OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims:  To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity ( a _w) and sub-inhibitory doses of the fungicide carbendazim.
Methods and Results:  A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20–28°C), a _w (0·94–0·98), carbendazim level (0–450 ng ml⁻¹) and time (3–15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed.
Conclusions:  ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a _w, temperature and carbendazim doses.
Significance and Impact of the Study:  This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.     

Effect of modification of storage atmosphere on phospholipids and ultrastructure of cauliflower mitochondria

Differences in mitochondrial membrane composition and ultrastructure were studied after storage of cauliflower ( Brassica oleracea , L., Botrytis group) for 5 days at 25°C in air or under controlled atmospheres: 3% O₂, 21% O₂+ 15% CO₂ or 3% O₂+ 15% CO₂. In air, postharvest senescence involved a 20% decrease in mitochondrial phospholipid content. A large reduction in the relative abundance of phosphati-dylcholine (PC) and in the degree of unsaturation of PC and phosphatidyl ethanolamine (PE) was observed. However, the degree of unsaturation increased in cardiolipin (CL). Storage under 3% O₂ did not prevent phospholipid breakdown. Low O₂ prevented the relative decrease in PC observed during storage in air and the loss of linoleic acid from PC, but not from PE. This relative protection offered by the low O₂ atmosphere was lost under 3% O₂+ 15% CO₂. The high CO₂ atmospheres caused twice as much loss in phospholipids as that observed during storage in air. Extensive loss of mitochondrial protein, a marked decrease in phospholipid to protein ratio, and electron micrograph observations suggest structural alterations in the presence of high CO₂.     

Accumulation and utilization of triacylglycerol and polysaccharides in Liposcelis bostrychophila (Psocoptera, Liposcelididae) selected for resistance to carbon dioxide

Abstract: Two populations of the psocid, Liposcelis bostrychophila Badonnel, were exposed to two CO₂-enriched atmospheres (35% CO₂ + 21% O₂, and 55% CO₂ + 21% O₂, balance N₂) for 30 generations. Controls were reared in normal atmospheres. The reserves of triacylglycerol and polysaccharides were evaluated in adults of the two experimental and the control populations in generations F₁₅ and F₃₀. The utilization rate of triacylglycerol and polysaccharides in the CO₂-enriched atmospheres were also determined in generation F₃₀. The results indicated that the reserves of triacylglycerol and polysaccharides increased significantly during selection for CO₂ resistance; the higher the resistance level, the greater the reserves. Exposure of these populations to controlled atmosphere was associated with a steady utilization of the reserves. By contrast, the unselected population responded to controlled atmospheres by accelerated utilization of triacylglycerol and polysaccharides. Comparison of the utilization rates during CO₂ exposure showed that triacylglycerol is the main energy source, and polysaccharides contribute to metabolic energy supply only to a small extent.     

Growth, CO2 consumption and H2 production of Anabaena variabilis ATCC 29413-U under different irradiances and CO2 concentrations

Aims:  The objective of this study is to develop kinetic models based on batch experiments describing the growth, CO₂ consumption, and H₂ production of Anabaena variabilis ATCC 29413-U^TM as functions of irradiance and CO₂ concentration.
Methods and Results:  A parametric experimental study is performed for irradiances from 1120 to 16100 lux and for initial CO₂ mole fractions from 0·03 to 0·20 in argon at pH 7·0 ± 0·4 with nitrate in the medium. Kinetic models are successfully developed based on the Monod model and on a novel scaling analysis employing the CO₂ consumption half-time as the time scale.
Conclusions:  Monod models predict the growth, CO₂ consumption and O₂ production within 30%. Moreover, the CO₂ consumption half-time is an appropriate time scale for analysing all experimental data. In addition, the optimum initial CO₂ mole fraction is 0·05 for maximum growth and CO₂ consumption rates. Finally, the saturation irradiance is determined to be 5170 lux for CO₂ consumption and growth whereas, the maximum H₂ production rate occurs around 10 000 lux.
Significance and Impact of the Study:  The study presents kinetic models predicting the growth, CO₂ consumption and H₂ production of A. variabilis . The experimental and scaling analysis methods can be generalized to other micro-organisms.     

The impact of elevated ozone and carbon dioxide on young Acer saccharum seedlings

The effects of high O₃ (200 nl l⁻¹ during the light period) and high CO₂ (650 μl l⁻¹ CO₂, 24 h a day) alone and in combination were studied on 45-day-old sugar maple ( Acer saccharum Marsh.) seedlings for 61 days in growth chambers. After 2 months of treatment under the environmental conditions of the experiment, sugar maple seedlings did not show a marked response to the elevated CO₂ treatment: the effect of high CO₂ on biomass was only detected in the leaves which developed during the treatment, and assimilation rate was not increased. Under high O₃ at ambient CO₂, assimilation rate at days 41 and 55 and Rubisco content at day 61 decreased in the first pair of leaves; total biomass was reduced by 43%. In these seedlings large increases (more than 2-fold) in glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and in anaplerotic CO₂ fixation by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were observed, suggesting that an enhanced reducing power and carbon skeleton production was needed for detoxification and repair of oxidative damage. Under high O₃ at elevated CO₂, a stimulation of net CO₂ assimilation was observed after 41 days but was no longer observed at day 55. However, at day 61, the total biomass was only reduced by 21% and stimulation of G6PDH and PEPC was less pronounced than under high O₃ at ambient CO₂. This suggests that high CO₂ concentration protects, to some extent, against O₃ by providing additional carbon and energy through increased net assimilation.     

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems

The effects of 80% oxygen–20% carbon dioxide (O₂–CO₂) and 80% nitrogen–20% carbon dioxide (N₂–CO₂) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at −1 and 4 °C. In both N₂–CO₂ and O₂–CO₂ atmospheres, lactobacilli were predominant over Brochothrix , pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O₂–CO₂ atmospheres did not yield total viable counts in excess of 10⁵ cfu cm⁻² on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N₂–CO₂ atmospheres yielded maximum total viable counts of approximately 10⁷ cfu cm⁻² and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at −1 °C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O₂ on rancidity than by the high levels of lactobacilli.     

Control of Aspergillus section Flavi growth and aflatoxin accumulation by plant essential oils

Aims:  The antifungal effect of Pimpinella anisum (anise), Pëumus boldus (boldus), Mentha piperita (peppermint), Origanum vulgare (oregano) and Minthosthachys verticillata (peperina) essential oils against Aspergillus section Flavi (two isolates of Aspergillus parasiticus and two isolates of Aspergillus flavus ) was evaluated in maize meal extract agar at 0·982 and 0·955 water activities, at 25°C.
Methods and Results:  The percentage of germination, germ-tube elongation rate, growth rate and aflatoxin B₁ (AFB₁) accumulation at different essential oils concentrations were evaluated. Anise and boldus essential oils were the most inhibitory at 500 mg kg⁻¹ to all growth parameters of the fungus. These essential oils inhibited the percentage of germination, germ-tube elongation rate and fungal growth. AFB₁ accumulation was completely inhibited by anise, boldus and oregano essential oils. Peperina and peppermint essential oils inhibited AFB₁ production by 85–90% in all concentrations assayed.
Conclusions:  Anise and boldus essential oils could be considered as effective fungitoxicans for Aspergillus section flavi .
Significance and Impact of the Study:  Our results suggest that these phytochemical compounds could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.     

Incubation time and water activity effects on ochratoxin A production by Aspergillus section Nigri strains isolated from grapes

AIMS: The objective of this study was to determine the temporal ochratoxin A (OTA) accumulation profile of Aspergillus section Nigri at different water activity (aw) levels. METHODS AND RESULTS: Two Aspergillus carbonarius and two Aspergillus niger aggregate strains isolated from grapes were tested in vitro for OTA accumulation at 25 degrees C on synthetic nutrient medium, over periods of 20 days at different aw levels. Results were modelled by a multiple linear regression and response surface predictive models were obtained. High levels of aw favoured OTA production by these moulds. Maximum amounts of OTA were found at the earlier growth states (5 days for A. carbonarius and 7-13 days for A. niger aggregate). CONCLUSIONS: Provided that A. section Nigri, and mainly A. carbonarius, play the main role in OTA presence in grapes, it would be critical to adjust the harvest and processing time to significantly reduce the chances for OTA accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochratoxin A production by A. section Nigri has been shown for the first time to occur optimally after as little as 5 days on a grape-like medium.     

Antifungal effect of gaseous nitric oxide on mycelium growth, sporulation and spore germination of the postharvest horticulture pathogens, Aspergillus niger, Monilinia fructicola and Penicillium italicum

Aim:  To evaluate the antifungal activity of nitric oxide (NO) against the growth of the postharvest horticulture pathogens Aspergillus niger , Monilinia fructicola and Penicillium italicum under in vitro conditions.
Methods and Results:  Different volumes of NO gas were injected into the Petri dish headspace to obtain the desired concentrations of 50–500  μ l l⁻¹ . The growth of the fungi was measured for 8 days of incubation in air at 25°C . All concentrations of NO were found to produce an antifungal effect on spore germination, sporulation and mycelial growth of the three fungi, with the most effective concentration for A. niger and P. italicum being 100 and 500  μ l l⁻¹ for M. fructicola .
Conclusions:  Short-term exposure to a low concentration of NO gas was able to inhibit the subsequent growth of A. niger , M. fructicola and P. italicum .
Significance and Impact of the Study:  NO gas has potential use as a natural fungicide to inhibit microbial growth on postharvest fruit and vegetables.     

Influence of soil O2 and CO2 on root respiration for Agave deserti

Respiration measured as CO₂ efflux was determined at various soil O₂ and CO₂ concentrations for individual, attached roots of a succulent perennial from the Sonoran Desert, Agave deserti Engelm. The respiration rate increased with increasing O₂ concentration up to about 16% O₂ for established roots and 5% O₂ for rain roots (fine branch roots on established roots induced by wetting of the soil) and then remained fairly constant up to 21% O₂. When O₂ was decreased from 21 to 0%, the respiration rates were similar to those obtained with increasing O₂ concentration. The CO₂ concentration in the root zone, which for the shallow-rooted A. deserti in the field was about 1 000 μl l^-1, did not affect root respiration at concentrations up to 2 000 μl l^-1, but higher concentrations reduced it, respiration being abolished at 20 000 μl l^-1 (2%) CO₂ for both established and rain roots. Upon lowering CO₂ to 1 000 μl l^-1 after exposure to concentrations up to 10000 μl l^-1 CO₂, inhibition of respiration was reversible. Uptake of the vital stain neutral red by root cortical cells was reduced to zero, indicating cell death, in about 4 h at 2% CO₂, substantiating the detrimental effects of high soil CO₂ concentrations on roots of A. deserti . This CO₂ response may explain why roots of desert succulents tend to occur in porous, well-aerated soils.     

Effects of CO2 enrichment on mineral accumulation and nitrogen relations in a submersed macrophyte

1. Six- to eight-week greenhouse experiments with independent control of pH and dissolved CO₂ evaluated the potential for CO₂ enrichment to stimulate the accumulation of Al, Fe, P and N in shoots of Vallisneria americana , particularly at pH 5. These minerals were provided only as they occurred in natural lake sediments.
2. The effect of CO₂ enrichment at pH 5 v pH 7.3 on growth and tissue N concentration was also determined.
3. CO₂ enrichment at pH 5 effected 5.5- and 7-fold increases in total shoot accumulation of Al and Fe, respectively. In a two-way factorial experiment, CO₂ enrichment yielded 6- to 11-fold greater total shoot P accumulation in plants grown on less and more fertile sediments, respectively.
4. In a three-way factorial experiment, CO₂ enrichment stimulated Vallisneria growth, especially at pH 5, and resulted in a 31–58% reduction in tissue [N] for different pH × sediment combinations. These are greater reductions than previously reported. It also increased total shoot N accumulation up to 6-fold, and there were significant interactions with pH and sediment source: the CO₂ enrichment effect on shoot N accumulation was greater at pH 5 than at pH 7.3, and it was greater with the more fertile sediment at pH 5.
5. Water chemistry (pH and/or [CO₂]) and sediment fertility thus both indirectly influenced the accumulation of sediment-derived minerals in macrophyte shoots within the water column.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Effects of CO2 enrichment on mineral accumulation and nitrogen relations in a submersed macrophyte

1. Six- to eight-week greenhouse experiments with independent control of pH and dissolved CO₂ evaluated the potential for CO₂ enrichment to stimulate the accumulation of Al, Fe, P and N in shoots of Vallisneria americana , particularly at pH 5. These minerals were provided only as they occurred in natural lake sediments.
2. The effect of CO₂ enrichment at pH 5 v pH 7.3 on growth and tissue N concentration was also determined.
3. CO₂ enrichment at pH 5 effected 5.5- and 7-fold increases in total shoot accumulation of Al and Fe, respectively. In a two-way factorial experiment, CO₂ enrichment yielded 6- to 11-fold greater total shoot P accumulation in plants grown on less and more fertile sediments, respectively.
4. In a three-way factorial experiment, CO₂ enrichment stimulated Vallisneria growth, especially at pH 5, and resulted in a 31–58% reduction in tissue [N] for different pH × sediment combinations. These are greater reductions than previously reported. It also increased total shoot N accumulation up to 6-fold, and there were significant interactions with pH and sediment source: the CO₂ enrichment effect on shoot N accumulation was greater at pH 5 than at pH 7.3, and it was greater with the more fertile sediment at pH 5.
5. Water chemistry (pH and/or [CO₂]) and sediment fertility thus both indirectly influenced the accumulation of sediment-derived minerals in macrophyte shoots within the water column.     

Species-specific responses to atmospheric carbon dioxide and tropospheric ozone mediate changes in soil carbon

We repeatedly sampled the surface mineral soil (0–20 cm depth) in three northern temperate forest communities over an 11-year experimental fumigation to understand the effects of elevated carbon dioxide (CO₂) and/or elevated phyto-toxic ozone (O₃) on soil carbon (C). After 11 years, there was no significant main effect of CO₂ or O₃ on soil C. However, within the community containing only aspen ( Populus tremuloides Michx.), elevated CO₂ caused a significant decrease in soil C content. Together with the observations of increased litter inputs, this result strongly suggests accelerated decomposition under elevated CO_2. In addition, an initial reduction in the formation of new (fumigation-derived) soil C by O₃ under elevated CO₂ proved to be only a temporary effect, mirroring trends in fine root biomass. Our results contradict predictions of increased soil C under elevated CO₂ and decreased soil C under elevated O₃ and should be considered in models simulating the effects of Earth's altered atmosphere.     

Carbon dioxide and ethylene interactions in tulip bulbs

The effect of CO₂ on ethylene-induced gummosis (secretion of polysaccharides), weight loss and respiration in tulip bulbs ( Tulipa gesneriana L.) was investigated. A pretreatment with 1-MCP prevented these ethylene-induced effects, indicating that ethylene action must have been directed via the ethylene receptor. Treatment with 0.3 Pa ethylene for 2 days caused gummosis on 50% of the total number of bulbs of cultivar Apeldoorn, known to be sensitive for gummosis. Addition of CO₂ (10 kPa) reduced the ethylene-induced gummosis to 18%. In a second experiment the influence of ethylene and CO₂ on respiration and FW loss of bulbs of the cultivar Leen van der Mark was studied. A range of ethylene partial pressures (0.003–0.3 Pa) was applied continuously for 29 days. Ethylene caused a transient peak in O₂ consumption rate during the first days after the start of application. The relation between O₂ consumption rate and ethylene partial pressure could be described by Michaelis-Menten kinetics. Respiratory peaks were reduced by CO₂. This inhibition by CO₂ could not totally be due to competition with ethylene at the receptor binding-site, as was indicated by the use of an O₂ consumption model. Pre-treatment of bulbs with 1-MCP and subsequent exposure to CO₂ showed that CO₂ could influence respiration irrespective of any interaction with ethylene. Ethylene and CO₂ both stimulated weight loss. The effect of combined treatments of ethylene and CO₂ on weight loss was at least as strong as the sum of the separate effects, which implies that competition between ethylene and CO₂ at the receptor binding-site was unlikely.     

Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Aims:  The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants.
Methods and Results:  The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l⁻¹ of FaeB was selected (12-fold more than the A. niger overproducing strain).
Conclusions:  This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days.
Significance and Impact of the Study:  This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.     

Interaction of elevated CO2 and O3 on growth, photosynthesis and respiration of three perennial species grown in low and high nitrogen

Seedlings of three species native to central North America, a C₃ tree, Populus tremuloides Michx., a C₃ grass, Agropyron smithii Rybd., and a C₄ grass, Bouteloua curtipendula Michx., were grown in all eight combinations of two levels each of CO₂, O₃ and nitrogen (N) for 58 days in a controlled environment. Treatment levels consisted of 360 or 674 μmol mol^-1 CO₂, 3 or 92 nmol mol^-1 O₃, and 0.5 or 6.0 m M N. In situ photosynthesis and relative growth rate (RGR) and its determinants were obtained at each of three sequential harvests, and leaf dark respiration was measured at the second and third harvests. In all three species, plants grown in high N had significantly greater whole-plant mass, RGR and photosynthesis than plants grown in low N. Within a N treatment, elevated CO₂ did not significantly enhance any of these parameters nor did it affect leaf respiration. However, plants of all three species grown in elevated CO₂ had lower stomatal conductance compared to ambient CO₂-exposed plants. Seedlings of P. tremuloides (in both N treatments) and B. curtipendula (in high N) had significant ozone-induced reductions in whole-plant mass and RGR in ambient but not under elevated CO_2. This negative O₃ impact on RGR in ambient CO₂ was related to increased leaf dark respiration, decreased photosynthesis and/or decreased leaf area ratio, none of which were noted in high O₃ treatments in the elevated CO₂ environment. In contrast, A. smithii was marginally negatively affected by high O_3.     

Rice production in a changing climate: a meta-analysis of responses to elevated carbon dioxide and elevated ozone concentration

Rice is arguably the most important food source on the planet and is consumed by over half of the world's population. Considerable increases in yield are required over this century to continue feeding the world's growing population. This meta-analysis synthesizes the research to date on rice responses to two elements of global change, rising atmospheric carbon dioxide concentration ([CO₂]) and rising tropospheric ozone concentration ([O₃]). On an average, elevated [CO₂] (627 ppm) increased rice yields by 23%. Modest increases in grain mass and larger increases in panicle and grain number contributed to this response. The response of rice to elevated [CO₂] varied with fumigation technique. The more closely the fumigation conditions mimicked field conditions, the smaller was the stimulation of yield by elevated [CO₂]. Free air concentration enrichment (FACE) experiments showed only a 12% increase in rice yield. The rise in atmospheric [CO₂] will be accompanied by increases in tropospheric O₃ and temperature. When compared with rice grown in charcoal-filtered air, rice exposed to 62 ppb O₃ showed a 14% decrease in yield. Many determinants of yield, including photosynthesis, biomass, leaf area index, grain number and grain mass, were reduced by elevated [O₃]. While there have been too few studies of the interaction of CO₂ and O₃ for meta-analysis, the interaction of temperature and CO₂ has been studied more widely. Elevated temperature treatments negated any enhancement in rice yield at elevated [CO₂], which suggests that identifying high temperature tolerant germplasm will be key to realizing yield benefits in the future.