Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Diamondback moth egg susceptibility to rainfall: effects of host plant and oviposition behavior

Oviposition patterns of the diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), differ between common cabbage (Brassica oleracea L. var. capitata) and Chinese cabbage (Brassica rapa L. var. pekinensis) (Brassicaceae) host plants. This study shows that the moth prefers to oviposit on adaxial rather than abaxial leaf surfaces and petioles of both host plants. More eggs were laid in leaf veins than on leaf laminas of both host plants, especially in Chinese cabbage, where 94.6% of eggs were laid in veins. On Chinese cabbage, very few eggs were laid in clusters (≥2 eggs), whereas on common cabbage approximately 30% of eggs were laid in groups of 2 or more eggs. Removal of wax from common cabbage leaves dramatically increased the number of eggs laid singly on the leaf lamina of treated plants, suggesting that leaf waxes affect how eggs are distributed by ovipositing DBM. Eggs were most susceptible to removal by rainfall from the plant surface immediately (<1 h) after oviposition and when close to hatching (>72h old) whereas they were least susceptible 24 h after oviposition. Eggs laid on common cabbage plants were more susceptible to simulated rainfall than eggs laid on Chinese cabbage plants. On common cabbage plants, egg susceptibility to rainfall on different plant parts ranked adaxial leaf surfaces>petioles = abaxial leaf surfaces>stem, but there was no difference in egg susceptibility to rainfall on the various plant parts of Chinese cabbage. Furthermore, on common cabbage plants, eggs laid on both adaxial and abaxial leaf surfaces were afforded significant protection from the effects of rainfall by leaves higher in the plant canopy. On common cabbage plants, oviposition patterns reduce the potential impact of rainfall on eggs, possibly reducing the effect of this important abiotic mortality factor in the field.     

Isolation of anti-fungal phenolic compounds from petioles of two Hevea brasiliensis (rubber) genotypes and their effect on Phytophthora meadii

Phenolic compounds were present in greater amounts in non‐infected petioles of genotypes of Hevea brasiliensis that are resistant to Phytophthora leaf disease than in genotypes that are susceptible. Phenolic compounds extracted from petioles of either susceptible (PB86) or resistant (RRIC100) genotypes, before or after infection with Phytophthora meadii, had anti‐fungal properties. Artificially infected petioles of PB86 had phenolic acids, triterpenoids or flavonoids, whereas healthy petioles contained only triterpenoids or flavonoids. However, healthy or infected petioles of RRIC100 contained only trace amounts of the above compounds and of vanillin (3‐methoxy‐4‐hydroxybenzaldehyde). Vanillin and umbelliferone (7‐hydroxycoumarin) were shown to suppress zoospore germination of P. meadii on glass slides and to inhibit its growth in pea broth and V‐8 juice agar. Vanillin was slightly more active than umbelliferone. Resistance of RRIC100 to Phytophthora was suspected as being related to the polymerisation of phenolic compounds to form lignin, which may suppress further spread of the pathogen's mycelium into healthy tissues. Formation of lignin from phenolic aldehydes as a barrier to disease spread may be a critical factor in resistance.     

The Nature of Systemic Invasion of Stem and Leaves of Sunflowers by Plasmopara helianthi Novot. var. helianthi Novot. with Mechanism of Sporulation and Zoospore Release

During the systemic development of Plasmopara helianthi Novot. var. helianthi Novot. the hyphae in the stem advance especially through the intercellular spaces of loose parenchyma forming haustoria in adjacent cells. The hyphae which reach leaf blades through petioles continue their growth along the main veins intercellularly in non-vascular tissues enclosing the vascular bundles. At the same time, the hyphae spread to intercellular spaces of spongy parenchyma where the growth is limited by the veinlets resulting in angular chlorotic lesions. Under humid conditions the sporangiophores arising from the aggregated hyphae in a substomatal cavity emerge through the stomatal pore on the lower side of the leaf and zoosporangia are borne terminally on sporangiophores. Soon after biflagellated zoospores are liberated into distilled water from the zoosporangia, they retract their flagellae and then lyse.     

Physiological Responses of Rice Plants Supplied with Silicon to Monographella albescens Infection

This study investigated the effect of silicon (Si) on the resistance of rice plants of the cv. ‘Primavera’ cultivar that were grown in a nutrient solution with 0 (?Si) or 2 mm (+Si) Si to leaf scald, which is caused by Monographella albescens. The leaf Si concentration increased in the +Si plants (4.8 decag/kg) compared to the ?Si plants (0.9 decag/kg), contributing to a reduced expansion of the leaf scald lesions. The extent of the cellular damage that was caused by the oxidative burst in response to the infection by M. albescens was reduced in the +Si plants, as evidenced by the reduced concentration of malondialdehyde. Higher concentrations of total soluble phenolics and lignin‐thioglycolic acid derivatives and greater activities of peroxidases (POX), polyphenoloxidases (PPO), phenylalanine ammonia‐lyases (PAL) and lipoxygenases (LOX) in the leaves of the +Si plants also contributed to the increased rice resistance to leaf scald. In contrast, the activities of chitinases and β‐1,3‐glucanases were higher in the leaves of the ?Si plants probably due to the unlimited M. albescens growth in the leaf tissues, as indicated by the larger lesions. The results of this study highlight the potential of Si in decreasing the expansion of the leaf scald lesions concomitantly with the potentiation of phenolic and lignin production, and the greater activities of POX, PPO, PAL and LOX rather than simply acting only as a physical barrier to avoid M. albescens penetration.     

Suppression by Red Light Irradiation of Corynespora Leaf Spot of Cucumber Caused by Corynespora cassiicola

The effect of red light irradiation on development of Corynespora leaf spot of cucumber plants (Cucumis sativus L. cv. Hokushin) caused by Corynespora cassiicola (Berk. & Court.) was investigated in greenhouses. In a greenhouse without red light (?Red), lesions enlarged, coalesced, and finally covered the entire leaves of cucumber. In a greenhouse with red light (+Red), however, lesion appearance was delayed relative to that under ?Red and its development was also significantly suppressed. Such difference in disease development was also observed in cucumbers grown under +Red and ?Red in the same greenhouse. Disease suppression under red light was also observed in glasshouse‐grown C. cassiicola‐inoculated cucumbers. Red light did not inhibit the infection behaviour of the pathogen. Our results indicated that the delay and suppression of Corynespora leaf spot of cucumber under +Red were due to induction of resistance in cucumber, and not to differences in environmental conditions or fungal population between the two greenhouses. Red light‐induced resistance might contribute to the development of new methods for controlling Corynespora leaf spot of greenhouse‐grown cucumber.     

A high affinity pyruvate decarboxylase is present in cottonwood leaf veins and petioles: A second source of leaf acetaldehyde emission?

Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K_m value for pyruvate was 42 μM. This K_m is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K_m = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.     

The roles of ascospores and conidia of Pyrenopeziza brassicae in light leaf spot epidemics on winter oilseed rape (Brassica napus) in the UK

Ascospores of Pyrenopeziza brassicae were produced in apothecia (cup‐shaped ascomata) on oilseed rape debris. The conidia, which were morphologically identical to the ascospores, were produced in acervular conidiomata was greater than for lesions caused by ascospores. In June 2000, on the ground under a crop with light on the surface of living oilseed rape tissues. Ascospores were more infective than conidia on oilseed rape leaves. The proportion of lesions caused by conidia located on leaf veins leaf spot, numbers of petioles with apothecia decreased with increasing distance into the crop from the edge of pathways. Air‐borne ascospores of P. brassicae were first collected above debris of oilseed rape affected with light leaf spot on 5 October 1998 and 18 September 1999,12 or 23 days, respectively, after the debris had been exposed outdoors. P. brassicae conidia were first observed on leaves of winter oilseed rape on 6 January 1999 and 15 February 2000, respectively, after plots had been inoculated with debris in November 1998 and October 1999. In 1991/92, numbers of ascospores above a naturally infected crop were small from January to April and increased in June and July. P. brassicae conidia were first observed in February and the percentage plants with leaves, stems or pods with light leaf spot increased greatly in May and June. In 1992/93, in a crop inoculated with debris, numbers of airborne ascospores were small from October to January and increased from April to June. P. brassicae conidia were first observed on leaves in late November and light leaf spot was seen on stems and pods in March and June 1993, respectively.     

Sensitivity to ethylene: the key factor in submergence-induced shoot elongation of Rumex

Rosettes of flooding-resistant Rumex palustris plants show a submergence-induced stimulation of elongation, which is confined to the petioles of young leaves. This response increases the probability of survival. It is induced by ethylene that accumulates in submerged tissues. Flooding-intolerant Rumex acetosella plants do not show this response. We investigated whether differences in shoot elongation between the species, between old and young leaves and between the petiole and leaf blade of a R. palustris plant result from differences in internal ethylene concentration or in sensitivity to the gas. Concentrations of free and conjugated ACC in petioles and leaf blades of R. palustris indicated that ethylene is synthesized throughout the submerged shoot, although production rates varied locally. Nevertheless, no differences in ethylene concentration were found between submerged leaves of various ages. In contrast, dose-response curves showed that only elongation of young petioles of R. palustris was sensitive to ethylene. In R. acetosella, elongation of all leaves was insensitive to ethylene. We conclude that variation in ethylene sensitivity rather than content explains the differences in submergence-induced shoot elongation between the two Rumex species and between leaves of R. palustris.     

Effects of soil water level on the development of adult plant resistance to powdery mildew in barley

Barley grown in dry soil developed greater adult plant resistance (APR) to powdery mildew (Erysiphe graminis DC. f. sp. hordei Mérat) than barley grown in wet soil. Conidial germination and appressorium formation were less, and fungal development between formation of appressoria and elongating secondary hyphae on upper leaves was inhibited, when adult plants were grown in dry soil. Mildew colonies expanded more slowly on leaves of adult plants than on leaves of seedlings, especially if adult plants had grown in dry soil. APR was reduced if plants, previously grown in dry soil, were well watered more than 32 h before inoculation. Conidia originating from plants grown in dry soil had a lower solute potential and greater ability to infect plants grown in dry but not wet soil than conidia originating from plants grown in wet soil. APR could not be attributed simply to increased cell wall or cuticle thickness, nor to lowered leaf solute potentials, as has sometimes been suggested for powdery mildew diseases. Increasing plant age and water stress induced increases in cell wall and cuticle thickness, but these changes did not always coincide with changes in disease resistance. Increasing plant age and water stress also lowered leaf solute potentials but fungal solute potentials were lower than leaf solute potentials and, more importantly, were lower than leaf water potentials. Thus, fungal growth was not limited by the availability of water from the host during penetration and hyphal establishment. It is suggested that resistance levels may be determined not by the thickness of epidermal structures, nor by lowering of solute potential per se, but by specific substances harmful to the fungus which accumulate in either cell wall, cuticle or sap, and whose concentration is dependent on the age and water stress of leaves.     

Benefits associated with the domatia mediated tritrophic mutualism in the shrub Coprosma lucida

The fitness benefits of plant structural adaptations that increase the effectiveness of fungivores against leaf pathogenic fungi are poorly understood. In a 12‐month field experiment, we investigated the effect of domatia on mite density, the role of these mites in limiting leaf fungi, and the associated effects on plant fitness in the endemic New Zealand shrub, Coprosma lucida. The presence of domatia on mite density was controlled using combinations of domatia blocking, sham blocking, mite addition and mite control using miticide. Limiting access to domatia reduced mite density and increased the proportion of leaves without mites. Mite families represented were predominantly fungivorous/detritivorous (97.2%), and predaceous (2.6%); herbivorous mites were absent. Mites significantly reduced fungal hyphae, fungal spores and pollen, but the effect was surface‐(upper/lower) and density‐dependent with the greatest reduction in fungi occurring over low mite densities. Fungal hyphae reduced leaf longevity, but were associated with increased production of new leaves. Hyphae density on old leaves was negatively correlated with the number of domatia produced on new leaves. New leaves in the mite reduction treatment had slightly reduced levels of carbon but not nitrogen. High levels of fungal infection on the lower surface increased the number of fruit fascicles per shoot, however on the upper surface where fungi were reduced by mites, hyphae density was negatively related to reproduction. The data support a limited interpretation of a fitness benefit for plants with domatia. While domatia increased mite density, control of fungi by mites occurred at lower average densities than supported by plants without functioning domatia. We suggest the primary function of leaf domatia in this mutualism is to increase the probability of a leaf‐level beneficial mite presence rather than to maximise mite density. Many mites are not necessarily better than few mites, but some mites are better than none.     

Action of mephosfolan on anthocorid predators of Phorodon humuli

In glasshouse and field studies, soil drenches of the systemic insecticide mephosfolan were applied to hop plants to control the aphid Phorodon humuli, and to study the toxicity to anthocorid predators. The mortality of Anthocoris nemorum and A. nemoralis eggs in the leaf tissues increased with the dosage of mephosfolan while A. confusus eggs, which were laid in the petioles and veins, appeared to be unaffected at the dosages used, up to 2 g a.i./plant. Mortality of A. nemorum eggs, which were laid mainly in the leaf margins, was higher than in A. nemoralis eggs, which were laid mainly in the interveinal areas. For both species mortality was highest for the eggs laid in the leaf margins, and susceptibilities appeared similar. It is suggested that mephosfolan becomes unevenly distributed within the leaves, being most concentrated in the leaf margins and least concentrated in the veins and petioles. No species showed any preference when offered mephosfolan-treated and untreated leaves for oviposition, and the oviposition rates were unaffected by various mephosfolan dosages. Oviposition rates, larval mortality and duration of larval development were unaffected when specimens of each species were reared from eclosion to maturity on a diet of either mephosfolan-killed or heat-killed P. humuli.     

Isolation of scopoletin from leaves of Hevea brasiliensis and the effect of scopoletin on pathogens of H. brasiliensis

Scopoletin (7-hydroxy-6-methoxy coumarin) which inhibited the conidial germinationof Corynespora cassiicola was isolated from the uninfected mature leaves ofHevea brasiliensis. Scopoletin was not detected in uninfected immature rubber leaves. The immature leaves produced scopoletin after being infected with C. cassiicola. The concentration of scopoletin in infected leaves was higher than in uninfected mature leaves. Scopoletin also inhibited the conidial germination of other fungal pathogens of H. brasiliensis. However, no correlation was observed between scopoletin accumulation and clonal resistance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Trenching Behavior by Caterpillars of the Euphorbia Specialist, Pygarctia roseicapitis: A Field Study

Six-hour continuous observations on the arctiid caterpillar, Pygarctia roseicapitis, were made under natural conditions in the field, and all foraging-related activities were recorded. Individuals invariably cut veins of the latex-rich host plants before feeding distal to the cuts. Seven percent of the time was spent in this vein-cutting behavior, and 40% of the time was spent feeding. Most vein-cutting events were on petioles, allowing up to six feeding bouts on the resultant wilted leaf. Both leaves and plants were often abandoned before the resource was fully utilized. The results are discussed in relation to vein-cutting behavior by other insect herbivores and the possible trade-offs in such strategies.     

A Histological Evaluation of Induced Resistance to Phytophthora infestans (Mont.) de Bary in Potato Leaves

Systemically induced resistance against Phytophthora infestans in the potato cultivar ‘Bintje’ was studied histologically at the light microscopy level on the leaf surface, in the epidermis and in the mesophyll of challenged potato leaves. Systemic disease resistance was induced by a local pre-infection with the same fungus. On the leaf surface of induced plants, the germination of cysts was enhanced. In the epidermis of induced plants, papilla deposition increased and penetration decreased, whereas in the mesophyll of induced plants the spread of hyphae was reduced. It is suggested that the reduction of disease severity in induced plants is the result of the combined action of several successive defence reactions.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.     

Pythium aphanidermatum Causing Pythium Rot on Lampranthus spectabilis in Korea

Pythium aphanidermatum was found to be associated with rotting of leaves and stems in Lampranthus spectabilis in the summers of 2013 and 2014. Infected plants were initially characterized by water‐soaked and discoloured tissue, which are soon covered with cottony aerial hyphae. Subsequently, infected tissues wilted, leaves and stems appeared desiccated, and infected plants died. Pathogenicity of a representative isolate was proved, fulfilling Koch's postulates. Sequence of the internal transcribed spacer region of ribosomal DNA from the isolate shared 100% identity with that of P. aphanidermatum. To our knowledge, this is the first report of P. aphanidermatum causing leaf and stem rot on L. spectabilis in Korea.     

Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF’s function in growth and leaf development.     

Fungal colonization and decomposition of Castanopsis sieboldii leaves in a subtropical forest

Fungi play a crucial role in the decomposition of lignin in fallen leaves but few studies have examined the functional roles of ligninolytic fungi associated with the decomposition of fallen leaves on tropical forest soils. This study examined fungal populations responsible for lignin decomposition in Castanopsis sieboldii leaves in a subtropical evergreen broad-leaved forest in southern Japan. Fallen leaves of C. sieboldii are characterized by the occurrence of bleached portions attributable to fungal colonization of leaf tissues and decomposition of lignin. The bleached area accounted for 29.7%, on average, of the total area of C. sieboldii fallen leaves in the study site. Leaf mass per unit area (LMA) and lignin content were lower in the bleached area than in the surrounding nonbleached area of the same leaves, indicating that removal of lignin enhanced mass loss from leaf tissues and created small-scale heterogeneity of decomposition within single leaves. An unidentified species of Lachnocladiaceae (Basidiomycetes) was isolated frequently from the bleached area and caused selective decomposition of lignin in leaves under pure culture conditions, indicating that this fungus was responsible for the bleaching. The greater hyphal length of basidiomycetes in the bleached area than in the nonbleached area supported the finding that this Lachnocladiaceae sp. was associated with the bleaching. The relatively rapid decomposition of C. sieboldii leaves on the subtropical forest soil is partly attributable to colonization of the litter by this Lachnocladiaceae sp.     

Are leaves ‘freewheelin'? Testing for a Wheeler‐type effect in leaf xylem hydraulic decline

A recent study found that cutting shoots under water while xylem was under tension (which has been the standard protocol for the past few decades) could produce artefactual embolisms inside the xylem, overestimating hydraulic vulnerability relative to shoots cut under water after relaxing xylem tension (Wheeler et al. 2013). That study also raised the possibility that such a ‘Wheeler effect’ might occur in studies of leaf hydraulic vulnerability. We tested for such an effect for four species by applying a modified vacuum pump method to leaves with minor veins severed, to construct leaf xylem hydraulic vulnerability curves. We tested for an impact on leaf xylem hydraulic conductance (K_x) of cutting the petiole and minor veins under water for dehydrated leaves with xylem under tension compared with dehydrated leaves after previously relaxing xylem tension. Our results showed no significant ‘cutting artefact’ for leaf xylem. The lack of an effect for leaves could not be explained by narrower or shorter xylem conduits, and may be due to lesser mechanical stress imposed when cutting leaf petioles, and/or to rapid refilling of emboli in petioles. These findings provide the first validation of previous measurements of leaf hydraulic vulnerability against this potential artefact.