Exploring the use of chamomile (Matricaria chamomilla L.) bioactive compounds to control flixweed (Descurainia sophia L.) in bread wheat (Triticum aestivum L.): Implication for reducing chemical herbicide pollution

Intensive chemical herbicide use has resulted in human health and environmental issues. This study evaluated the phytotoxic potential of chamomile extract as a bioherbicide to minimize chemical herbicide use in wheat production. Treatments including four concentrations (0, 50, 100, and 150 mL/L) of three different chamomile plant parts (root, shoot, and root + shoot) extracts were applied to flixweed as a major weed in wheat production. Except for 50 mL/L of root extract, other concentrations of chamomile extracts decreased the germination rate of flixweed. Germnaiton rate of wheat increased with chamomile extracts except at 150 mL/L concentration of shoot extract at which the germination rate of flixweed and wheat reduced by 71.7 and 35.4%, respectively, compared to respective controls. Compared to wheat, malondialdehyde and proline in flixweed were increased fivefold in flixweed and compared to the control, ranged from 84–473 and 240–1422%, respectively. Chamomile extract also declined cell viability much quicker in flixweed than in wheat reflecting on greater inhibitory effect for flixweed control. Chamomile shoot extract reduced seedling weight and vigor index of flixweed by 63.75 and 59.4%, respectively, compared to the respective control. Results of liquid chromatography mass spectrometry of chamomile extract indicated polyphenols, flavonoids, terpenoids, and bioactive phenolic coumarins, glycosylated derivatives, quercetin and its derivatives, herniarin, umbelliferone, P-cymene, chamazulene, farnesol, amitrole, 1,8-cineole, and limonene were effective in inhibiting the germination and growth of flixweed. We concluded that 150 mg/L of chamomile shoot extract could be used as a bioherbicide to sustainably suppress flixweed in wheat production.     

Analysis of terpenoid biosynthesis pathways in German chamomile (Matricaria recutita) and Roman chamomile (Chamaemelum nobile) based on co-expression networks

German chamomile and Roman chamomile are the two most widely known chamomile species due to the medicinal properties of volatile compounds from their flowers. We determined the volatile compound content of different organs of these two chamomiles, and found that main volatile compounds in German chamomile were terpenoids and those in Roman chamomile were esters. Furthermore, 24 tissues from two chamomiles were sequenced and analyzed by gene co-expression network. The results showed higher terpene synthase expression levels and more modules correlated with sesquiterpenoids in German chamomile, which may explain its high sesquiterpenoid content. In both chamomiles, unigenes in volatile compound-correlated modules were significantly enriched in pathways related to plant-pathogen interactions and circadian rhythm, demonstrating that volatile compounds of chamomiles are influenced by these factors. There were ten times more unigenes related to plant-pathogen interactions in German chamomile than in Roman chamomile, which indicates German chamomile has higher resistance to pathogens.     

Nitrogen deficiency induced changes of free amino acids and coumarin contents in the leaves of Matricaria chamomilla

Through o-hydroxycinnamic acids, the biosynthesis of coumarins is connected with aromatic amino acid metabolism and nitrogen uptake. Therefore the quantitative changes in levels of some free amino acids and coumarins (herniarin and its glucosidic precursors (Z) - and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids; umbelliferone) in the leaf rosettes of chamomile (Matricaria chamomilla L.) subjected to nitrogen deficiency were studied. Nitrogen content decreased in the leaf rosettes and in the roots of N-deficient plants during the course of the experiment, but these plants produced significantly higher root biomass. Among secondary metabolites, the sum of 2-β-D-glucopyranosyloxy-4-methoxycinnamic acids increased sharply, herniarin increased slowly and the content of umbelliferone was low in N-deficient plants. We have concluded that nitrogen deficiency is not an inducing factor for stress accumulation of herniarin and umbelliferone. A decrease in levels of all detected amino acids, besides histidine, was found. Within aromatic amino acids, tyrosine was the most abundant. The content of free phenylalanine was significantly lower in both, control and N-deficient plants when compared to the content of tyrosine. In this view, the increase of herniarin glucosidic precursors is apparently due to enhancing phenylalanine ammonia-lyase activity under nitrogen deficiency and nitrogen-free carbon skeletons are shunted in to the phenylpropanoid metabolism, including biosynthesis of (Z)-and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids.     

Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress

The influence of low (3 μM) and high (60 and 120 μM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 μM Cd. Cd accumulation was approximately seven- (60 μM Cd treatment) to eleven- (120 μM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation.     

Effect of dew temperature,post-inoculation condition,and pathogen dose on suppression of scentless chamomile by Colletotrichum truncatum

Scentless chamomile, a noxious weed in western Canada, has a high natural tolerance to many herbicides. Colletotrichum truncatum, a host specific fungal pathogen, is suppressive to scentless chamomile when applied inundatively. A broadcast application was used at 200 L ha^?1 to evaluate biocontrol potential of this pathogen under a range of dew temperatures (DT), post-inoculation conditions (PIC), and pathogen doses (PD). A DT between 20 and 25°C was more conducive to infection, resulting in higher levels of disease and weed suppression as compared to 15 and 30°C. Under similar post-inoculation temperature regimes, disease was only slightly more severe in growth chambers than in the greenhouse. There was a positive linear relationship between the PD and weed suppression. An inoculum concentration >1×10⁸ spores mL^?1 reduced plant fresh weight by approximately 50% when compared to untreated controls. These results indicate that biocontrol of scentless chamomile using C. truncatum in the semi-arid Canadian prairies will likely encounter frequent non-favourable field conditions.     

Supercritical carbon dioxide extraction of chamomile flowers: extraction efficiency, stability, and in-line inclusion of chamomile-carbon dioxide extract in beta-cyclodextrin

The extraction of chamomile flowers using supercritical carbon dioxide was investigated with respect to extraction efficiency and compared with solvent extraction. The stability of matricine, a sensitive constituent of the essential oil of chamomile, in these extracts was studied during storage at different temperatures over 6 months. Matricine was stable at -30 degrees C. A slight decrease (80-90% recovery) occurred at +5 degrees C, whereas complete decomposition of matricine took place within 3-4 months at room temperature and at +30 degrees C, respectively. An in-line inclusion of chamomile constituents in beta-cyclodextrin (beta-CD) during the extraction process was assessed and inclusion rates between 40 and 95% were obtained depending on the amount of beta-CD and the type of chamomile constituent. No further stabilization of matricine in the carbon dioxide extract/beta-CD complexes was achieved. High residual water contents in the complexes even after freeze-drying were identified as accelerating the decomposition. In addition, the extractability of flavonoids, such as apigenin and apigenin-7-glucoside, was determined. Apigenin-7-glucoside, the more hydrophilic substance, was not extractable with pure carbon dioxide and showed a recovery of 11% using methanol modified carbon dioxide (18%, w/w) at 60 degrees C and 380 bar. Extraction conditions in the two-phase region of the binary mixture carbon dioxide-methanol (70 degrees C, 100 bar) led to a drastic change in fluid polarity and hence extractability increased to 92-95%.     

Headspace-solid-phase microextraction fast GC in combination with principal component analysis as a tool to classify different chemotypes of chamomile flower-heads (Matricaria recutita l.)

Headspace-solid-phase microextraction gas chromatography-principal component analysis (HS-SPME GC-PCA) is proposed as a complementary or alternative method to essential oil (EO) GC-PCA in order to discriminate between flower-heads of chamomile of different chemotypes. Ninety-two EOs and the headspaces sampled by HS-SPME of the corresponding chamomile flower-heads were examined by conventional GC and fast GC (F-GC) and the results submitted to statistical analysis by PCA. HS-SPME F-GC-PCA showed itself to be a rapid technique by which to distinguish chamomile flower-head chemotypes a produced results in agreement with the accepted EO classification. Using this method, the analysis time was reduced from at least 4.5 h with EO conventional GC to less than 1 h with HS-SPME F-GC. This approach can thus successfully be used as an analytical decision maker in order to reduce the number of time-consuming EO conventional GC analyses by limiting them to those samples that cannot unequivocally be classified. The EO conventional GC and HS-SPME F-GC results of PCA were very uniform, but they did not provide quantitative correlations between the components as determined by the two methods. A different statistical approach and a larger number of samples will be needed in order to correlate components in the headspace sampled by SPME and those in the corresponding EO quantitatively through a function.     

Colletotrichum sp: A potential candidate for biocontrol of scentless chamomile (Matricaria perforata) in western Canada

Based on an assessment of 706 fungal isolates obtained from Canada and Europe, a group of Colletotrichum sp. isolates, tentatively identified as C. truncatum, was moderately efficacious for biocontrol of scentless chamomile (Matricaria perforata). In this study, 19 C. truncatum isolates, 11 from Canada and eight from Europe, were compared for virulence, crop safety, and minimum dew requirement for infection to narrow the selection of candidates. Applied at 1×10⁶ spores mL^-1, these isolates expressed variable virulence under controlled environments, with slightly higher variations observed on the Canadian isolates. There was also a slight difference in host specificity among the isolates tested; most isolates caused disease only on chamomile species (M. perforata and M. recutita) but two Canadian isolates also infected lentil, flax, or both. At 20°C, most isolates required more than 20 h dew for maximum infection. This requirement can be an impediment for using this fungus as a biocontrol agent in western Canada where the climate is semi-arid. Treatment of scentless chamomile at the 10-leaf stage with the herbicide metribuzin 48 h prior to fungal inoculation increased weed control to 72%, compared to 40 and 47% by the herbicide and fungus applied alone. However, a similar treatment using the herbicide bentazon did not enhance the weed control significantly as compared to the herbicide alone.     

Intercropping Cover Crops with Pineapple for the Management of Rotylenchulus reniformis

Effect of cover crops intercropped with pineapple (Ananas comosus) on Rotylenchulus reniformis population densities and activity of nematode-trapping fungi (NTF) were evaluated in two cycles of cover crop and pineapple. Sunn hemp (Crotalaria juncea), rapeseed (Brassica napus), African marigold (Tagetes erecta), or weeds were intercropped with pineapples. Beds planted with sunn hemp or rapeseed had lower population densities of R. reniformis than African marigold, weeds, or pineapple plots during cover crop growth, and the subsequent pineapple-growing periods. Rapeseed was a good host to Meloidogyne javanica and resulted in high population densities of M. javanica in the subsequent pineapple crop. Fireweed (Erigeron canadensis) occurred commonly and was a good host to R. reniformis. Bacterivorous nematode population densities increased (P ≤ 0.05) most in sunn hemp, especially early after planting. Nematode-trapping fungi required a long period to develop measurable population densities. Population densities of NTF were higher in cover crops than weeds or pineapples during the first crop cycle (P < 0.05). Although pineapple produced heavier fruits following sunn hemp than in the other treatments (P < 0.05), commercial yields were not different among rapeseed, weed, and sunn hemp treatments.     

Accumulation of tetracoumaroyl spermine in Matricaria chamomilla during floral development and nitrogen deficiency

The new natural polyamine conjugate 1N,5N,10N,14N-tetracoumaroyl spermine (tetracoumaroyl spermine) recently isolated from chamomile (Matricaria chamomilla L.) flower heads is applicable for the treatment of several human disorders such as depression and anxiety. High variability in the level of tetracoumaroyl spermine is found in commercial tisanes. Accumulation of tetracoumaroyl spermine was tested during floral development, and nitrogen deficiency was chosen as its putative limiting environmental factor. It was observed that tetracoumaroyl spermine is present mainly in tubular flowers, reaching its maximal content during the 3rd phase of flowering when the corollae of tubular flowers start to open. The later observed decrease could result from a release of pollen that also contains a considerable amount of tetracoumaroyl spermine. It is likely that tetracoumaroyl spermine plays an important role in pollen development, and so, despite overall N-deficiency in the plants, tetracoumaroyl spermine is accumulated at the same or even higher rate than in the flowers of the N-sufficient control.     

Methyl jasmonate and Echinothrips americanus regulate coumarin accumulation in leaves of Matricaria chamomilla

Matricaria chamomilla synthesize (Z)- and (E)-2-ß-d-glucopyranosyloxy-4-methoxy cinnamic acid (GMCA), the biosynthetic precursors of herniarin. During leaf development the content of (E)-GMCA decreased and no changes were found in the content of other compounds evaluating in the experiments. Methyl jasmonate elicits an increase in (E)-GMCA and herniarin content in young growing leaves as well as mature and senescing leaves 48 h after treatment. In contrast, attack by the thrips, Echinothrips americanus, causes increase not only in GMCAs and herniarin content but also in the amount of coumarin, umbelliferone. The results confirmed that coumarin-like metabolites can be considered as plant defence compounds in biotic and abiotic stress conditions.     

Impact of competition from wheat and below-ground herbivory on growth and reproduction of scentless chamomile, Tripleurospermum perforatum (Mérat) Laínz

Abstract: This study investigated the effect of competition from wheat and below ground herbivory on the growth and reproduction of scentless chamomile, Tripleurospermum perforatum (Mérat) Laínz (Asteraceae), a target plant for classical biological control. Field cages, in which scentless chamomile was planted, were established in 1994 near Neuenburg (Rhine Valley, Germany), and two weevil species, Diplapion confluens Kirby and Coryssomerus capucinus (Beck), were released alone (40 adults each) or together in a substitutive design (20 adults each). Wheat was sown alone or in combination with both herbivores as an additional stress factor. To test the cage effect on plant growth, an uncaged control was set up. At the end of the experiment, caged control plants were about 20 cm (18%) higher, and allocated more biomass to shoots and less to roots and seeds than uncaged plants. Competition from wheat reduced the number of shoots per T. perforatum plant from 7.6 to 3.2. As a consequence, the biomass of scentless chamomile plants that competed with wheat was reduced by 39% and reproductive output by 49%, compared with plants that were grown without competition. At the end of the experiment, 70–80% of T. perforatum plants had been attacked by an average of two to three D. confluens and 60–70% of plants by one to two C. capucinus . At these densities that were lower than expected, the two herbivores alone or in combination had no significant effect on any of the plant parameters measured. However, in the presence of wheat, herbivory further reduced the number of shoots per plant. We conclude that control of scentless chamomile using insect biocontrol agents will be more likely to be successful if agent densities are high, and agents are released in habitats where scentless chamomile is competing with other plant species.     

Larval cannibalism in aphidophagous ladybirds: Influencing factors, benefits and costs

Spray retention on scentless chamomile and round-leaved mallow was characterized in relation to droplet size and travel speed. The effect of varying retention on biocontrol efficacy was studied using Colletotrichum truncatum and Colletotrichum gloeosporioides f. sp. malvae, the bioherbicide agents for the two weeds, respectively. In retention studies, a tracer dye solution was applied using a cabinet sprayer fitted with a fine, medium, or coarse nozzle at travel speeds of 0.5, 1.0, and 2.0 km/h that resulted in approximate application volumes of 500–2000 L/ha. Retention efficiency, a ratio between the volume retained on plants and actual volume applied, was calculated for each application. In general, finer sprays achieved higher retention efficiency on whole plants of both weeds. On an average, fine sprays resulted in 68% greater retention than coarse sprays on scentless chamomile and 22–59% on round-leaved mallow. Faster travel speeds increased spray retention only slightly. Applying the biocontrol agents at 500 L/ha with different droplet sizes showed different effects on weed control on scentless chamomile and round-leaved mallow. C. truncatum applied with fine droplets was more efficacious than treatments with coarser droplets on scentless chamomile. On round-leaved mallow, however, the efficacy of C. gloeosporioides f. sp. malvae did not appear to be substantially affected by the droplet size. This varying effect may be due to reduced retention on the vertical stems of round-leaved mallow, which is critical to biocontrol of this weed by C. gloeosporioides f.sp. malvae.     

Spray retention for liquid and mycoherbicide inoculum in three weed-biocontrol systems

Spray retention is often used to measure herbicide delivery to optimize application parameters, but little is known about retention characteristics of mycoherbicide inoculum applied for weed biocontrol. This study examined inoculum retention of three mycoherbicide agents, Pyricularia setariae, Colletotrichum truncatum and C. gloeosporioides f. sp. malvae, on their respective weed targets: green foxtail, scentless chamomile and round-leaved mallow. Conidium suspensions of these fungal pathogens containing a sodium-fluorescein tracer dye were applied at 500, 1000 and 2000 L ha^-1 using a cabinet sprayer, and the liquid volume and number of conidia retained on the plants were quantified. On all three weed species, liquid and conidium retention showed a high degree of correlation at varying application volumes although slight differences existed depending on the weed species. Based on the analysis of regression slopes, liquid retention reflected conidium retention most closely on green foxtail but slightly overestimated the number on scentless chamomile and round-leaved mallow, possibly due to different plant morphology and spray run-off at extremely high application volumes. Liquid retention can generally be used as an indicator in studying effects of spray quality on mycoherbicide retention for improved delivery and biocontrol in these weed-biocontrol systems.     

Acoustic traits of bat-pollinated flowers compared to flowers of other pollination syndromes and their echo-based classification using convolutional neural networks

Bat-pollinated flowers have to attract their pollinators in absence of light and therefore some species developed specialized echoic floral parts. These parts are usually concave shaped and act like acoustic retroreflectors making the flowers acoustically conspicuous to the bats. Acoustic plant specializations only have been described for two bat-pollinated species in the Neotropics and one other bat-dependent plant in South East Asia. However, it remains unclear whether other bat-pollinated plant species also show acoustic adaptations. Moreover, acoustic traits have never been compared between bat-pollinated flowers and flowers belonging to other pollination syndromes. To investigate acoustic traits of bat-pollinated flowers we recorded a dataset of 32320 flower echoes, collected from 168 individual flowers belonging to 12 different species. 6 of these species were pollinated by bats and 6 species were pollinated by insects or hummingbirds. We analyzed the spectral target strength of the flowers and trained a convolutional neural network (CNN) on the spectrograms of the flower echoes. We found that bat-pollinated flowers have a significantly higher echo target strength, independent of their size, and differ in their morphology, specifically in the lower variance of their morphological features. We found that a good classification accuracy by our CNN (up to 84%) can be achieved with only one echo/spectrogram to classify the 12 different plant species, both bat-pollinated and otherwise, with bat-pollinated flowers being easier to classify. The higher classification performance of bat-pollinated flowers can be explained by the lower variance of their morphology.     

High nitrogen supply affects the metabolism of Matricaria chamomilla leaves

N-status of the two Matricaria chamomilla cultivars grown in the presence of high potassium nitrate concentration was evaluated and compared with ammonium nitrate supply. After 5 days of potassium nitrate treatment the visible increase of dry mass together with total chlorophyll accumulation were observed. In both cultivars, ammonium nitrate application led to increased accumulation of N-containing compounds in chamomile leaves. NH₄NO₃ nitrogen supply influenced activity of nitrate reductase positively. In vivo nitrate reductase activity reached maximum in lower nitrate supply and decreased in higher nitrate availability significantly. Among the most abundant leaf secondary metabolites, the high nitrate availability both KNO₃ and NH₄NO₃ significantly increased umbelliferone level. The highest potassium nitrate dose (60 mmol per plant) caused an osmotic stress accompanied with lower tissue water content and turgor loss. In such condition the decrease in (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid, herniarin and dicycloethers, as well as PAL activity was observed. On the other hand, strong increase of umbelliferone is likely a stress response and is related to its antioxidant activity.     

Comparative evaluation of the activity of plant infusions against Helicobacter pylori strains by three methods

The aim of the study was to assess the activities of six plant infusions against Helicobacter pylori strains using a comparative screening assay (CSA), agar-well diffusion method (AWDM) and microscopy. Green tea, St John’s wort (SJW), rooibos, peppermint, chamomile and lime flower aqueous infusion concentrations were chosen to mimic those in herbal teas/tisanes. CSA concentrations were 4.5 mg ml^?1 for chamomile and 6.8 mg ml^?1 for the other agents. AWDM amounts were 0.4 mg/well for the chamomile and 0.6 mg/well for the other agents. Using CSA, ≥8 × 10⁴ colony forming unit reduction was found in >60 % of the strains by the green tea (81.5 %), SJW (75.9 %) and rooibos (63.0 %) within 2 h. Similarly, by AWDM, the activity against >60 % of the strains was found by the green tea, SJW and rooibos. Gram staining results were alike, showing mostly/only coccoids in >66 % of the strains by the same three agents within 2 h. Lime flowers showed the lowest activity by all methods. In conclusion, CSA allows comparing the activities of many agents against numerous strains. To our knowledge, these are the first data about rooibos and lime flower activities against H. pylori. All the three methods revealed that the most active agents were the green tea, SJW and rooibos, which also possess additional beneficial properties, e.g. antioxidant, anti-inflammatory and antitumor effects, therefore these plants may have a beneficial use as prophylactic agents against or adjuvants in the therapy of H. pylori infection.     

Identification of microRNAs,phasiRNAs and Their Targets in Pineapple

MicroRNAs (miRNAs) are small non-coding RNAs that regulate their target mRNA levels by directing cleavage or repressing its translation. Besides its outstanding nutritional and medicinal significances, pineapple serves as a model for studying genome evolution in cereal crops as well as obligate crassulacean acid metabolism (CAM) photosynthesis. Thus, studying miRNAs in pineapple is critical for better understanding their roles in this plant species. Here we carried out computational and experimental analysis of miRNAs and phased small interfering RNAs (phasiRNAs) in pineapple by analyzing small RNA profiles from flowers, fruits and leaves. The analyses have identified 131 conserved miRNAs that could be grouped into 37 families and 16 novel miRNAs. Three TAS3 loci and forty five 21 nucleotide (nt) PHAS loci, and seventy three 24 nt PHAS loci were also identified. The putative targets of the identified miRNAs and phasiRNAs were predicted. The presented results provide a comprehensive view of small regulatory RNAs and their targets in pineapple.     

Perianth organization and intra-specific floral variability

Floral symmetry and fusion of perianth parts are factors that contribute to fine-tune the match between flowers and their animal pollination vectors. In the present study, we investigated whether the possession of a sympetalous (fused) corolla and bilateral symmetry of flowers translate into decreased intra-specific variability as a result of natural stabilizing selection exerted by pollinators. Average size of the corolla and intra-specific variability were determined in two sets of southern Spanish entomophilous plant species. In the first set, taxa were paired by family to control for the effect of phylogeny (phylogenetically independent contrasts), whereas in the second set species were selected at random. Flower size data from a previous study (with different species) were also used to test the hypothesis that petal fusion contributes to decrease intra-specific variability. In the phylogenetically independent contrasts, floral symmetry was a significant correlate of intra-specific variation, with bilaterally symmetrical flowers showing more constancy than radially symmetrical flowers (i.e. unsophisticated from a functional perspective). As regards petal fusion, species with fused petals were on average more constant than choripetalous species, but the difference was not statistically significant. The reanalysis of data from a previous study yielded largely similar results, with a distinct effect of symmetry on variability, but no effect of petal fusion. The randomly-chosen species sample, on the other hand, failed to reveal any significant effect of either symmetry or petal fusion on intra-specific variation. The problem of low-statistical power in this kind of analysis, and the difficulty of testing an evolutionary hypothesis that involves phenotypic traits with a high degree of morphological correlation is discussed.