Inhibition of fungal appressorium formation by pepper (Capsicum annuum) esterase

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.     

A Microscopic Characterization of the Infection of Green and Red Pepper Fruits by an Isolate of Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is a common pathogenic fungus in many plants. To investigate the specificity of the isolate C. gloeosporioides to green fruits, fungal behaviours and anthracnose development on green and red pepper fruits were compared using light and stereo microscopic techniques. When the isolate of C. gloeosporioides was inoculated on both green and red fruits, conidial germination, appressoria, and infection hyphae were observed on both fruits within 24 h after inoculation. The fungal invasion and colonization continued to the epidermal cells of green fruits, but not to those of red ones. Initial anthracnose symptoms were detected only on green fruits at 2 days after inoculation resulting in typical sunken necrosis within 5 days after inoculation. Thus the specificity of the isolate to green fruits may be due to successful invasion and colonization of the infection hyphae from appressoria into the epidermal cells through epicuticular layers of green pepper fnats, but not on red ones.     

Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in Magnaporthe grisea, Is Inhibited by Self-Inhibitors and Requires Surface Attachment

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of Magnaporthe grisea, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in Colletotrichum gloeosporioides and Colletotrichum trifolii differentiation. After calmodulin cDNA and genomic DNA from M. grisea were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the M. grisea genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of M. grisea appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.     

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.     

酚类物质对两种潜伏炭疽菌生长和繁殖的影响

通过测定丹宁、间苯二酚、邻苯二酚、绿原酸和咖啡因,作者对潜伏侵染在香蕉果实中的芭蕉炭疽菌Colletotrichum musae(Berk & Curt)Arx和芒果果实中的胶孢炭疽菌C.gloeosporioides Penz.的分生孢子萌发,附着胞和分生孢子的形成,以及菌丝体生长的影响进行了研究。结果表明,这几种酚类物质,在一定浓度下,可抑制两种炭疽菌的生长和发育,其中邻苯二酚对芭蕉炭疽菌的作用浓度最低,而间苯二酚对胶孢炭疽菌的作用浓度最低,丹宁则对两种菌的作用浓度最高,在一定浓度下影响着附着胞的形成。     

营养物质对两种炭疽菌潜伏侵染的影响

通过不同浓度的几种糖类对潜伏侵染在青香蕉果实中的colletotrichum musae和芒果果实中的colletotichum gloeosporioides的菌体的影响进行了测定,结果表明,高浓度淀粉可极显著地提高两种炭疽菌的孢子萌发率,并有利于附着胞和分生孢子的形成。单糖和二糖在较低浓度时有利于孢子萌发和产孢,不利于附着胞形成。未成熟果实的坚硬结构和高淀粉含量为病菌提供了以附着胞形式潜伏侵染在寄主中的条件。     

玉米大斑病菌钙调磷酸酶A亚基的克隆与特征分析

根据已知病原真菌钙调磷酸酶A亚基(Calcineurin A, CNA)丝苏蛋白磷酸酶保守结构域设计引物, 从玉米大斑病菌cDNA中扩增出CNA基因片段。利用cDNA末端快速克隆(Rapid amplification of cDNA ends, RACE)手段获得该基因全长cDNA序列(GenBank登录号: EF 407562)。Southern杂交结果显示, 该基因在玉米大斑病菌基因组只有1个拷贝。CNA特异性抑制剂Cyclosporin A对玉米大斑病菌分生孢子和附着胞发育有抑制作用, 抑制作用与抑制剂浓度呈正相关, 相同浓度的抑制剂对附着胞形成的抑制作用大于对孢子萌发的抑制。经CsA(5µg/mL)处理后, 玉米大斑病菌分生孢子不能侵入玉米叶片, 初步表明CNA基因参与玉米大斑病菌的致病过程。     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

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.     

几种真菌发酵液对致病疫霉的抑制作用

测定了８种真菌发酵液在５种不同浓度下对致病疫霉菌丝生长、游动孢子静止、静止胞萌发、附着胞形成和侵入丝形成等不同阶段的影响。结果表明,供试真菌不同浓度的发酵液,对致病疫霉上述各个阶段均有一定程度的抑制作用,并均随发酵液浓度增加,抑制作用逐渐增强,浓度为１００％时,抑制作用均达到最高。其中,立枯丝核菌发酵液的抑制作用最强,浓度为１００％时,对致病疫霉菌丝生长的抑制率达到９０．４％,而静止胞萌发率仅为２．４％,附着胞及侵入丝均未见形成。     

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.     

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).     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature,polyamines and polyamine synthesis inhibitor

Prunus dulcis L. ‘Mamaei’ is grown widely in souhtwest of Iran. It blooms in early spring when temperatures are still low. Based on our knowledge there are no reports in the literature regarding pollen behavior of this cultivar under specified condition. Thus, the possible factors for low germination percentage in this cultivar have not been reported. The effect of three different temperatures (10, 25, or 35 °C), polyamines (putrescine, spermidine, and spermine) and polyamine synthesis inhibitor, methylglyoxals-bis (guanyl-hydrazone) (MGBG) on in vitro pollen germination and pollen tube growth were investigated in P. dulcis L. ‘Mamaei’. All temperatures and chemicals significantly affected both pollen germination percentage and pollen tube growth. In general, different polyamines stimulated the pollen germination percentage compared to the control at all temperatures, but increasing the temperature, particularly to 35 °C, had demonstrated inhibitory effects on pollen germination. At a concentration of 0.05 mM putrescine and spermidine and 0.005 and 0.025 mM spermine revealed longer pollen tube growth than that of the control at 10 °C, while higher concentrations tended to inhibit pollen tube growth. At 25 °C, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mM putrescine and 0.005 mM spermine, which slightly stimulated pollen tube growth. Pollen germination and pollen tube growth were inhibited by MGBG at all temperatures and in all concentrations.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.