Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.     

Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.     

Differential susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 10⁸ conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (10⁸ conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (10⁷ blastospores/ml, grown for 3–4 days in Sabouraud dextrose + 0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence.     

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host‐specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell‐free filtrate, for which activity of elastase, N‐acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N‐glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.     

Effects of combining the fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhiziumanisopliae</Emphasis> on the mortality of the tick <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> (ixodidae) in relation to seasonal changes

The effect of formulation, fungal concentration, type and seasonal changes on the mortality of the tick Amblyomma variegatum was investigated. A previous study demonstrated high pathogenicity of strains of the fungi Beauveria bassiana and Metarhizium anisopliae against the ticks Rhipicephalus appendiculatus and Amblyomma variegatum (Kaaya et al. J Invertebr Pathol 1996; 67: 15–20). The present study was undertaken to explore possible additive or synergistic effects of the two fungi on A. variegatum. The effects of oil and water formulations at different concentrations of each fungus and combination of the two on the mortality of A. variegatum in the laboratory and in the field during the wet and dry seasons were determined and compared. The oil formulation performed better in all assays, with highest tick mortality of 92% occurring during the wet season at conidia concentration of 1 × 10¹⁰ conidia/ml of the mixed fungi compared to 49% for the water formulation at similar conidia concentration. However, at the same conidial concentration during the dry season, mortalities in the field were relatively low with the mixture of the fungi recording 24% and 17% tick mortality for the oil and water formulation respectively. The effect of infecting the ticks with a cocktail of the two fungi was inconclusive under more controlled conditions in the laboratory but field results under both wet and dry seasons indicated significant differences between the separate and mixed fungi infections. The results demonstrate a potential of cocktail formulations in the control of ticks and possibly of other arthropod pests.     

A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

Gas permeability of plant cuticles

Cuticles from the adaxial surface of Citrus aurantium L. leaves and from the pericarp of Lycopersicon esculentum L. and Capsicum annuum L. were isolated enzymatically and their oxygen permeability was determined. Isolated cuticles were mounted between a gaseous and an aqueous compartment with the physiological outer side of the membrane facing the gaseous compartment. Permeability for oxygen was characterized by permeability (P) and diffusion (D) coefficients. P and D were independent of the driving force (gradient of oxygen concentration) across the cuticle, thus, Henry's law was obeyed. P values for the diffusion of oxygen varied between 3·10^-7 (Citrus), 1.4·10^-6 (Capsicum), and 1.1·10^-6 (Lycopersicon) m·s^-1. Extraction of soluble lipids from the cuticles increased the permeability. By treating the cutin matrix and the soluble lipids as resistances in series, it could be demonstrated that the soluble lipids were the main resistance for oxygen permeability in Citrus cuticles. However, in Lycopersicon and Capsicum, both the cutin matrix and the soluble lipids determined the total resistance. P values were not affected by either the proton concentration (pH 3–9) or the cations (Na⁺, Ca²⁺) present at the morphological inner side of the cuticles. It is concluded that the water content of cuticles does not affect the permeability properties for oxygen. Partition coefficients indicated a high solubility of oxygen in the cuticle of Citrus. The data suggest a solubility process in the cuticle of Citrus with respect to oxygen permeation.Abbreviations CM cuticular membrane - MX cutin polymer matrix - SCL soluble cuticular lipids     

The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C(16:1), C(18:1), C(18:2), C(18:3) and C(20:1). There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C(14:0), C(16:1) and C(20:0) in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C(16:0), C(18:0), C(18:1) and C(18:2)) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.     

The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Boophilus microplus Infection by Beauveria amorpha andBeauveria bassiana: SEM Analysis and Regulation of Subtilisin-like Proteases and Chitinases

Beauveria bassiana is a well-known broad-range arthropod pathogen which has been used in biological control of several pest insects and ticks such as Boophilus microplus. Beauveria amorpha has both endophytic and entomopathogenic characteristics, but its capacity for biological control has still not been studied. During the processes of host infection, B. bassiana and B. amorpha produce several hydrolytic extracellular enzymes, including proteases and chitinases, which probably degrade the host cuticle and are suggested to be pathogenicity determinants. To access the role of these enzymes during infection in the tick B. microplus, we analyzed their secretion during fungus growth in single and combined carbon sources, compared to complex substrates such as chitin and B. microplus cuticle. Chitin and tick cuticle-induced chitinase in both fungus and protease was induced only by tick cuticle. SEM analysis of B. amorpha and B. bassiana infecting B. microplus showed apressorium formation during penetration on cattle tick cuticle.     

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin)

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.     

Development of pilot-scale fermentation and stabilisation processes for the production of microsclerotia of the entomopathogenic fungus Metarhizium brunneum strain F52

Using 100 L stirred-tank bioreactors, we evaluated the effect of fermentation parameters and drying protocols on the production and stabilisation of microsclerotia (MS) of the entomopathogenic fungus Metarhizium brunneum (formerly M. anisopliae F52). Results showed that stirred-tank bioreactors can be used to mass produce stable MS of Metarhizium and that culturing and drying protocols significantly affected MS yield and stability. Length of fermentation (4–7 days) for Metarhizium cultures had no significant impact on biomass accumulation, MS formation or the storage stability of the air-dried MS granules. Although cultures of Metarhizium grown on media with a carbon-to-nitrogen (C:N) ratio of 30:1 produced significantly more biomass when compared to cultures grown in media with a C:N ratio of 50:1, MS formation and desiccation tolerance following drying were similar. After storage for 1 year at 4°C, conidia production by air-dried MS granules from 50:1 media was significantly higher compared to MS granules from 30:1 media. The addition of diatomaceous earth (DE) to cultures of Metarhizium prior to drying at rates of 0–60 g L^?1 had no significant effect on MS desiccation tolerance but did impact conidia production. Air-dried MS granules without DE produced significantly more conidia g^?1 during the first 4 months of storage, but after 1 year, conidia production was similar regardless of DE content of the MS granule. Microsclerotial granules with higher moisture levels (2.6–5.0% w/w) produced significantly more conidia immediately after drying and MS granules with low moisture (0–2.5% w/w) produced more conidia after 12 months storage.     

Downstream processing of Beauveria bassiana and Metarhizium anisopliae-based fungal biopesticides against Riptortus pedestris: solid culture and delivery of conidia

We established a fungal production platform by focusing on substrates of solid culture for conidial productivity and thermotolerance, and focusing on surfactants to effectively deliver fungal conidia to the Riptortus pedestris cuticles. First, to produce thermotolerant fungal conidia, 2 of each Beauveria bassiana and Metarhizium anisopliae isolates were cultured on 13 cereal substrates for 10 days. Overall, five substrates (millet, non-glutinous Italian foxtail millet, barley, glutinous Italian foxtail millet, and brown rice) produced greatest number of conidia with thermotolerant conidia. When the selected substrates were mixed with minerals, zeolite, perlite or vermiculite to reduce the amount of cereal grains, vermiculite combination showed relatively high conidial production compared to the other mineral combinations. Next, to efficiently deliver the fungal conidia to the cuticles of R. pedestris, six surfactants, CO-2.5, CO-12, LE-7, PE-61, TED-3, and siloxane were each combined with the fungal conidia. The 0.01% combination showed significantly increased insect mortality, which varied depending on isolate. Virulence tests against R. pedestris were performed with conidial suspensions of isolates to assess their virulence. As a result, isolates showed the highest virulence when a virulence test was conducted at 25°C, rather than 20°C, 30°C and 35°C. This work suggests that the combination of cereal grain substrates and vermiculite could be considered for economic conidial production with high thermotolerance, and the CO-12 surfactant is the most suitable for effective delivery to target insects, followed by the information on optimal temperature for virulence against R. pedestris.     

Increased heat tolerance afforded by oil-based conidial formulations of Metarhizium anisopliae and Metarhizium robertsii

The thermotolerance of oil-based conidial formulations of Metarhizium anisopliae s.l. (IP 46) and Metarhizium robertsii (ARSEF 2575) were investigated. Conidia of IP 46 or ARSEF 2575 were suspended in different adjuvants and exposed to 45?±?0.2°C for 4, 6, 8 or 24?h; their viability was then assessed after 48?h incubation at 27?±?1°C. Conidia heated in pure mineral or vegetable oil exhibited mean relative viability exceeding 70% after 8?h of heat exposure, whereas low germination (≤20%) was observed when conidia were heated in water (Tween 80^® 0.01%), carboxymethyl cellulose gel or emulsifiable oils (Graxol^® or Assist^®) and exposed to heat for 6 or 8?h. In addition, conidia of IP 46 suspended in either pure mineral or canola oil and exposed to heat for 48?h had moderate viability, 57% or 41%, respectively. Unstable oil-in-water emulsions showed a higher percentage of conidia incorporated into oil micellae, while the stable emulsions had higher percentage of conidia outside the oil micellae. The thermotolerance of conidia formulated in stable emulsions, however, did not differ from that of conidia formulated in unstable emulsions. The present study highlights possibilities to alleviate the deleterious effects of heat stress towards Metarhizium spp. conidia applied for controlling arthropod pests and vectors through oil-based formulations.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Decomposition and sorption characterization of plant cuticles in soil

The sorption of organic compounds by plant cuticular matter has been extensively investigated; however, little has been studied regarding the effect of plant cuticle degradation on their role in the sorption of organic compounds in soils. The sorption of phenanthrene was studied in soil samples which had been incubated for up to 9 months with three different types of plant cuticle isolated from tomato fruits, pepper fruits and citrus leaves. The main change in the diffuse reflectance Fourier-transform infrared (DRIFT) spectra during incubation of the cuticles was related to cutin decomposition. The peaks assigned to methyl and ethyl vibration and C=O vibration in ester links decreased with decomposition. In general, with all samples, the phenanthrene sorption coefficients calculated for the whole incubated soils (K _d) decreased with incubation time. In contrast, the carbon-normalized K _d values (K _oc) did not exhibit a similar trend for the different cuticles during incubation. The origin of the cuticle also affected the linearity of the sorption isotherms. With the tomato and citrus cuticle samples, the Freundlich N values were close to unity and were stable throughout incubation. However with the green pepper cuticle, the N values exhibited a significant decrease (from 0.98 to 0.70). This study demonstrates that the structural composition of the plant cuticle affects its biodegradability and therefore its ability to sorb organic compounds in soils. Of the residues originating from plant cuticular matter in soils, the cutan biopolymer and lignin-derived structures appear to play a dominant role in sorption as decomposition progresses. Responsible Editor: Alfonso Escudero.