In vitro screening of some plant extracts against fungal pathogens of mulberry ( Morus spp.)

Twenty one plant species were screened in vitro for their fungitoxic properties against four fungal pathogens viz., Phyllactinia corylea (Powdery mildew), Peridiopsora mori (Brown rust) and Pseudocercospora mori (Black leaf spot) by slide germination method and Myrothecium roridum (Brown leaf spot) by poisoned food technique. Conidial germination of P. corylea was significantly reduced in 5% (w/v) ethanolic extracts all tested plant. Extract of Cassia tora and Cassia sophera completely inhibited conidial germination of P. corylea. Other effective plant extracts inhibited >?90% germination were Allium sativum (99.56%), Ocimum sanctum (97.80%), Moringa oleifera (97.32%). Conidial germination of Pseudocercospora mori was completely inhibited in extract of A. sativum and D. metel. More than 90% inhibition was observed with extract of Holarrhena antidysentrica (93.10%), Adhatoda Zeylanica (91.40%) and Calotropis gigentia (90.40%). Urediniospore germination of Peridiopsora mori was significantly reduced in 19 plant extracts. Extract of A. sativum and D. metel completely inhibited urediniospore germination P. mori. Other effective plant extracts, which inhibited >?90% germination were Calotropis gigentia (99.40%), Targets patula (98.96%), Azadirachta indica (98.85%), Mirabilis jalapa (95.50%) and Chromoleana odorata (90.51%). Maximum inhibition (33.33%) of colony growth of M. roridum was observed with amendment of 5% solvent extracts of D. metel followed by A. sativum (25%), Chromoleana odorata (20%) and Eucalyptus citriodora (16.66%).     

Inhibition of thigmostimulated cell differentiation with RGD-peptides inUromyces germlings

Summary Germlings of the plant pathogenic fungusUromyces appendiculatus sense and respond to topographical signals of various substrata by undergoing a cell differentiation process that culminates in a structure termed an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as integrins that often exhibit specific affinities to the tripeptide sequence Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in the presence of buffered synthetic peptides containing the amino acid sequence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5–2.0 mM), were inhibited from developing appressoria. Two non-RGD peptides (GGGG and RGES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affected by any of the peptides. Furthermore, 0.5 m diameter micropipettes that are normally inductive for appressorium formation when positioned between the germling apex and the substratum did not induce appressorium formation when coated with the RGD peptide. Silanized micropipettes left uncoated or coated with RGES were inductive for appressorium formation. Those observations lead to the hypothesis that an integrin-like protein may be involved in the process of signaling for initiation of appressorium formation inUromyces. An RGDSPC-affinity column was used to isolate proteins fromUromyces germlings with affinity to the RGD sequence. Elution with RGD or EDTA, but not with RGES, yielded at least 12 proteins of which one protein (95 kDa) expressed affinity on immunoblots to two different antibodies of ₁-integrin; one to the carboxyl-terminus of a synthetic peptide of integrin from chicken, and the other from the amino terminus of integrin from human placenta.Abbreviations ECM extracellular matrix - EDTA ethylenediamine-tetraacetic acid - MAb monoclonal antibody - PAb polyclonal antibody - PVDF polyvinylidene difluoride     

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet‐like leaf tissues) and gemmae (small, flat, sheet‐like leaf tissues that propagate asexually via bud‐like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP‐02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP‐4N) and examined their germination and subsequent appressorium formation under a high‐fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP‐4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP‐4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP‐4N fungal structures between thalli and gemmae were observed. At 4 h post‐inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post‐inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP‐02 conidia were not destroyed and formed normal, well‐lobed appressoria on the surface of M. polymorpha gemmae.     

Involvement of extracellular matrix and integrin-like proteins on conidial adhesion and appressorium differentiation in Magnaporthe oryzae

Conidial adhesion and appressorium formation of Magnaporthe oryzae on the rice surface are important early events in the infection process. As an initiative step to understand the mechanisms underlying these cellular processes at a biochemical level, the effect of a human fibronectin antibody (HFA) and RGD peptides on conidial adhesion and appressorium formation was evaluated. HFA inhibited conidial adhesion and appressorium formation in a dosage-dependent manner. RGD peptides also inhibited these cellular events. Conidial adhesion and appressorium formation inhibited by RGD peptides were restored by chemicals involved in the cyclic AMP-dependent signaling pathway. These results suggest that extracellular matrix proteins might be involved in conidial adhesion and appressorium formation through integrin-like receptor mediation and modulation of cAMP-dependent signaling in the cells.     

Differential Responses of Host and Non-host Substrata on Germination of Ascospores of Sclerotinia sclerotiorum

Effect of host.(Cicer arietinum L.) and some non-host (Allium cepa L., A. Sativum L., Ocimum sanictum L., Azadirachta indica Juss., Zingiber officinale Roscoe. And Curcuma longa L.) substrata on the germination of ascospores io Sclerotinia sclerotiorum (Lib.) de Bary has been observed. Maximum germination was noticed on flower petals of gram (C.arietinum) with minimum time (2.5 h) for germ tube initiation. Among the non-host substrate germination was completely inhibited on ginger rhizome peeling whereas delayed germination (after 12h) and lowest germination percentage (48%) as compared with other non-hosts, were observed on turmeric rhizome peeling. It is suggested that ginger extract may be effective in controlling stem rot and wilt of gram incited by S. sclerotiorum in the field.     

Role of cuticular lipids and water-soluble compounds in tick susceptibility to Metarhizium infection

The arthropod cuticle acts as a physiochemical barrier protecting the organism from pathogens' entry. Entomopathogenic fungi actively penetrate the cuticles of arthropod hosts and are therefore directly affected by cuticle composition. Previously we have observed that Metarhizium spp. developing on resistant ticks ultimately die without penetrating tick's cuticle, suggesting that the cuticles of resistant ticks have antifungal compounds. In the present study, lipids and water-soluble cuticular components were extracted from engorged female tick cuticles, of one susceptible and one resistant tick species to Metarhizium spp. While conidia exposed to lipids from the susceptible tick, Rhipicephalus annulatus, germinated and differentiated into appressorium, conidia exposed to lipids from the resistant tick, Hyalomma excavatum, were inhibited. Soluble cuticular component extracts from both susceptible and resistant ticks stimulated conidial germination but not appressorium differentiation. A comparative analysis of the fatty acid profile in lipid extract of each tick exhibited similar compositions, but the relative abundance of C16:0, C18:0, C18:1ω9C and C20:0 was 2–5 times higher in the extracts from resistant ticks. All of these fatty acids inhibited conidial germination in vitro at 1% and 0.1% w/v concentration, but C20:0 stimulated appressorium differentiation at low concentration. This is the first report demonstrating a possible link between the presence of antifungal compounds in a specific concentration in tick cuticle and tick resistance to infection.     

Chemical Signals from Avocado Surface Wax Trigger Germination and Appressorium Formation in Colletotrichum gloeosporioides

The surface wax of the host, avocado (Persea americana) fruit, induced germination and appressorium formation in the spores of Colletotrichum gloeosporioides. Waxes from nonhost plants did not induce appressorium formation in this fungus, and avocado wax did not induce appressorium formation in most Colletotrichum species that infect other hosts. Bioassays of the thin-layer chromatographic fractions of the avocado wax showed that the fatty alcohol fraction was the main appressorium-inducing component. Testing of authentic n-C8 to n-C32 fatty alcohols revealed that C24 and longer-chain alcohols induced appressorium formation. Gas-liquid chromatography/mass spectrometry analysis of free fatty alcohols revealed that avocado wax contains a high content of very long chains. Waxes from nonhost plants containing an even higher content of the very long-chain alcohols did not induce appressorium formation. Waxes from nonhost plants strongly inhibited appressorium induction by avocado wax. Thus, a favorable balance between appressorium-inducing very long-chain fatty alcohols and the absence of inhibitors allows the fungus to use the host surface wax to trigger germination and differentiation of infection structures in the pathogen.     

In vitro evaluation of the antifungal activity of Allium sativum bulb extract against Trichophyton rubrum,a human skin pathogen

The aqueous extract of Allium sativum bulbs showed an antifungal effect against the fungal skin pathogen, Trichophyton rubrum, isolated from infected patients. For a given concentration (200 mg of bulbs/1 ml), the volume of the aqueous garlic extract loaded on to the discs, and the diameter of the inhibitory zone formed around the disc on the fungal lawn showed positive correlation. Extract-included microbial assay confirmed the antifungal effect of Allium sativum. The extract was not heat stable, it lost its antifungal property above 60 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

The Infection of Soybean Leaves by Phakopsora pachyrhizi during Conditions of Discontinuous Wetness

The ability of Phakopsora pachyrhizi to cause infection under conditions of discontinuous wetness was investigated. In in vitro experiments, droplets of a uredospore suspension were deposited onto the surface of polystyrene. After an initial wetting period of either 1, 2 or 4 h, the drops were dried for different time intervals and then the wetness was restored for 11, 10 or 8 h. Germination and appressorium formation were evaluated. In in vivo experiments, soybean plants were inoculated with a uredospore suspension. Leaf wetness was interrupted for 1, 3 or 6 h after initial wetting periods of 1, 2 or 4 h. Then, the wetting was re‐established for 11, 10 or 8 h, respectively. Rust severity was evaluated 14 days after inoculation. The germination of the spores and the formation of the appressoria on the soybean leaves after different periods of wetness were also quantified in vivo by scanning electron microscopy. P. pachyrhizi showed a high infective capacity during short periods of time. An interruption of wetness after 1 h caused average reductions in germination from 56 to 75% and in appressorium formation from 84 to 96%. Rust severity was lower in all of the in vivo treatments with discontinuous wetness when compared to the control plants. Rust severity was zero when the interruption of wetness occurred 4 h after the initial wetting. Wetting interruptions after 1 and 2 h reduced the average rust severity by 83 and 77%, respectively. The germination of the uredospores on the soybean leaves occurred after 2 h of wetness, with a maximum germination appearing after 4 h of wetness. Wetness interruption affected mainly the spores that had initiated the germination.     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Germination Inhibitor in Citrus unshu Fruit Peelings: (+)-Abscisyl β-D-Glucopyranoside

(+)-Abscisyl β-D-glucopyranoside was isolated from mature Citrus unshu peelings as a germination inhibitor component. This is the first report of the direct isolation of (+)-abscisyl β-D-glucopyranoside from this source.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Perilipin LDP1 coordinates lipid droplets formation and utilization for appressorium-mediated infection in Magnaporthe oryzae

Lipid droplets (LDs) serve as one of the major reservoirs in conidia of Magnaporthe oryzae and are quickly utilized during appressorium formation. Here, we identified a gene, LDP1, encoding a perilipin that is important for LD formation and utilization during appressorium maturation. LDP1 is highly expressed in conidium and immature appressorium. Disruption mutants of LDP1 were significantly reduced in virulence, due to appressorial turgor reduction and difficulty in penetration. LDs were significantly reduced in the Δldp1 mutant, indicating LDP1 was required for LDs formation. LDP1 was colocalized with the LDs in conidium and immature appressorium but was gradually separated during appressorium maturation. A typical intracellular triacylglycerol lipase, TGL1-2, was clearly separated with LDs in conidium and immature appressorium but was well colocalized with LDs during appressorium maturation. The subcellular localization of TGL1-2 was affected by LDP1. These data suggested that LDP1 was bound to LDs for protecting from utilization in conidia and at the early appressorium stage but was separated from LDs for lipase entering and degradation. LDP1 was phosphorylated by CPKA at Thr96, which was essential for its localization and functions. These data indicate perilipin LDP1 can coordinate LD formation and utilization for appressorium-mediated infection of M. oryzae.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.