Studies on octylphenoxy surfactants. Part 2: Effects on foliar uptake and translocation

The effects of octylphenol (OP) and four of its ethoxylated derivatives on uptake into, and distribution within, maize leaf of 2-deoxy-glucose (2D-glucose), atrazine and o, p′-DDT are reported. The surfactants and OP (2 g litre^?1 in aqueous acetone) increased the uptake, at both 1.5 and 24 h, of the three model compounds (applied at 1 g litre^?1) having water solubilities in the g, mg and μg litre^?1 ranges. The uptake of 2D-glucose was positively correlated with the hygroscopicity of the surfactants. The uptake of DDT and atrazine increased with the uptake of the surfactants, being inversely related to their hydrophile:lipophile balance (HLB). Uptake of 2D-glucose and atrazine was enhanced at high humidity, the relative enhancement for atrazine increasing with increasing ethylene oxide (EO) content of the surfactants. A significant proportion of the atrazine and DDT entering the leaf was recovered from the epicuticular wax, the amount of atrazine recovered from the wax increasing with the EO content of the surfactants. The proportion of the surfactants taken up which was recovered from the epicuticular wax was minimal at an EO content of 12.5–16 mole equivalents. The appearance of the deposits on the leaf surface differed markedly among the surfactants, with similar trends for all three chemicals and without visible evidence for infiltration of the stomatal pores. The total quantities of glucose and atrazine translocated were increased by all surfactants but that of DDT was not, despite increases in uptake of up to 7.5-fold. Relative translocation (export from treated region of leaf as a percentage of chemical penetrating beyond the epicuticular wax) was reduced in all cases in the presence of surfactant. Up to 30% of the applied [¹⁴C]chemicals was not recovered from the treated leaf after 24 h. The reduced recovery of 2D-glucose, but not that of atrazine and DDT, was largely attributable to movement out of the treated leaf, with approximately 70% of the chemical taken up being translocated basipetally. Loss of atrazine and DDT was a result of volatilisation. There was no evidence that either [14C]2 D-glucose or [¹⁴C]atrazine was metabolised to [¹⁴C]carbon dioxide.     

Wax matters: absence of very‐long‐chain aldehydes from the leaf cuticular wax of the glossy11 mutant of maize compromises the prepenetration processes of Blumeria graminis

Conidial germination and differentiation, the so‐called prepenetration processes, of the barley powdery mildew fungus (Blumeria graminis f.sp. hordei) are triggered in vitro by very‐long‐chain aldehydes, minor constituents of barley leaf wax. However, until now it has not been demonstrated that these cuticle‐derived molecules also play a significant role in the initiation and promotion of the fungal prepenetration processes in vivo, on the surface of a living plant leaf. In the maize (Zea mays) wax mutant glossy11, which is completely devoid of cuticular very‐long‐chain aldehydes, germination and appressorial differentiation of B. graminis were strongly impeded. Spraying the mutant leaf surface with aldehyde‐containing wild‐type wax or pure n‐hexacosanal (C₂₆‐aldehyde) fully restored fungal prepenetration, whereas maize wild‐type leaf surfaces coated with n‐docosanoic acid exhibited reduced conidial germination rates of 23%, and only 5% of the conidia differentiated infection structures. In vitro studies were performed to further corroborate the extensive prevention of fungal germination and differentiation in response to artificial surfaces coated with aldehyde‐deficient maize wax. Because of its phenotype affecting the B. graminis prepenetration processes, the glossy11 mutation of maize may become a valuable molecular target and genetic tool that could provide a means of developing basal powdery mildew resistance in the globally important crops wheat and barley.     

The rapid and accurate determination of germ tube emergence site by Blumeria graminis conidia

The development of appressoria by germinating Blumeria graminis conidia depends on its germ tubes making contact with the host surface. Low angle, low temperature scanning electron microscopy showed that 80% or more of first-formed germ tubes of f. spp. hordei, tritici and avenae conidia emerged from close to the host leaf surface, and so made contact with it allowing them to become functional primary germ tubes. Light microscopy of f. sp.hordei confirmed this result and, in addition, showed that germ tubes frequently emerged close to, and contacted, various hydrophobic and hydrophilic artificial substrata. Geometric models of conidium-substratum interfaces were developed and a “surface point method” was derived to predict the frequency with which contact would result if germ tube emergence was a random phenomenon. However, observed contact frequencies were far higher (c. three to eight times) than predicted. Thus, the germ tube emergence site was determined as a response to substratum contact. In part, this appeared to be a non-specific response. Nevertheless, germ tube contact frequencies were greater on the curved surface of leaf epidermal cells than on planar surface, suggesting that specific recognition of leaf surface characteristics may promote directional emergence. The area of contact required to stimulate directional germ tube emergence was very small: contact with a microneedle tip or with a spiders’ suspension thread was sufficient for many conidia. Similarly, on leaves, the only contact is between the tips of a limited number of conidial wall projections and the edges of epicuticular leaf wax plates. Micromanipulation to roll conidia so that their original site of contact with a leaf was rotated away from it, led to the majority of first-formed germ tubes growing away from the leaf, i.e. emerging close to the site of original contact. The experiments indicated that the site of germ tube emergence is determined within 1 min of deposition. This implicated the release of conidial extracellular materials in recognition of the conidium-leaf surface contact site.     

The role of the abaxial leaf surface waxes of Lolium spp. in resistance to Erysiphe graminis

Field and glasshouse observations of Lolium spp. grasses indicated that the lower, abaxial, leaf surface was rarely infected by powdery mildew ( Erysiphe graminis ) even when the upper, adaxial, surface was densely colonized. Experiments showed that conidia of two strains of E. graminis , one from Lolium and one from Avena , germinated equally well on both surfaces of Lolium and Avena leaves, but that the subsequent growth and development of germlings was impaired on the lower surface of Lolium leaves, so that most formed only multiple short germ tubes or an abnormal long tube, and only c. 25% or fewer formed infection structures. This contributes to the apparent resistance of the lower Lolium leaf surface to powdery mildew and may help to explain why the disease is relatively unimportant in UK ryegrass crops, since infection structures develop at a high frequency on only 50% of the leaf area, i.e. the upper surface. Scanning electron microscopy showed that the epicuticular waxes on the lower Lolium leaf surface form amorphous sheets. This contrasts with the crystalline plate waxes seen on the upper surface of Lolium leaves and on both surfaces of oat leaves. However, when the lower Lolium leaf surface was washed with chloroform to remove epicuticular wax, normal germling and infection structure development was obtained on the wax-free surface. This suggests that the sheet waxes prevent the pathogen gaining access to features of the cuticular membrane which trigger normal germling development.     

Attachment and adhesion of conidia of Stagonospora nodorum to natural and artificial surfaces

Attachment and adhesion of conidia of a wheat-isolate of Stagonospora nodorum to leaf and artificial surfaces was studied. Attachment of conidia was a non-viable process, separate from adhesion, that occurred rapidly and irreversibly. Attachment involved conidial-surface carbohydrates and was partially influenced by surface hydrophobicity. The subsequent adhesion, via the secretion of extracellular matrix from conidia, was a viable process that induced the complete cover of conidia in response to wheat leaf surface components containing epi-cuticular wax and to a lesser extent to barley but inducing only partial covering on glass. Results suggest that specific surface components from the compatible host promote rapid attachment and adhesion of S. nodorum conidia.     

Comparative studies of safeners for the prevention of EPTC injury in maize

The herbicide safener N-dichloroacetyl-1-oxa-4-aza-spiro-4,5-decane (AD-67) is of similar efficiency as the extensively used N.N-diallyl-2,2-dichloroacetamide (R-25788) and the structurally related 3-(dichloroacetyl)-2,2-dimethyl-1,3-oxa-zolidine (AD-2) in reducing EPTC [S-ethyl-N,N-dipropyl (thiocarhamate)] injury to maize (Zea mays L. cv. KSC 360). EPTC treatment produces growth retardation and deformities and inhibits CO₂ fixation. It does not reduce epicuticular lipids appreciably but affects wax arrangement on the leaf surface. When EPTC is applied together with one of these safeners, these injuries are not observed. All three safeners act similarly. Each prevents the herbicide-induced aggregation of epicuticular wax of maize, thereby protecting the plants against the formation of areas where the underlying cuticle layers are exposed and increase in transpiration.     

Relations between glasshouse climate and dry weight of petals,epicuticular wax,cuticle, pre-harvest flowering period and susceptibility toBotrytis cinerea of gerbera and rose flowers

Studies were conducted on the effects of seasonal levels of relative humidity, temperature, and total radiation, on dry weight of petals, on fresh weight of epicuticular wax and of cuticle of petals, on numbers ofBotrytis cinerea lesions in petals, and on preharvest flowering periods in gerbera and rose. No temporal relationships or significant correlations were found among dry weight of petals, weight of wax and cuticle of petals, and numbers of lesions on the petals. Temperature, relative humidity and total radiation did not correlate significantly with dry weight of petals, or with fresh weights of wax and cuticle of petals, except for a positive correlation between relative humidity and cuticle weight in the gerbera cultivar Delphi. No relation was found between weight of epicuticular wax and cuticle of petals and susceptibility of gerbera and rose petals toB. cinerea. The thickness of wax and cuticle on flowers did not seem to be an important factor influencing the susceptibility of flowers toB. cinerea. The seasonal pattern in number of lesions produced on the flowers byB. cinerea was related to the effects of relative humidity and radiation on infectivity of conidia of the pathogen on the flower surface but not to the effects on the susceptibility of flowers.     

Effect of low temperatures on 2,4-D behaviour in maize plants

The foliar surface of 4-leaf maize plants was found to be poorly wettable and retained 106 μl g^?1 dry matter when sprayed with a U46D (2,4-D formulation) blank. The third leaf retained 141 μl g^?1. A 7-day cold spell (17/9°C) increased retention per unit dry matter by 53% (135% on the third leaf). Cold stress lowered epicuticular wax quantity by 29% on the third leaf. Contact angles of formulated 2,4-D lay between 115 and 125° and were not significantly affected by cold stress. 2,4-D rapidly entered into maize third leaf (66% in 24 h) but migration from it was less than 1.5%. 2,4-D was readily degraded in maize (80% in 72 h). The most abundant metabolite was probably an ester conjugate; little of the hydroxy derivatives were found. Cold stress reduced 2,4-D degradation, and 72 h after treatment the amount of undegraded 2,4-D was 78% higher in cold-stressed maize plants. It was concluded that 2,4-D selectivity in maize results from low spray retention per unit dry matter and active degradation of penetrated herbicide. Cold stress affects both factors.     

Wettability of soybean (Glycine max L.) leaves by foliar sprays with respect to developmental changes

BACKGROUND: Leaf wettability considerably defines the degree of retention of water and agrochemical sprays on crop and non‐target plant surfaces. Plant surface structure varies with development therefore the goal was to characterise the wettability of soybean leaf surfaces as a function of growth stage (GS). RESULTS: Adaxial surfaces of leaves developed at GS 16 (BBCH) were 10 times more wettable with water than leaves at the lower canopy (GS 13). By measuring contact angles of a liquid having an intermediate surface tension on different leaf patches, an illustrative wetting profile was elucidated, showing to what degree wetting varies (from > 120° to < 20°) depending on leaf patch and GS. While the critical surface tension of leaf surfaces at different GSs did not correlate with the observed changes, the slope of the Zisman plot accurately represented the increase in wettability of leaves at the upper canopy and lateral shoots (GSs 17 to 19, 21 and 24). The discrimination given by the slopes was even better than that by water contact angles. SEM observations revealed that the low wettability observed at early GSs is mainly due to a dense layer of epicuticular wax crystals. The Zisman plot slope does not represent the changes in leaf roughness (i.e. epicuticular wax deposition), but provides an insight into chemical and compositional surface characteristics at the droplet–leaf interface. CONCLUSIONS: The results with different wettability measurement methods demonstrated that wetting is a feature that characterises each developmental stage of soybean leaves. Positional wettability differences among leaves at the same plant and within the same leaf are relevant for performance, selectivity and plant compatibility of agrochemicals. Implications are discussed. Copyright © 2011 Society of Chemical Industry     

Analysis of foliar uptake of pesticides in barley leaves: Role of epicuticular waxes and compartmentation

Uptake of pesticides into barley leaves was measured under controlled conditions. Leaves detached from plants were submerged in aqueous solutions of ¹⁴C-labelled (2,4-dichlorophenoxy)acetic acid, triadimenol, bitertanol and pentachlorophenol. Uptake was biphasic. A short (30-min) period with high rates of uptake was followed by uptake that proceeded more slowly and was steady over hours. Compartmentation of pesticides was studied by desorbing pentachlorophenol from leaves previously loaded with [¹⁴C]pentachlorophenol. From the uptake and desorption kinetics it was concluded that penetration of pesticides proceeds as follows: the compounds are first sorbed at the surface of epicuticular wax aggregates where they are in contact with the donor solutions. Solutes then diffuse through the surface wax aggregates into the cuticle. Equilibrium between donor solutions, surface wax and cuticle is established in about 30 min. After this time the amounts of solutes in these compartments no longer increase. Uptake after this time represents penetration into the leaf cells. This fraction of the pentachlorophenol is retained irreversibly, while that sorbed in wax and cutin can be desorbed again. All compounds were sorbed in cuticular waxes and partition coefficients wax/water were determined. On a mass basis only 5 to 10% of the amounts sorbed in cutin are sorbed in wax. This comparatively low solubility in wax contributes to the barrier properties of cuticular waxes. The other determinant of permeability is the very low mobility of solutes in cuticular waxes.     

Factors affecting the foliar absorption and redistribution of pesticides. 1. Properties of leaf surfaces and their interactions with spray droplets

Foliar uptake into eleven plant species, grown under controlled environment, has been determined for spray deposits of glyphosate, 2, 4-D and prochloraz applied as solutions in aqueous solvents in the presence and absence of an ethoxylated nonylphenol surfactant. Over 24 h, uptake of glyphosate did not exceed 6% of applied chemical into any species whereas uptake of 2, 4-D and prochloraz differed between species and was modified significantly by the addition of surfactant. Uptake of prochloraz was increased consistently by adding surfactant, but the response of 2, 4-D was variable. Increased uptake was attributed mainly to surfactant-enhanced wetting of the leaf surfaces. Uptake of prochloraz per unit wetted area increased in the presence of surfactant but that of 2, 4-D decreased. Uptake of either chemical did not correlate with the presence of specialised leaf surface structures, cuticular morphology or distribution of the chemicals within the dried deposits. The dried chemicals were distributed either uniformly or as annuli as a result of complex interactions between the active ingredient, surfactant and the leaf surfaces. Regression analysis indicated that the epicuticular wax and cuticular membrane were the major sinks for both 2, 4-D and prochloraz during the 24-h period.     

Leaf wax layer may prevent appressorium differentiation but does not influence orientation of the leaf rust fungus Puccinia hordei on Hordeum chilense leaves

The infection process of most rust fungi start with spore germination, directional growth of the germ tube towards a stoma, differentiation of an appressorium over the stoma, and penetration into the substomatal cavity. In the South American wild barley Hordeum chilense Roem. & Schult., wide variation occurs in the degree to which several rust fungal species are able to form appressoria over the stomata. Apparently, features of the plant may hamper early stages of the infection process. Such an early defence is called avoidance. In order to find out how germ tube growth is directed towards stomata, and whether the cuticular wax layer plays a role in this orientated growth and in appressorium differentiation, several orientation and differentiation parameters of Puccinia hordei germ tubes were measured on H. chilense leaves with and without the wax layer. Orientated growth of the germ tubes started upon contact with the epidermal cell junctions. The growth of lateral branches of the germ tube over the first epidermal cell junction that it meets, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube to stomata. Removal of the cuticular wax layer did not result in loss of germ tube orientation. This suggests that the leaf wax layer has no role in the guidance of germ tubes. On high avoidance accessions, removal of the wax layer allowed appressoria to develop over stomata that would otherwise be overgrown. No effect of the cell widths in stomatal complexes was found on the chance that stomata were overgrown. This suggests that the overgrowth of stomata on H. chilense leaves by P. hordei germ tubes is mainly caused by the wax covering of the stomatal apparatus.     

Normal germling development by Erysiphe graminis on cereal leaves freed of epicuticular wax

Experiments were conducted to test the hypothesis that recognition of the physical structure of epicuticular leaf waxes by Erysiphe graminis may be important to the development of normal germlings and the formation of functional appressoria. Comparisons of germination rates and characteristics of germling development by E. graminis f.sp. avenae , and in one experiment by f.sp. hordei , were made between intact cereal leaves and leaves from which the epicuticular waxes had been stripped away.
Overall, fungal development was very similar on intact and wax-free leaves: although germination rates were slightly, but significantly, lower, and lengths of appressorial germ tubes slightly greater, on stripped than intact leaves, a very similar proportion of germlings formed apparently normal appressoria in both cases. This was true for f.sp. avenae on first- and fifth-formed leaves of susceptible and adult plant resistant oats, and on barley and wheat first leaves, and for f.sp. hordei on first leaves of barley, oat and wheat. The appressoria formed on stripped leaves not only appeared normal, but also formed haustoria with at least the same frequency as on intact leaves; in several experiments, a higher proportion formed haustoria in stripped than intact leaves. Wax removal did not affect the adult plant resistance of oat cv. Maldwyn, which limits haustorium formation by appressoria, indicating that epicuticular wax was not involved in this resistance. It is concluded that the physical structure of epicuticular wax is not involved in the recognition processes leading to normal germling development.     

Secondary spread of Zygophiala wisconsinensis on the surface of apple fruit

Although the fungus Zygophiala wisconsinensis is a prominent component of the sooty blotch and flyspeck (SBFS) disease complex of apple, few studies have characterized in situ morphological adaptations of this or other SBFS species that accompany colonization of the epicuticular wax layer of fruit. Scanning electron microscope was used to monitor stages in the formation of sclerotium-like bodies on the wax surface, as well as ramifications from these structures to form secondary sclerotium-like bodies. At a mature stage in the formation of sclerotium-like bodies, the matrix of hyphae, conidiophores, and conidia between these bodies collapsed, resulting in formation of the independent clusters of typical colonies of species in the flyspeck mycelial type. Formation of conidiophores and secondary conidia in situ were also described for the first time for any flyspeck species. Observation of conidiophores on naturally and artificially inoculated apple peels with light microscope raises the possibility that formation of secondary conidia may be an important means of spatial dispersion of Z. wisconsinensis in apple orchards and the conidia might function in secondary spread.     

Molecular characterization of a Stagonospora nodorum lipase gene LIP1

A triglyceride lipase gene (LIP1) was cloned from Stagonospora nodorum, the causal agent of wheat glume blotch. LIP1 encodes a 561 amino acid preproprotein with a predicted N‐terminal signal peptide. Its expression was up‐regulated during plant infection and in culture media supplemented with saturated fatty glycerides. The recombinant Lip1 protein possessed lipolytic activity against a broad range of lipid substrates. When applied to wheat leaves, recombinant Lip1 decreased the hydrophobicity of the leaf surface, probably by liquefaction of epicuticular wax. Pretreatment of wheat leaves with Lip1 decreased the rate of conidial adhesion from 69·5% to 22·7% and from 58·9% to 28·4% in two independent assays based on different protocols. LIP1 replacement strains showed decreased lipolytic activity on culture media relative to the wild‐type strain, and adhesion of the conidia to the wheat leaf surface was impaired in the gene replacement strains. In two experiments, adhesion rates were 54·3% and 41·6% in the LIP1 replacement strains, as opposed to 77·7% and 66·6%, respectively, in the wild‐type. Collectively, the data demonstrate that the secreted lipase Lip1 is important for the adhesion of S. nodorum infection to wheat leaves.     

Studies on the role of microorganisms in the metabolism of dieldrin in the epicuticular wax layer of blowflies Calliphora erythrocephala

Microorganisms which inhabit the surface of blowflies, Calliphora erythorcephala, were identified as Bacillus spp., Micrococcus spp., Proteus spp., Streptococcus spp., and yeasts. These microorganisms were able to metabolize [¹⁴C]dieldrin incorporated into their culture media. Micrococcus spp. converted up to 29% of the applied dose of dieldrin to its more polar metabolites while the lowest ability to breakdown dieldrin was associated with Streptococcus spp. which metabolized nearly 0.8% of the applied dose. The addition of sesamex into microbial cultures partly inhibited the breakdown of dieldrin. Cuticular microflora of blowflies were controlled by the topical application of a mixture of gentamicin and amphotericin B. Pretreatment of blowflies with these antibiotics blocked dieldrin metabolism, to a certain degree, in the epicuticular wax layer. The formation of some of the metabolites, which were uniquely confined to the external lipid layer of blowflies, was found to be due to the activity of certain microorganisms. Therefore, microorganisms living on the surface of blowflies were found to play a role in the metabolism of topically applied [¹⁴C]dieldrin. There was a loss in the recovery of the dose of dieldrin applied to blowflies with the lapse of time. This was principally due to its volatilization from treated insects. The volatilization of [¹⁴C]dieldrin from different surfaces was also studied. The loss of the applied dose of dieldrin from various surfaces was in the order of glass > aluminum > fiberglass > DEAE-cellulose paper > silica gel G.     

Extracellular Matrix, Esterase and the Phytotoxin Prehelminthosporol in Infection of Barley Leaves by Bipolaris sorokiniana

Light microscopy and cryo-scanning electron microscopy showed that hyphae of Bipolaris sorokiniana adhered to the wax surfaces of barley leaves by means of an extensive extracellular matrix (ECM). Prehelminthosporol, the major non-host specific phytotoxin formed by B. sorokiniana was immunolocalized in large amounts in the ECM surrounding the hyphae. Similarly, esterase activity involved in degradation of the cuticular wax surface was found in the ECM. Therefore, it appears that the ECM is not only important for adhesion of the fungus to its host, but also functions as a sink of phytotoxins and lytic enzymes important for infection of the host plant.     

First touch: An immediate response to surface recognition in conidia of Blumeria graminis

Conidia of the plant pathogen Blumeria graminis recognize and respond to features of certain substrata. Appropriate surfaces induce release of an extracellular proteinaceous matrix from the body of the conidium. Contact with a hydrophobic substratum elicits almost immediate release of the matrix at the contact interface. In this investigation we present, for the first time, evidence that recognition of the substratum by conidia can stimulate uptake of anionic, low-molecular-weight materials before germination. This facilitated transport could be a mechanism for recognition of the host and determination of the direction of growth of the emerging germ tube toward the host leaf surface.     

Epicuticular Wax and White Pine Blister Rust Resistance in Resistant and Susceptible Selections of Eastern White Pine (Pinus strobus)

ABSTRACT Epicuticular wax on needles was evaluated for its influence on Cronartium ribicola infection of resistant and susceptible selections of Pinus strobus. Environmental scanning electron microscopy comparisons revealed that needles from a resistant selection of eastern white pine, P327, had a significantly higher percentage of stomata that were occluded with wax, fewer basidiospores germinating at 48 h after inoculation, and fewer germ tubes penetrating stomata than needles from a susceptible selection H111. In addition, needles from seedlings that failed to develop symptoms 6 weeks after inoculation, from a cross between P327 and susceptible parent H109, had a significantly higher percentage of stomata occluded with wax compared with needles from seedlings that developed symptoms. In experiments where epicuticular waxes were removed from needles before seedlings were infected, resistant seedlings without wax developed approximately the same number of infection spots (as measured by spot index) as susceptible seedlings with wax intact. Gas chromatography/mass spectrometry comparisons of extracted epicuticular waxes revealed several peaks that were specific to P327 and not found in susceptible H111 suggesting biochemical differences in wax composition. These results implicate the role of epicuticular waxes as a resistance mechanism in P. strobus selection P327 and suggest a role for waxes in reducing spore germination and subsequent infection through stomatal openings.