Epicuticular Wax Research Articles

Physicochemical characterization of epicuticular coating of apples sold in Abuja Nigeria

Most of the apples consumed across Nigeria are imported from South Africa, Europe and United States of America. Despite the epicuticular hydrophobic wax naturally produced by the fruit which makes it resistant to external spoilage factors, extra coating is usually employed by producers for protection against biotic and abiotic attack during storage in order to extend shelf-life. Nowadays, Nigerians have raised concerns on the nature, level and safety of these wax coatings in apples available in Nigerian market. The results of organoleptic, physicochemical and Fourier transfer infrared spectroscopy (FTIR) analyses suggested that the epicuticular waxes of apples in Abuja markets are mainly from petrochemical sources with adulterants from waxes of vegetable sources or beeswax. The mineral concentration of Cu, K, Mg, Mn, Zn and Pb in waxes per average sized apples (mg/g) ranged from 1.73 to 15.66, 10.80 to 1357, 26.46 to 173.12, 0.11 to 0.22, 2.05 to 41.80 and 3.78 to 11.66 mg/g, respectively. The results indicated hundred (100%) of the samples contained copper (0.02 mg/g) and zinc (0.05 mg/g), and sixty percent (60%) contained lead above WHO permissible limit (0.01 mg/g). It is however recommended that the layer of coating should be to reduce apple growers or sellers to the barest minimum to avoid over-coating and the amount of waxes consumed per apple. Waxes can be removed from apple by dipping the apple in hot water for a few seconds to remove the wax or washing with pure white vinegar and rinsing in warm water. Key words: Apples, waxes, beeswax, paraffin wax, mineral, Fourier transfer infrared spectroscopy (FTIR).

Fruit and seed micromorphology and its systematic significance in tribe Sorbarieae (Rosaceae)

We investigated fruit and seed morphology in tribe Sorbarieae and one traditionally included genus (Lyonothamnus), which revealed that these characteristics are taxonomically significant for generic delimitation in the tribe Sorbarieae, and two distinct groups in the genus Sorbaria were identified. The following fruit and seed micromorphological characteristics were described: size, shape, texture, the presence/absence of epicuticular wax and, trichomes, testa cell size, and surface ornamentation. Multicellular capitate glands with stalks were only observed on the follicle surfaces of Lyonothamnus. Simple unbranched unicellular non-glandular trichomes were found on the follicles of Chamaebatiaria millefolium, Sorbaria grandiflora, S. sorbifolia complex, and Spiraeanthus schrenkianus. However, Sorbaria arborea complex, S. kirilowii, and S. tomentosa complex had platelet epicuticular wax on their glabrous follicles. Moreover, four types of testa ornamentation were identified: type I, colliculate (Lyonothamnus); type II, reticulate, with straight anticlinal walls and smooth to micro-papillate periclinal walls (Chamaebatiaria); type III, reticulate, with undulate anticlinal walls and smooth periclinal walls (Spiraeanthus); and type IV, reticulate testa ornamentation and divided into two subtypes based on anticlinal and periclinal cell wall patterns (Sorbaria). These studied characteristics identified using principal component analysis may have diagnostic importance among the taxa in the tribe. Our fruit and seed micromorphological results provide strong evidence that genus Lyonothamnus could be positioned in the tribe Lyonothamneae, not Sorbarieae. Moreover, these data supported the preliminary phylogenetic hypothesis that Sorbaria could be divided into two groups.

The semidominant mutation w5 impairs epicuticular wax deposition in common wheat (Triticum aestivum L.).

The semidominant EMS-induced mutant w5 affects epicuticular wax deposition and mapped to an approximately 194-kb region on chromosome 7DL. Epicuticular wax is responsible for the glaucous appearance of plants and protects against many biotic and abiotic stresses. In wheat (Triticum aestivum L.), β-diketone is a major component of epicuticular wax in adult plants and contributes to the glaucousness of the aerial organs. In the present study, we identified an ethyl methanesulfonate-induced epicuticular wax-deficient mutant from the elite wheat cultivar Jimai22. Compared to wild-type Jimai22, the mutant lacked β-diketone and failed to form the glaucous coating on all aerial organs. The mutant also had significantly increased in cuticle permeability, based on water loss and chlorophyll efflux. Genetic analysis indicated that the mutant phenotype is controlled by a single, semidominant gene on the long arm of chromosome 7D, which was not allelic to the known wax gene loci W1-W4, and was therefore designated W5. W5 was finely mapped to an ~ 194-kb region (flanked by the molecular markers SSR2 and STARP11) that harbored four annotated genes according to the reference genome of Chinese Spring (RefSeq v1.0). Collectively, these data will broaden the knowledge of the genetic basis underlying epicuticular wax deposition in wheat.

Changes in Cuticle Components and Morphology of 'Satsuma' Mandarin (Citrus unshiu) during Ambient Storage and Their Potential Role on Penicillium digitatum Infection.

To elucidate the role of fruit cuticle in fungal infection, changes in cuticle composition and morphology of ‘Satsuma’ mandarin during ambient (at 25 °C) storage and their role in Penicillium digitatum infection were investigated. Results showed that the epicuticular wax yield increased from 1.11 μg cm−2 to 4.21 μg cm−2 during storage for 20 days and then decreased to 1.35 μg cm−2 as storage time prolonged to 40 days. Intracuticular wax content of fruits stored for 20 days showed a peak value that was 1.7-fold higher than that of fruits stored for 40 days. The contents of cutin monomers of fruits showed a decreased trend during storage, while their proportions in the cutin stayed stable. Acids were identified as the most abundant components in epicuticular wax independently of the storage time, followed by alkanes and terpenoids. Terpenoids were found as the predominant components in intracuticular wax during the whole storage, followed by alkanes and acids. The flattened platelets crystals of fruits at harvest changed into small granule-like wax ones after 10 days of storage then gradually distributed across the surface of the fruits as stored for 40 days. Results of in vitro tests showed that mycelial growth of Penicillium digitatum could be promoted by epicuticular wax and conidial germination could be inhibited by cutin at different storage stages. These results shed new light on the chemical basis for cuticle involvement in fungal infection.

Refining the genetic architecture of flag leaf glaucousness in wheat

Key messageThe cuticle is the plant’s barrier against abiotic and biotic stresses, and the deposition of epicuticular wax crystals results in the scattering of light, an effect termed glaucousness. Here, we dissect the genetic architecture of flag leaf glaucousness in wheat toward a future targeted design of the cuticle.The cuticle serves as a barrier that protects plants against abiotic and biotic stresses. Differences in cuticle composition can be detected by the scattering of light on epicuticular wax crystals, which causes a phenotype termed glaucousness. In this study, we dissected the genetic architecture of flag leaf glaucousness in a panel of 1106 wheat cultivars of global origin. We observed a large genotypic variation, but the geographic pattern suggests that other wax layer characteristics besides glaucousness may be important in conferring tolerance to abiotic stresses such as heat and drought. Genome-wide association mapping identified two major quantitative trait loci (QTL) on chromosomes 3A and 2B. The latter corresponds to the W1 locus, but further characterization revealed that it is likely to contain additional QTL. The same holds true for the major QTL on 3A, which was also found to show an epistatic interaction with another locus located a few centiMorgan distal to it. Genome-wide prediction and the identification of a few additional putative QTL revealed that small-effect QTL also contribute to the trait. Collectively, our results illustrate the complexity of the genetic control of flag leaf glaucousness, with additive effects and epistasis, and lay the foundation for the cloning of the underlying genes toward a more targeted design of the cuticle by plant breeding.

Particulate matter on foliage of Betula pendula, Quercus robur, and Tilia cordata: deposition and ecophysiology

Trees in urban and industrial areas significantly help to limit the amount of particulate matter (PM) suspended in the air, but PM has a negative impact on their life. The amount of PM gathered on leaves depends on quantity, size, and morphology of leaves and can also be increased by the presence of epicuticular waxes, in which PM can become stuck or immersed. In this study, we determined the ability of PM to accumulate on leaves in relation to the species of tree and PM source. We tested saplings of three common European tree species (Betula pendula, Quercus robur, and Tilia cordata) by experimentally polluting them with PM from different sources (cement, construction, and roadside PM), and then assessing the effects of PM on plant growth and ecophysiology. In all studied species, we have found two types of PM accumulation: a layer on the leaf surface and an in-wax layer. Results showed that the studied species accumulate PM on their leaf blade, reducing the efficiency of its photosynthetic apparatus, which in a broader sense can be considered a reduction in the plants’ normal functioning. Saplings of Q. robur suffered the least, whereas B. pendula (especially photosynthetic rate and conductivity) and T. cordata (especially increase in leader shoot length) exhibited greater negative effects. The foliage of B. pendula collected the most PM, followed by Q. robur, and then T. cordata, regardless of the dust’s source. All tested species showed a tendency for higher wax production when growing under PM pollution stress. We believe that, potentially, B. pendula best enhances the quality of the PM-contaminated environment; however, faster leaf fall, reduced productivity, and worse quality of wood should be considered in urban forest management.

Anti-inflammatory activity of the epicuticular wax and its isolated compounds catechin and gallocatechin from Eugenia brasiliensis Lam. (Myrtaceae) leaves

This study describes anti-inflamatory activity of epicuticular wax (ECW) extracted from leaves of Eugenia brasiliensis Lam., and its isolated compounds, catechin and gallocatechin in a pleurisy model in mice. The results for exudation and cell migration showed that the extract and isolated compounds from the plant were effective in inhibiting these inflammatory parameters. The ECW demonstrated a dose-dependent effect on the inhibition of cell migration. The results for NOx levels reveals that the isolated compounds from Eugenia brasiliensis were effective in inhibiting the production of this inflammatory mediator, however, ECW were not able in inhibiting the production of NO in the pleural cavity, at the tested doses. The results here obtained, helps to reinforce the scientifically hypothesis that E. brasiliensis has an important anti-inflammatory activity, and that this effect may be, at least in part, by the presence of the compounds catechin and gallocatechin.

Epicuticular wax and its effect on canopy temperature and water use of Sorghum

Epicuticular wax is thought to enable sorghum [Sorghum bicolor (L.) Moench] plants to cope with drought. Increased reflectivity of solar radiation and reduced conductance of water vapor are mechanisms responsible for aiding plants in water conservation. Increased reflectivity should lead to a decrease in canopy temperature and water use, whereas decreased conductance should lead to an increase in canopy temperature because of a decrease in evaporative cooling. It is not clear how these competing effects exert control over water use in a crop such as sorghum. To better understand the role of epicuticular waxes on the energy balance of sorghum, experiments were conducted to determine the effects of waxes on field-scale energy fluxes using near-isogenic lines of grain sorghum having different levels of epicuticular wax loading. Waxes caused an overall 2% increase in albedo, and about 86% of the reflected energy between 400 and 1100 nm was from near-infrared wavelengths. This is at variance with recent reports that suggest that waxes can substantially increase the reflectivity of sorghum. Instead, our results indicated that the amount of reflected radiant energy due to waxes was small. When water was non-limiting, waxes caused a 22% decrease in canopy conductance compared to a 2% increase in albedo on average. Consequently, at the expense of higher canopy temperatures, waxes caused a 5% reduction in latent heat flux when water was not limiting. Without rain, water became limiting to plants with lower wax load sooner than it did for plants with higher wax load. The high wax load canopy then was on average 0.4 °C cooler, had 24% greater canopy conductance and 13% greater latent heat fluxes compared to the low wax one. Results suggested that the primary mechanism through which waxes affect the energy balance of sorghum is by means of reduced conductance of water vapor.

Effect of the epicuticular wax level of leaf lamina on the behaviour of leaf hopper <em>Deltocephalus menoni</em> (Hemiptera: Cicadellidae); A vector of sugarcane white leaf disease

Sugarcane White Leaf Disease (WLD) is one of the major threats to the cane sugar industry in Sri Lanka and Deltocephalus menoni is the only recorded vector in local conditions. Epicuticular waxes (EW) play an important role in protecting plants against herbivore attacks. Therefore, this study was conducted with the objective of determining the effect of EW level on the behavioral characteristics of D. menoni in ten different sugarcane varieties. EW level of the selected varieties, behavioral characteristics of D. menoni and the level of WLD infection under natural conditions were studied. The effect of surface waxes on feeding behavior of D. menoni was also investigated under in-vitro conditions using agarosesucrose. Pearson correlation coefficient test was performed to detect the associations between EW and insect behavioral characteristics. Varieties with higher level of EW showed a significantly lower D. menoni feeding and disease infection. There was a significantly positive correlation (r = 0.78, P = <0.0001) between the level of feeding and rate of disease infection in natural environment. Hence it is quite possible that wax may play a significant role in feeding of D. menoni on sugarcane. In vitro study also further confirmed the relationship of EW and feeding behaviour by recording high feeding preference in agarose-sucrose diet containers with less wax content. Therefore, sugarcane accessions having high level of EW could be incorporated into directional breeding of varieties to increase the resistance against WLD.

Amounts and Types of Epicuticular Leaf Waxes among Onion Accessions Selected for Reduced Damage by Onion Thrips

Onion thrips (Thrips tabaci) is the main insect pest of onion (Allium cepa), and feeding damage routinely causes serious yield losses. Lower amounts of epicuticular waxes on onion leaves have been associated with fewer onion thrips and less feeding damage, and research is needed to assess the relationships between amounts and composition of epicuticular waxes and feeding damage by onion thrips. This study used gas chromatography mass spectroscopy to determine amounts and types of epicuticular waxes on the foliage of onion accessions that had been field-selected for lower damage from onion thrips. Hentriacontanone-16 (H16), octaconasol-1, and triacontanol-1 were the most prevalent waxes on the foliage of these selections. Amounts of H16 were significantly lower on selections visually classified as having glossy or semiglossy foliage. Semiglossy selections were identified with similar amounts of total epicuticular wax as waxy phenotypes, due primarily to lower amounts of H16 and higher amounts of other waxes. These semiglossy selections suffered significantly less feeding damage from onion thrips in a field evaluation, supporting the identification of unique wax profiles toward the development of thrips-resistant onion.

Genetic Analyses of Epicuticular Waxes Associated with the Glossy Foliage of ‘White Persian’ Onion

Onion (Allium cepa) plants with lower amounts of epicuticular waxes on foliage suffer less damage from the insect pest Thrips tabaci (onion thrips). Glossy onion accumulates significantly less epicuticular wax compared with wild-type “waxy” onion, and a single recessive locus (gl) has been proposed to condition this phenotype. Genetic analyses of types and amounts of epicuticular waxes were completed using two segregating families from the cross of the glossy inbreds B9885 and B9897 (both originally selected from the onion cultivar White Persian) with waxy inbred B8667 and semiglossy (intermediate amounts of waxes) inbred B5351, respectively. F2 progenies were grown in greenhouses and scored visually for foliar phenotypes, and amounts and types of epicuticular waxes were determined using gas chromatography-mass spectrometry (GCMS). For one F2 family from the cross of glossy B9885 by waxy B8667, visually scored glossy vs. waxy foliage fit a 1:3 ratio and the phenotype mapped to chromosome 8 of onion. This same region on chromosome 8 was significantly associated with amounts of the ketone hentriacontanone-16 (H16) and fatty alcohols 1-octacosanol (Oct1) and 1-triacontanol (Tri1). Visually scored F2 progeny from the cross of glossy B9897 × semiglossy B5351 did not fit expected models for one or two recessive loci. Significant quantitative trait loci (QTL) were revealed on chromosomes 5 and 8 controlling amounts of H16. Epistasis was detected between regions on chromosomes 1 and 8, and a 100-fold increase of H16 was conditioned by homozygous genotypes for the B5351 region on chromosome 1 and the B9885 region on chromosome 8. The three QTL model explained 41% of the phenotypic variation for amounts of H16 at logarithm of odds of 16.6. Amounts of Oct1 and Tri1 in the B9897 × B5351 family were associated with a major QTL on chromosome 1, explaining 37% to 46% of the phenotypic variation, respectively. This research demonstrates that glossy foliage of ‘White Persian’ onion is conditioned by a recessive locus on chromosome 8 for which we propose the name glwp. These results are important for selection of onion with unique profiles of epicuticular waxes to reduce losses resulting from onion thrips.

The sorghum epicuticular wax locus Bloomless2 reduces plant damage in P898012 caused by the sugarcane aphid

AbstractThe sugarcane aphid [Melanaphis sacchari] has been rapidly spreading in the southern United States with devastating impacts on sorghum [Sorghum bicolor (L.) Moench]. Previous studies have shown the sorghum epicuticular wax mutants, in particular a bloomless2 (bm2) mutant and a sparse bloom mutant, were resistant to greenbugs [Schizaphis graminum], a type of aphid. In this study we sought to determine if five different genotypes, which all have mutations in the Bm2 locus, differ for sugarcane aphid resistance as compared to their respective wild‐type plants (P954035, P898012, and Tx7078). Two greenhouse studies with artificial infestation and one field study under natural infestation were conducted. Aphid numbers on the second leaf from the top and the lowest green leaf, plant damage, and plant growth stage were assessed at weekly intervals. The Greenhouse Test 2 and the Field test were the most effective at determining plant resistance and susceptibility to the sugarcane aphid. Bm2 mutants in the P898012 background had greater aphid damage than their wild type plants. These findings suggest that the Bm2 locus has a role in reducing aphid damage in the P898012 background yet has a limited role in the Tx7078 and P954035 backgrounds.

Disordered wax platelets on Tradescantia pallida leaves create golden shine.

Plants have various strategies to protect themselves from harmful light. An example of such a protective mechanism is the growth of epicuticular nanostructures, such as a layer of hair or wax crystals. Most nanostructures are optimised to screen UV radiation, as UV light is particularly damaging for cellular tissue. We find that, contrary to the commonly found UV reflectance, the epicuticular wax crystals on Tradescantia leaves reflect strongly in the higher visible wavelength regime. Thus, they give the leaves a golden shine. We characterize the optical appearance of Tradescantia pallida 'purpurea' leaves by angularly resolved spectroscopy and compare the results to finite difference time domain simulations. We find that it is the disordered assembly of the wax platelets that is the crucial parameter to obtain the observed reflected intensity increase for higher wavelengths.

Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus in Cultivated Papaya and its Wild Relatives

Papaya ring spot virus (PRSV) disease has been the major impediment in papaya cultivation. The disease is transmitted through three aphid vectors and field tolerance towards this disease varies among Carica papaya cultivars as well as within the Vasconcellea genus. Leaf morphological traits are known to have influence on the probing preferences of aphids. Hence, this study was conducted to know whether the leaf parameters could contribute to the incidence of PRSV possibly by influencing the probing or feeding behaviour of aphid vectors. Leaf parameters viz., leaf thickness, leaf epicuticular wax content, presence and type of trichomes, trichome density were correlated with disease incidence at field conditions. The result revealed that leaf thickness along with epicuticular wax content had significant negative correlation with disease incidence. Similarly, trichome density had negative impact on disease incidence at 99.92% significance level. High epicuticular wax content and high trichome density in V. cauliflora and V. cundinamarcensis were found to be negatively associated with low to very low infection indicating that these parameters may have limited the vector transmission significantly.

Correlation of Leaf Parameters with Incidence of Papaya Ring Spot Virus in Cultivated Papaya and its Wild Relatives

Papaya ring spot virus (PRSV) disease has been the major impediment in papaya cultivation. The disease is transmitted through three aphid vectors and field tolerance towards this disease varies among Carica papaya cultivars as well as within the Vasconcellea genus. Leaf morphological traits are known to have influence on the probing preferences of aphids. Hence, this study was conducted to know whether the leaf parameters could contribute to the incidence of PRSV possibly by influencing the probing or feeding behaviour of aphid vectors. Leaf parameters viz., leaf thickness, leaf epicuticular wax content, presence and type of trichomes, trichome density were correlated with disease incidence at field conditions. The result revealed that leaf thickness along with epicuticular wax content had significant negative correlation with disease incidence. Similarly, trichome density had negative impact on disease incidence at 99.92% significance level. High epicuticular wax content and high trichome density in V. cauliflora and V. cundinamarcensis were found to be negatively associated with low to very low infection indicating that these parameters may have limited the vector transmission significantly.

Effect of controlled-temperature storage as pre-treatment on selective recovery of waxes from flax and wheat straw

ABSTRACT Flax and wheat straw were stored at −20°C, 4°C, or 20°C for up to 28 days as a pre-treatment to wax extraction performed using n-hexane or by immersion in liquid nitrogen. The epicuticular wax separation yield improved significantly after 28 days of storage at 4°C for both flax and wheat samples. Controlled-temperature storage resulted in a loss of selectivity in regard to epicuticular wax recovery from flax straw. Alterations within the epicuticular waxes that occurred during controlled-temperature storage were analyzed using scanning electron microscopy. Thermal analysis of the waxes using DSC yielded findings that were in concordance with the chemical results.

Pericarp features of wild perennial Helianthus L. species as a potential source for improvement of technical and technological properties of cultivated sunflower

Although the anatomy of the sunflower achene has been extensively studied, not enough attention is given to the research of the pericarp characteristics as potentially important parameters in the breeding programme. Therefore, we considered that it was important to make very detail comparative analysis of morpho-anatomical characteristics of achene as well as miromorphological and n-alkane characteristics of epicuticular wax of the 18 wild perennial Helianthus species. For anatomical analyses cross-sections were obtained from the middle part of pericarp using cryotechnique procedure. Micromorphological observation, morphological and anatomical analysis of pericarp were performed using Light and Scanning Electron Microscopy. The composition of the epicuticular n-alkanes was characterized by GC-FID and GC/MS analyses. Results showed differences in the distribution of phytomelanin pigment among the species. Denser phytomelanin network in the species H. salicifolius, H. resinous, H. tuberosus, and H. hirsutus may be connected with better resistance to insects and mechanical damages. Species H. resinosus, H. giganteus, and H. hirsutus are characterized by the highest percentage of cuticle and sclerenchyma thickness in the pericarp which can be a predictor of mechanical resistance to biotic and abiotic factors. Species H. strumosus, H. maximiliani, and H. grosseserratus are characterized with thin pericarp, poorly developed cuticle and epicuticle wax, as well as thin sclerenchyma tissue. In the pericarp waxes, C29 was the dominant alkane but several samples had n-alkanes longer than C33.These long-chain n-alkanes may be related to drought tolerance in some species. Obtained results of pericarp characteristics can be useful for improving technical and technological properties of the achene as well as for preservation of collection of wild sunflower species.

Factors Associated with Variation in Cuticular Hydrocarbon Profiles in the Navel Orangeworm, Amyelois transitella (Lepidoptera: Pyralidae).

Cuticular hydrocarbons (CHCs) are the main components of the epicuticular wax layer that in many insects functions as a barrier against desiccation. CHCs also play many other roles, including serving as sex pheromones, kairomones, primer pheromones, and colony-, caste-, species- and sex-recognition signals. In insects, CHC profiles can vary depending upon age, species, sex, and strain. Understanding factors associated with variation in hydrocarbon profiles is important for identifying potential vulnerabilities relating to pest ecology and life histories and for developing tools for pest monitoring and management strategies. In this study, we assessed potential sources of variation in CHC profiles in the navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae), an economically important pest of nut crops in California. Using coupled gas chromatography-mass spectrometry, we characterized and compared CHC profiles between adults of pyrethroid-resistant (R347) and susceptible (ALMOND) strains. We further compared CHC profiles from adults differing in age (1, 3, 5, and 7 d post-eclosion) and sex. Hydrocarbon profiles comprised 47 different CHCs in detectable quantities that ranged from C17 to C43 in chain length and included straight-chain alkanes and a variety of mono-, di-, and tri-methylalkanes. Adults from resistant populations had greater quantities of CHCs in total than those from susceptible strains, but relative quantities of individual components were similar. The six most abundant compounds were n-pentacosane, n-heptacosane, n-nonacosane, n-hentriacontane, 11,25+ 13,23 + 15,21-dimethylpentatriacontane, and 13,23+ 11,25 + 9,17-dimethylheptatriacontane. Post-eclosion, total CHCs increased with adult age, with males producing greater quantities than females at all ages. Our results show that CHC profiles vary depending on age, sex, and strain and suggest that CHC profiles may be useful as biomarkers to differentiate between insecticide- resistant and susceptible populations.

Characterization of epicuticular wax structures on leaves of urban plant species and its association with leaf wettability

Epicuticular wax (EW) protects the plant’s integrity and acts as a barrier against biotic and abiotic stresses. The micro-structured three-dimensional EW’s and presence of leaf trichomes influence the wettability of a leaf surface. In this study, leaves of 96 perennial urban plant species were examined to determine an association between epicuticular wax structure (EWS) types and leaf wettability and investigate their seasonal variation. The EWS types were identified using Scanning Electron Microscopy (SEM), while leaf wettability was analyzed by measuring the drop contact angle (DCA) on both the abaxial and the adaxial sides of leaves collected from a common garden in June and September 2016. Four distinct EWS types namely thin film, platelets, crusts, and tubules were observed on leaves of investigated plant species in both June and September. The EWS types varied significantly between functional plant types and plant families in both June and September. In June, the abaxial DCA ranged from 56° to 147°, and the adaxial DCA ranged from 56° to 136°. In September, the abaxial DCA ranged from 54° to 130°, and the adaxial DCA ranged from 51° to 125°. The effect of time, leaf side, and EWS type on leaf wettability were significant. Plant species which showed a change in EWS type or clustering from June to September did not show a more pronounced reduction in DCA compared to those species which exhibited a constant EWS type. Findings from our study illustrate that DCA is not a good indicator in determining the different EWS types due to overlapping DCA intervals between the identified EWS types. However, the identified EWS types remained fairly stable throughout the in-leaf season and do not require repeated measurements for characterization.

Rhizobacteria-induced systemic tolerance against drought stress in Sorghum bicolor (L.) Moench

Induction of systemic tolerance in sorghum [Sorghum bicolor (L.) Moench] against drought stress was studied by screening a large collection of rhizobacterial isolates for their potential to exhibit this essential plant growth-promoting trait. This was done by means of a greenhouse assay that measured the relative change in both plant height and -biomass (roots and shoots) between rhizobacteria-primed versus non-primed (naïve) plants under drought stress conditions. In order to elucidate the metabolomic changes in S. bicolor that conferred the drought stress tolerance after treatment (priming) with selected isolates, untargeted ultra-high performance liquid chromatography-high definition mass spectrometry (UHPLC-HDMS)-based metabolomics was carried out. Intracellular metabolites were methanol-extracted from rhizobacteria-primed and naïve S. bicolor roots and shoots. Extracts were analysed on a UHPLC-HDMS system and the generated data were chemometrically mined to determine signatory metabolic profiles and bio-markers related to induced systemic tolerance. The metabolomic results showed significant treatment-related differential metabolic reprogramming between rhizobacteria-primed and naïve plants, correlating to the ability of the selected isolates to protect S. bicolor against drought stress. The selected isolates, identified by means of 16S rRNA gene sequencing as members of the genera Bacillus and Pseudomonas, were screened for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity by means of an in vitro assay and the presence of the acdS gene was subsequently confirmed by PCR for strain N66 (Pseudomonas sp.). The underlying key metabolic changes in the enhanced drought stress tolerance observed in rhizobacteria-primed S. bicolor plants included (1) augmented antioxidant capacity; (2) growth promotion and root architecture modification as a result of the upregulation of the hormones gibberellic acid, indole acetic acid and cytokinin; (3) the early activation of induce systemic tolerance through the signalling hormones brassinolides, salicylic acid and jasmonic acid and signalling molecules sphingosine and psychosine; (4) the production of the osmolytes proline, glutamic acid and choline; (5) the production of the epicuticular wax docosanoic acid and (6) ACC deaminase activity resulting in lowered ethylene levels. These results unravelled key molecular details underlying the PGPR-induced systemic tolerance in sorghum plants, providing insights for the plant priming for abiotic stress.