Reduced Water Potential Research Articles

Control of purple spot of loquat fruit (Eriobotrya japonica) by means of mineral compounds

SummaryTreatments to control purple spot of loquat fruit (Eriobotrya japonica) were tested based on the hypothesis that the disorder appears as a consequence of water unbalance between flesh and epidermal tissues caused by their different ability for sugar accumulation. Calcium nitrate, calcium chloride, Ca‐EDTA, ammonium nitrate and potassium nitrate at a concentration of 150 mm applied 2 weeks before fruit colour break reduced significantly the proportion of purple‐spotted fruit, giving rise to a reduction of water potential of the epidermal tissue that allows it to retain water. The most favourable date of treatment was during the 2 weeks prior to fruit colour break.

Evaluating fungi from wood and canola for their ability to decompose canola stubble

The survival and impact on canola of Leptosphaeria maculans, the causal agent of blackleg, might be reduced by agents that decompose canola stubble. Fifty-six fungal isolates from wood or canola were evaluated for their ability to overcome several biological and (or) physical constraints to their effectiveness in decomposing and (or) eliminating L. maculans from canola stubble. Relative to fungi from canola, wood decay fungi were more tolerant of reduced water potential but somewhat less tolerant of lower temperature. Wood decay fungi were no better than those isolated from canola at decomposing sterile canola stubble and less able than Coprinus spp. and Cyathus olla in colonizing and surviving in nonsterile stubble. None of the isolates were effective in eliminating L. maculans from stubble pieces or causing significant decomposition of nonsterile stubble either under laboratory conditions or in the field. These results suggest that considerable effort would be required to find isolates effective in managing blackleg disease through stubble decomposition. Wood decay fungi would seem to have little potential as biological control agents in this pathosystem because of their inability to colonize and decompose nonsterile canola stubble.

Interspecific interactions between the rare tooth fungi Creolophus cirrhatus, Hericium erinaceus and H. coralloides and other wood decay species in agar and wood

Creolophus cirrhatus, Hericium erinaceus and H. coralloides were paired against over 20 other wood decay fungi from beech (Fagus sylvatica) covering a range of ecological strategies, on 2% malt agar (MA), 0.5% MA, 0.5% MA adjusted to -1.25 MPa by addition of KCl, 0.5% MA adjusted to pH 4 with KOH/H3PO4 and 0.5% MA under 5% O2/30% CO2 all at 20 degrees C. Creolophus cirrhatus and H. coralloides were also paired against 17 other species in wood, incubated at 20 degrees for 36-38 wk. They were average to good combatants, deadlocking with or replacing over half of the antagonists, H. erinaceus being slightly more combative than the other two species. Outcomes in wood were representative of those in agar. Abiotic conditions altered the outcomes slightly, reduced water potential favouring C. cirrhatus and H. erinaceus. Overall extension rate of C. cirrhatus and H. erinaceus often increased in the presence of antagonists. There was prolific production of fruit bodies in controls and interactions, though notably not under altered gaseous regime. Results are discussed in an ecological context.

Germination responses of Caragana korshinskii Kom. to light, temperature and water stress

Caragana korshinskii Kom. is a very important shrub species for vegetation rehabilitation in northern China for its high ecological and economic values. Experiments were conducted to determine its germination responses to (i) different temperature regimes under light and/or dark conditions, (ii) different light intensities, and (iii) different water potentials combined with varied constant temperatures. Under alternating temperatures (from 5:15 to 25:35°C), final percent germinations of Caragana korshinskii were quite similar. In dark conditions, constant temperatures resulted in lower final percent germinations than alternating temperatures. At a controlled temperature regime of 10:20°C, neither final percent germinations nor germination rates showed significant differences among varied light intensities. As water potentials were reduced from 0 (distilled water) to −0.6 MPa, final percent germinations increased slightly and reached the peak at approximately −0.6 MPa, however, the increment was not significant. Beyond −0.6 MPa, further water potential reduction led to decreased final percent germinations and few seeds could germinate at −1.4 MPa. Water stress also strongly inhibited germination at very high or low temperatures. The experimental results suggested that middle May might be a suitable time for aerial seeding for this species.

Germination of winterfat ( Eurotia lanata (Pursh) Moq.) seeds at reduced water potentials: testing assumptions of hydrothermal time model

The hydrothermal time model not only quantifies seed germination progress as affected by temperature and water potential, but also has ecological and biological significance. Assumptions of the hydrothermal time model were tested using two non-dormant seed collections of winterfat ( Eurotia lanata) with two seed size classes. Winterfat is a native shrub with superior forage quality. Germination rates (GR) of subpopulations were estimated from germination time courses over a water potential range from 0 to −1.33 MPa at 2, 5, 10, 15, 20, and 25 °C. Parameters of the hydrothermal time model were estimated from the relationships between GR and temperature at various water potentials and between GR and ψ at various temperatures. Model assumptions were tested using these estimated parameters. Results indicate that base temperature ( T b(50)) and base water potential ( ψ b(50)) of the 50% subpopulation were not independent of temperature and water potential. The ψ b(50) was lowest at intermediate temperatures between 10 and 15 °C, while T b decreased linearly with increasing water availability. The estimated shift rates of T b(50) with ψ were between 2.18 and 3.81 °C MPa −1 for the two collections and large seeds had a greater shift rate than small seeds. Hydro time ( θ H) was constant among subpopulations only at optimal temperatures. A linear increase of θ H with subpopulation was found at lower temperatures, especially at 2 °C. There were no significant differences in ψ b(50) between large and small seeds, but significant differences were observed in hydrothermal time requirement ( θ HT(50)), which was lower at intermediate temperatures than at either lower or higher temperatures. The predictability of the hydrothermal time model was improved especially at low temperatures when θ HT(50) was allowed to change with temperature as measured by a modified R 2 value. Changes in other parameters with temperature or water potential did not further improve the predictability of the hydrothermal time model. Therefore, further efforts in improving the hydrothermal time model should focus on variations in θ HT.

Use of the hydrothermal time model to analyse interacting effects of water and temperature on germination of three grass species

The temperate grass species, red fescue (Festuca rubrassp.litoralis), perennial ryegrass (Lolium perenne) and Kentucky bluegrass (Poa pratensis), are often sown in mixtures for turfgrass. Differences in germination response to environmental conditions often result in different establishment success. The three species were compared in a study of imbibition at reduced water potential (Ψ) and in a study of the effects of Ψ and temperature on germination. In all three species, imbibition rate and extent were reduced with decreasing Ψ, causing a prolonged lag-phase before germination, particularly in Kentucky bluegrass. In perennial ryegrass and Kentucky bluegrass, water content at radicle emergence decreased non-linearly with decreasing Ψ, with a lower asymptote expressing the critical water content for germination. Seeds were germinated in factorial combinations of two temperatures (10 and 25°C) and nine Ψ levels (–2 to 0 MPa). Kentucky bluegrass required a longer thermal time to germinate than the other species and exhibited conditional dormancy at 25°C, which affected the use of the thermal time model. The effects of Ψ were described by a hydrotime model. Kentucky bluegrass had higher base water potential (Ψb) and required a longer hydrotime for germination than the other species. However, Ψbincreased with temperature in Kentucky bluegrass and red fescue, but decreased in perennial ryegrass, and the base temperatures also changed with Ψ. The interacting effects of Ψ and temperature affected the ability of the hydrothermal time model to predict germination performance across all temperature and Ψ conditions.

Effects of flooding and drought on stomatal activity, transpiration, photosynthesis, water potential and water channel activity in strawberry stolons and leaves

Transpiration, xylem water potential and water channel activity were studied in developing stolons and leaves of strawberry (Fragaria × ananassa Duch.) subjected to drought or flooding, together with morphological studies of their stomata and other surface structures. Stolons had 0.12 stomata mm−2 and a transpiration rate of 0.6 mmol H2O m−2 s−1, while the leaves had 300 stomata mm−2 and a transpiration rate of 5.6 mmol H2O m−2 s−1. Midday water potentials of stolons were always less negative than in leaves enabling nutrient ion and water transport via or to the strawberry stolons. Drought stress, but not flooding, decreased stolon and leaf water potential from −0.7 to −1 MPa and from −1 to −2 MPa, respectively, with a concomitant reduction in stomatal conductance from 75 to 30 mmol H2O m−2 s−1. However, leaf water potentials remained unchanged after flooding. Similarly, membrane vesicles derived from stolons of flooded strawberry plants showed no change in water channel activity. In these stolons, turgor may be preserved by maintaining root pressure, an electrochemical and ion gradient and xylem differentiation, assuming water channels remain open. By contrast, water channel activity was reduced in stolons of drought stressed strawberry plants. In every case, the effect of flooding on water relations of strawberry stolons and leaves was less pronounced than that of drought which cannot be explained by increased ABA. Stomatal closure under drought could be attributed to increased delivery of ABA from roots to the leaves. However, stomata closed more rapidly in leaves of flooded strawberry despite ABA delivery from the roots in the xylem to the leaves being strongly depressed. This stomatal closure under flooding may be due to release of stress ethylene. In the relative absence of stomata from the stolons, cellular (apoplastic) water transport in strawberry stolons was primarily driven by water channel activity with a gradient from the tip of the stolon to the base, concomitant with xylem differentiation and decreased water transport potential from the stolon tip to its base. Reduced water potential in the stolons under drought are discussed with respect to reduced putative water channel activity.

Desempenho de sementes de soja sob condições diferentes de potencial osmótico

Os cultivos sucessivos e as adubações necessárias acumulam resíduos minerais no solo, os quais alteram o potencial hídrico e afetam o desempenho fisiológico das sementes. Para avaliar a germinação e o vigor de sementes de soja, em condições variadas de potencial osmótico, conduziu-se um experimento em laboratório, no qual sementes de dois lotes da cultivar FEPAGRO RS 10, foram colocadas para germinar em papel-toalha umedecido com soluções de PEG 6000, NaCl, KCl e MgCl2, para a obtenção dos potencias osmótico de zero; -0,05; -0,10; -0,20; -040 e -0,80 MPa. As sementes foram avaliadas através do teste de germinação (primeira contagem e contagem final), comprimento e massa seca das plântulas. A análise dos resultados permitiu concluir que o decréscimo nos potenciais osmóticos induzidos pelos solutos utilizados reduziu o potencial fisiológico das sementes de soja, sendo o vigor mais afetado do que a germinação. O efeito da redução dos potenciais osmóticos sobre as sementes e plântulas depende da qualidade inicial das sementes, do tipo de sal e sua concentração. O PEG 6000 produz efeitos mais estressantes no desempenho das sementes de soja, do que os sais, quando há redução dos potenciais osmóticos até -0,80 MPa. O potencial osmótico de -0,80 MPa induzido por MgCl2 e PEG 6000 impede a germinação e a manifestação do vigor das sementes de soja.

Effect of water potential treatments on germination and seedling growth in some mungbean cultivars

Studies were conducted to find out the effect of reduced water potential treatments on germination and five seedling growth related characters viz., plumule length, radicle length, vigour index, promptness index and germination stress index in mung bean genotypes. Decreasing water potential or increasing moisture stress adversely affected germination and all seedling growth related characters in that every attribute was significantly reduced with every level of decreasing water potentials. The extent of reduction varied with the genotypes and the reduced water potential. However, the reduced water potential at -0.25MPa did not affect germination. Four genotypes recorded no germination at -1.00MPa and this would be critical level of water stress for mungbean. Five genotypes viz., KM 2, Vamban 1, Pusa 9072, K 1, and TARM 1 with higher germination stress index were found to possess higher level of tolerance to drought.

Relationship of water to adventitious rooting in stem cuttings of Populus species

Populus species, characterized by fast growth and easy vegetative propagation, are widely used in agroforestry practices. The substantial water requirement of poplars make them interesting subjects for water balance studies. No information exists on soil moisture requirements for initial root and shoot growth of Populus cuttings. This study on leafless hardwood cuttings of Populus x euramericana (Dode) Guinier cv. ‘Robusta’ examined the dynamics of water use during propagation, as influenced by two initial soil water potentials (−0.006 and −0.06 MPa). Differences in the initial water potential of the cuttings was achieved by three pretreatments i.e., fresh, soaked and dried. Initial Ψshoot was −1.45, −0.10 and −2.10 MPa in fresh, soaked and dried cuttings, respectively. Soil moisture had a major effect on rooting. Water-stressed cuttings took a longer time to root and had fewer roots. Pre-soaking of cuttings stimulated rooting, particularly under the drier soil moisture conditions. Initially the water potential of cuttings decreased with time and with the formation of roots it stabilized in all the pretreatments. The reduction in water potential of cuttings after planting was related to an increase in resistance to water flow in the xylem.

Circadian rhythm of stem and fruit diameter dynamics of Japanesepersimmon (Diospyrus kaki Thunb.) is affected by deficiency of water in saline environments.

Early diagnosis of water deficiency is essential to mitigate salt stress injury in plants. The effects of salt stress during the fruit growth stage on stem and fruit diameters of Japanese persimmon trees (Diospyrus kaki Thunb.) were measured by a micromorphometric technique under greenhouse conditions. This technique is less cumbersome and more precise in comparison to measurement of water potential in a small pressure chamber. The effect of stress was measured on photosynthetic rate, pre-dawn water potential, stomatal conductance, transpiration and Na+ and K+ contents of the stem and leaves. Salt stress was imposed by irrigating the plants with NaCl solution. Stem and fruit diameters of the plants given the control treatment started to decrease around 0600 h and reached a minimum at 1400 h. Salt stress did not change the diurnal pattern of response in stem and fruit diameter dynamics, but decreased the amplitude of the circadian rhythm by influencing both declining and recovery phases. The effect of salt stress on stem diameter appeared after 1 d of treatment, and on the third day in the fruit. Salt stress also reduced water potential, photosynthesis, transpiration and stomatal conductance, and increased concentrations of Na and K in the plant parts. Most of these effects were expressed after a lag period of 5 d of salt application. Utilization of micromorphometric techniques for early diagnosis of water deficiency in salt-prone environments is recommended based on results of this study.

The effect of water potential on accumulation of some essential elements in sugarbeet leaves, Beta vulgaris ssp. vulgaris

An investigation has been conducted on the effect of reduced water potential in nutrient solution on the accumulation of some essential macro- and micro nutrients in the aboveground pails of young sugarbeet plants. Plants of 8 different sugarbeet genotypes were exposed for 21 days to a nutrient solution whose water potential of 0.1 MPa was regulated by PEG. Contents of N, P, K Ca, Mg, Fe, Mn, Cu and Zn declined in all genotypes under water deficiency but the intensity of reduction varied among the genotypes. The results indicated that some harmful effects of water deficiency could be attributed to disturbances in plant mineral nutrition, especially the lack of N, P, and Mg, as well as to impaired ratios between the contents of particular elements, especially K/Ca.

Physiological and Morphological Traits Associated with Increased Water Use Efficiency in the Willow-leaf Peach

A chimeric, willow-leaf mutation of a standard peach [Prunus persica (L.) Batsch.] phenotype was evaluated for its water use efficiency (WUE). The willow-leaf phenotype had greater WUE than its standard-leaf parent under both nonstressed and well-watered conditions, and this was supported by isotopic carbon discrimination. Under water-stress conditions, willow-leaf trees developed less water potential gradient from the roots to the leaves. The mechanisms associated with increased WUE by the willow-leaf phenotype include a reduced water potential gradient within the plant and uncoupling of the leaf from the aerial environment. Willow-leaf peach trees in seedling populations, descended from a different willow-leaf parent, also had reduced carbon isotopic discrimination than did sibling standard-leaf seedlings.

THE RELATION OF MIDDAY STEM WATER POTENTIAL TO THE GROWTH AND PHYSIOLOGY OF FRUIT TREES UNDER WATER LIMITED CONDITIONS

Reductions in rates of both vegetative and fruit growth, and reductions in other physiological parameters such as leaf conductance and photosynthesis, are all well known responses of plants to water limited conditions. One simple hypothesis to explain the overall integration of these responses at the whole plant level is that a reduction in the availability of soil water to the roots will ultimately lead to a reduction in the water potential of the plant as a whole, and hence that a corresponding reduction in the turgor pressure of cells will occur. A reduction in cell turgor may lead, directly or indirectly, to many of the well known plant physiological responses to water limited conditions. A key observation that does not support this simple hypothesis is that under some water limited conditions, reductions in growth and stomatal conductance can occur in the absence of any detectable reduction in the water potential of fully exposed, transpiring leaves. This observation however, does not take into account the possibility that individual leaf stomatal responses may mask reductions in whole plant water potential. By using midday measurements of water potential made on non-transpiring leaves (midday stem water potential) we have found a clear relation between water potential and a number of physiological responses, such as fruit growth and leaf photosynthesis, across a range of fruit tree species growing under field conditions. In almond, overall tree size after two years of growth was linearly related to the average midday stem water potential measured during the same period. A similar linear relation was observed between fruit size and the seasonal average midday stem water potential measured in pear trees. In prune trees, despite an overall linear relation between water potential and light-saturated photosynthesis at the individual leaf level, evidence was found that whole canopy photosynthesis may be relatively unaffected by moderate levels of water stress (midday stem water potential around -1.5 MPa). We have found that moderate water stress is associated with improvements in fruit quality and economic return in prunes, and are currently using midday stem water potential to guide the management of RDI (regulated deficit irrigation) in this crop.

Accumulation of Compatible Solutes in Penicillium frequentans Grown at Reduced Water Activity and Biocontrol of Monilinia laxa

The biocontrol fungus Penicillium frequentans was grown on potato dextrose broth of either - 0.4 MPa water potential (unstressed) or reduced water potential (stressed) media modified with glycerol to - 7.0 MPa. The endogenous compatible solutes and sugars were quantified after 5, 10 and 15 days growth. Glycerol was the main compatible solute accumulated at reduced water potential, with a concomitant decrease in erythritol and mannitol, and the sugar, glucose. Maximum accumulation of glycerol occurred in 5-day-old cultures, with the content in P. frequentans biomass produced at reduced water potential being about 180 times higher than that produced in unmodified medium (- 0.4 MPa). There was no enhanced accumulation of the desiccation protectant trehalose. Homogenisation of cultures, for spray applications of the fungus, resulted in a partial loss of the solutes accumulated in the fungal biomass. P. frequentans controlled peach brown rot ( Monilinia laxa ) on peach twigs, with the disease index being significantly smaller when treated with the antagonist produced at reduced water potential. However, both inoculum types were able to reduce the length of lesions to a similar extent. This suggests that the inoculum of the antagonist produced in medium with freely available water (- 0.4 MPa) becomes rapidly adapted to the dry environment in the phyllosphere, counteracting the possible initial advantage of the inoculum produced at reduced water potential (- 7.0 MPa).

Leaf development, transpiration and ion uptake and distribution in sugarcane cultivars grown under salinity

The effects of salinity on leaf growth, initiation and senescence, on transpiration rates, on leaf water potential and on uptake and distribution of several ions were studied in two sugarcane cultivars differing in salinity sensitivity. Plants, growing in a growing mixture in pots, were exposed to salinized irrigation water for 68 days, starting 60 days after planting. EC values of the irrigation water were 1.0, 2.0, 4.0, 8.0 and 12 dS/m, obtained by using a mixture of NaCl and CaCl2. Plants were also grown in nutrient solution and were at a similar age when exposed to a salinity level of 3 dS/m for 30 days followed by 6.0 dS/m for an additional 30 days. Two Na:Ca ratios of 18:1 and 1:2 were used for salinization of the nutrient solution. Both leaf dry weight and area decreased with increasing salinity, but in the more salinity tolerant cultivar H69-8235, the decrease was moderate. Salinity hardly reduced average area per leaf in H69-8235, while the number of leaves declined sharply. This decline was caused by enhanced senescence of mature leaves and not by a decreased rate of leaf initiation. In the more sensitive cultivar, H65-7052, leaf area and initiation of new leaves were sharply reduced by salinity while leaf senescence was less affected. Leaf water potential decreased during the early stages of salinity exposure, and the reduction in water potential was larger in H69-8235. Salinity also decreased the rate of transpiration rate but to a lesser extent than leaf development and growth. The accumulation of Cl and Na in the TVD (top visible dewlap) leaf of the tolerant cultivar H69-8235 was greater than in the sensitive cultivar H65-7052. The concentration of Cl in the TVD leaf was more than 10 times that of Na in both cultivars. The concentration of both ions, but not of K, increased during the early stages of salinity exposure and then remained constant. A gradient in concentration of Cl and Na over the plant was found in both cultivars at all salinity levels, and was steepest between the TVD and younger leaves. No specific Na effect on leaf growth or transpiration could be detected. The accumulation of Cl and Na but not of K occurred primarily in the roots rather than in the leaves and stalks.

The genetical control of osmotolerance in fungi: A mutation analysis in the ascomycete Aspergillus nidulans

The ascomycete Aspergillus nidulans is remarkably osmotolerant and the wealth of genetical and physiological techniques available for this species make it an attractive model for a study of the basis of this character. A mutation assay estimates that around 200 genes can mutate to osmosensitivity and a number of these have now been identified as contributing to the ability to grow at reduced water potentials.

The Development of Seed Quality in Brassicas

Brassica crops have indeterminate growth and flower over an extended period of time. Harvested seed is therefore comprised of seed of varying degrees of physiological maturity and quality. Using population-based threshold models, broccoli (Brassica oleracea L. Group Italica), brussels sprouts (B. oleracea L. Group Gemmifera), red cabbage (B. oleracea L. Group Capitata), and rapeseed (B. napus L.) were characterized during seed development with respect to sensitivity to abiotic stress (reduced water potential) and shelf life. Using these models our data suggests that the physiological patterns of seed development are the same in all brassica crops we have tested to date. These population-based models can be used to provide a biological basis in which to evaluate cultural, postharvest and storage practices to ensure the production and maintenance of seed vigor.

Hydrothermal time analysis of tomato seed germination responses to priming treatments

Controlled hydration of seeds followed by drying (seed priming) is used to break dormancy, speed germination, and improve uniformity of radicle emergence. To date, empirical trials are used to predict optimal priming conditions for a given seed lot. Since priming is based upon seed water relations, it was hypothesized that the sensitivity of germination to reduced water potential before priming might be mechanistically related to, and therefore predictive of, priming responsiveness. Analyses of germination of 13 tomato (Lycopersicon esculentum Mill.) seed lots at two temperatures (15 °C and 20 °C) and three water potentials (0, −0.28 and −0.43 MPa) showed that seed lot germination responses could be quantitatively characterized by parameters derived from thermal time, hydrotime, and hydrothermal time models (R2=0.73–0.99). Six of the seed lots were primed at two temperatures (15 °C and 20 °C) and three water potentials (−1.0, −1.5 and −2.0 MPa) for various durations, dried, and their subsequent germination rates analysed according to hydropriming time and hydrothermal priming time models. The responses of germination rates to priming were characterized by hydropriming time (θHP) and hydrothermal priming time (θHTP) constants and the minimum water potential (ψmin) and temperature (Tmin) for achieving a priming effect. The values of ψmin and Tmin varied relatively little among tomato seed lots, and the generalized values of ψmin= −2.39 MPa and Tmin=9.10 °C accounted for 74% (15 °C), 57% (20 °C), and 62% (across both temperatures) of the increase in germination rates following priming. Nonetheless, while the hydrothermal time models described germination patterns both before and after priming, there was relatively little predictive relationship between them.

Acclimation of potato plants to polyethylene glycol-induced water deficit I. Photosynthesis and metabolism

The acclimation of photosynthesis and metabolism in response to water deficit is characterized using hydroponically grown potato plants (Solanum tuberosum cv. Désirée). Plants were subjected to a reduced water potential of the nutrient solution by adding 10% (w/v) PEG 6000. PEG-treated plants were retarded in growth. Leaves which had been fully developed before the PEG treatment and leaves grown during the PEG treatment showed different phenotypes and biochemical and physiological properties. Photosynthesis of all leaves decreased during the whole treatment. However, the decrease of photosynthesis in the two types of leaves had different causes indicated by differences in their metabolism. Leaves which were fully developed at the beginning of the PEG treatment began to wilt starting from the leaf rim. The apoplastic ABA content increased, coinciding with a decreased stomatal conductance. Increased energy charge of the cells indicated impaired chloroplastic metabolism, accompanied by a decrease of amounts of chloroplastic enzymes. The apoplastic and the symplastic ABA content were increased during water deficit and because ABA was concentrated in the cytosolic compartment it is suggested that ABA is involved in decreasing photosynthetic enzyme contents in old leaves. Young leaves, grown after the imposition of water deficit, were smaller than control leaves and had a curly surface. In young leaves apoplastic and cytosolic ABA contents were identical with control values. Carboxylation efficiency of photosynthesis was decreased, but the water use efficiency remained unchanged. Metabolic data of the photosynthetic pathways indicate a down-regulation of chloroplastic metabolism. It is concluded that in young leaves photosynthesis was non-stomatally limited. This limitation was not caused by ABA.