of the plant's mature leaves undamaged.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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No evidence of within leaf compensation for mite damage was found. In contrast, undamaged portions of damaged leaves showed a decline in photosynthesis compared with similar portions of undamaged leaves.
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ACKNOWLEDGMENTS |
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Received for publication March 18, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This article has been cited by other articles:
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  P. D. Nabity, J. A. Zavala, and E. H. DeLucia Indirect suppression of photosynthesis on individual leaves by arthropod herbivory Ann. Bot., February 1, 2009; 103(4): 655 - 663. [Abstract] [Full Text] [PDF]
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 A. A. Reddall, L. J. Wilson, P. C. Gregg, and V. O. Sadras Photosynthetic Response of Cotton to Spider Mite Damage: Interaction with Light and Compensatory Mechanisms Crop Sci., September 1, 2007; 47(5): 2047 - 2057. [Abstract] [Full Text] [PDF]
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