Calcium/Calmodulin-Dependent Signaling for Prepenetration Development in Colletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.     

A Target-Site-Specific Screening System for Antifungal Compounds on Appressorium Formation in Magnaporthe grisea

ABSTRACT Chemical fungicides are a major method of control for plant diseases in spite of potential negative effects on the environment and the appearance of resistant strains. Development of new chemical fungicides has been largely dependent upon in vivo efficacy tests in the greenhouse or in fields, which is in contrast to target-oriented in vitro screening systems widely used in the pharmaceutical field. To establish a target-site-specific screening system for antifungal compounds, specific inhibition on appressorium formation of the rice blast fungus Magnaporthe grisea was employed. For many plant-pathogenic fungi, including M. grisea, appressorium formation is an essential step to infect host plants. Among 1,000 culture filtrates of members of the class Actinomycetes and fungi, five (A5005, A5008, A5314, A5387, and A5397) from the class Actinomycetes showed differential inhibitory effects on appressorium formation of M. grisea in a dosage-dependent manner. Three (A5005, A5314, and A5387) of these were further fractionated into ethyl acetate and water fractions. The ethyl acetate fraction of A5005 and both the ethyl acetate and water fractions from A5314 and A5387 inhibited appressorium formation, while conidial germination remained little affected. Inhibition of appressorium formation by the ethyl acetate or water fraction was reversed by the exogenous addition of cyclic AMP. Significantly reduced numbers of conidia with appressoria were observed on rice leaves in the presence of culture filtrates. Furthermore, these culture filtrates also exhibited significant disease control of rice blast in the greenhouse. This rapid and target-oriented screening system could be adopted to screen candidate compounds for rice blast control and could be applicable for other appressorium-forming, plant-pathogenic fungi.     

玉米大斑病菌孢子萌发和附着胞形成的影响因素研究

 Factors of influence on conidium germination and appressorium formation of Setosphaeria turcica,such as light condition,conidial concentration,nutrient resources and pH value of conidial suspension were studied.There was no significant difference among light treatments.The optimal pH for conidium germination and appressorium formation was 5.0 to 7.0.The exogenous nutrient sources were not the indispensable factors for conidium germination,but 5% sugar solution were more favorable for appressorium formation than the others.Low conidial concentration in suspension (conidia ≤ 10⁴/mL) was propitious to conidium germination and appressorium formation,which were inhibited significantly in higher concentration.It was suggested that the phenomenon was due to the self-inhibitor,a kind of lipophilic substance,existing in the site of conidium germination.     

Antifungal activity and mode of action of Galla rhois-derived phenolics against phytopathogenic fungi

Antifungal activity and target sites of methanolic extract and its constituents from the gall (Galla rhois) caused by the Chinese sumac aphid, Schlechtendalia chinensis, on the nutgall sumac tree, Rhus javanica, were examined. In tests with six phytopathogenic fungi using a whole plant bioassay, the gall extract exhibited good antifungal activity. The biologically active constituents isolated from Galla rhois were characterized as the phenolics methyl gallate and gallic acid by spectroscopic analyses. Methyl gallate was highly suppressive to Magnaporthe grisea, Botrytis cinerea, and Puccinia recondita, whereas gallic acid exhibited good antifungal activity against M. grisea and Erysiphe graminis. These two compounds were ineffective against rice sheath blight caused by Rhizoctonia solani. Methyl gallate did not adversely affect conidial germination (94%) but significantly inhibited appressorium formation (7%) of M. grisea. Moderate and significant inhibition of conidial germination (64%) and appressorium formation (5%) of M. grisea, respectively, were observed with gallic acid. In complementation tests with M. grisea, cAMP and 1,16-hexadecanediol restored significantly and slightly appressorium formation inhibited by methyl gallate and gallic acid, respectively. These results indicate that methyl gallate and gallic acid acted on a cAMP-related signaling pathway regulating appressorium formation in M. grisea.     

Fruit Exocarp Phenols in Relation to Quiescence and Development of Monilinia fructicola Infections in Prunus spp.: A Role for Cellular Redox?

ABSTRACT Monilinia fructicola causes brown rot of Prunus species and usually remains quiescent on immature fruit but reactivates when fruit are mature. The dihydroxycinnamates caffeic acid and its quinate ester, chlorogenic acid, abundant in the exocarp of peach fruit, had no effect on fungal growth but markedly inhibited the production of the cell wall degrading enzymes polygalacturonase and cutinase in M. fructicola cultures. This inhibition was related to changes in the electrochemical redox potentials of the cultures, as measured with a redox electrode. Fungal culture filtrates had lower electrochemical redox potentials when the growth medium contained caffeic acid than in caffeic acid-free medium. Levels of total intracellular glutathione, the reduced form of which serves as a major cellular antioxidant, increased significantly in M. fructicola cells in response to external caffeic acid. The presence of caffeic acid, chlorogenic acid, or reduced glutathione in conidial suspensions of M. fructicola did not inhibit germination on flower petals and fruit, but inhibited appressorium formation from germinated conidia and subsequent brown rot lesion development. These results suggest that intracellular antioxidant levels in the pathogen can be influenced by phenols present in host tissue and that changes in the redox environment may influence gene expression and differentiation of structures associated with infection by the pathogen. The possible relationship of host phenols to quiescence and subsequent development of M. fructicola infections is discussed.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

利用绿色荧光蛋白GFP研究稻瘟病菌与水稻的互作

稻瘟病菌Magnaporthe oryzae严重威胁水稻的产量与质量,明确稻瘟病菌与水稻互作过程及机理,对防治稻瘟病具有重要意义。本研究利用稻瘟病菌常用致病菌株GUY11和ZB25,构建了绿色荧光蛋白GFP的过量表达菌株,并通过荧光显微观察菌株侵染寄主水稻过程中侵染结构的形成与发育,包括孢子萌发、附着胞形成、侵染钉形成、侵染菌丝增殖、坏死斑形成及产孢。另外,通过比较过量表达菌株对稻瘟病高抗水稻和易感水稻的侵染过程,发现侵染过程的差异主要集中于侵染钉的穿透和侵染菌丝的定殖。本研究为分析稻瘟病菌对寄主水稻的定殖规律提供了一种有效工具。     

Calcium/calmodulin-dependent signaling for prepenetration development in <Emphasis Type="Italic">Cochliobolus miyabeanus</Emphasis> infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca²⁺ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.     

Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola

ABSTRACT Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.     

Germination and appressiorial formation by uredospores ofUromyces viciae-fabae exposed to inhibitors of polyamine biosynthesis

An examination was made of the effects of three polyamine biosynthesis inhibitors on germination and appressorium formation by uredospores of the bean rust fungusUromyces viciae-fabea on artificial membranes. The ornithine decarboxylase inhibitor -difluoromethylornithine had no effect on uredospore germination, even when used at 2mM, whereas appressorium formation was reduced by 63% at 0.5 mM and by 99% at 2mM. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-andenosylmethionine decarboxylase, reduced germination when used at 0.025 mM, and at this concentration, appressorium formation was completed prevented. Uredospore germination was unaffected by as much as 3 mM cyclohexylamine, an inhibitor of spermidine esynthase, while appressorium formation was reduced at 1 mM and completely prevented at 3.3 mM. These results support previous suggestions that inhibitors of polyamine biosynthesis exert their main effect on the early stages of fungal development on the leaf surface.Abbreviantions CHA cyclohyxylamine - DFMO -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Antifungal activities of hyoscyamine and scopolamine against two major rice pathogens: <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The antifungal activities of hyoscyamine and scopolamine, major alkaloids extracted from the desert plant Hyoscyamus muticus, against two rice pathogens, Magnaporthe oryzae and Rhizoctonia solani, were studied. The minimum inhibitory concentration of hyoscyamine that resulted in distinctive inhibition (MIC₅₀) was 1 μg/ml for both fungi. Exposure to hyoscyamine caused the leakage of electrolytes from the mycelia of both fungi. Hyoscyamine (>1 μg/ml) irreversibly delayed or inhibited conidial germination and appressorium formation in M. oryzae grown on polystyrene plates. Hyoscyamine effectively inhibited the attachment of conidia to the surface of rice (Oryza sativa) leaves and inhibited appressorium formation on the leaves. A high concentration of scopolamine (1000 μg/ml) also delayed or inhibited conidial germination in M. oryzae, but conidial germination was restored after washing the conidia with water. Antifungal activity of hyoscyamine was reduced by scopolamine. Magnaporthe oryzae infection was significantly suppressed (by >95%) in leaves of intact rice plants treated with hyoscyamine (10 μg/ml). Moreover, 10 μg hyoscyamine/ml significantly reduced the disease severity index for sheath blight to ≤0.2, when compared with the disease index of control plants (>7.0). Hyoscyamine (>20 μg/ml) completely inhibited sclerotial germination and development of R. solani by delaying the initiation, maturation, and melanization of the sclerotia. These results suggest that tropane alkaloids may be useful for controlling blast and sheath blight diseases of rice and for studying the mechanisms that regulate conidial germination in M. oryzae and sclerotial germination and development in R. solani.     

Acibenzolar- S -methyl-induced resistance to sunflower rust (Puccinia helianthi) is associated with an enhancement of coumarins on foliar surface

Exogenous applications of acibenzolar- S -methyl (ASM) induced resistance to rust infection in sunflower, characterized by reduced infection frequency with no effect on latency period or pustule size, and no increase in host cell necrosis. Cytological studies showed that the reduced frequency of infection was due to a reduction in germination and appressorium formation, while stoma penetration, growth of infection hyphae and haustorium formation remained unaffected. Germination and germtube growth were not hampered by the direct application of ASM on urediospores. The data suggested that ASM had an effect on the production and secretion of fungitoxic compounds to the leaf surface that hamper rust urediospore germination and appressorium formation. This hypothesis is supported by the following experimental results: (i) an increase in the amount of accumulated and excreted coumarins and other phenolic compounds in ASM-treated plants, and (ii) a reduction of germination and of appressorium formation when ayapin, scopoletin, and leaf exudates collected from ASM-treated plants were applied exogenously.     

ZJUF0986活性代谢产物对稻瘟病菌致病性的影响

通过菌丝生长速率法和悬滴法测定ZJUF0986活性代谢产物对稻瘟病菌菌丝生长、孢子萌发和附着胞形成的影响。结果表明,ZJUF0986活性代谢产物对稻瘟病菌菌丝生长的有效中浓度EC50为18.55mg/L,与对照药剂三环唑的EC50(17.30mg/L)相比无显著性差异。活性代谢产物不仅影响孢子萌发,也显著降低附着胞的形成。浓度为10mg/L的活性代谢物可完全抑制孢子萌发及附着胞的形成;浓度为1.25mg/L时则明显延缓孢子萌发及附着胞的形成,处理48h后的孢子萌发率和附着胞形成率分别为62.17%和38.46%。以浓度为1.25mg/L活性代谢产物处理的稻瘟病菌孢子悬浮液接种离体大麦叶片,病原菌孢子在大麦叶片表面能部分萌发形成附着胞,但侵染栓形成延迟,致病性明显降低。     

水稻稻瘟病菌不同发育阶段对7种QoI类杀菌剂的敏感性

以嘧菌酯为对照药剂,研究了6种中国自主创制的QoI类杀菌剂对水稻稻瘟病菌Magnapothe grisea不同发育阶段的影响。结果表明,供试药剂对水稻稻瘟病菌菌丝扩展均表现出良好的抑制作用,EC50值为0.10 ~2.89 μg/mL;对病菌产孢量和孢子萌发的抑制作用明显,EC50值分别为0.10 ~4.92和0.75 ~7.14 μg/mL。在1.0 μg/mL的含药平板上,烯肟菌酯、嘧菌酯、丁香菌酯、SYP-2815对水稻稻瘟病菌产孢量的抑制率均大于90%;对病菌孢子芽管伸长也具有一定的抑制作用,EC50值为1.67 ~34.76 μg/mL。在1.0 μg/mL的含药平板上,7种QoI类药剂对水稻稻瘟病菌菌丝形态无明显影响,但可明显抑制其黑色素的产生。     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

Initiation of appressorial germ tube differentiation and appressorial hooking: distinct morphological events regulated by cAMP signalling in Blumeria graminis f.sp. hordei

Appressorial differentiation by conidia of the barley powdery mildew fungus, Blumeria graminis f.sp. hordei, is dependent on perception of multiple leaf-derived signals. Recently, we have demonstrated that cAMP signalling and PKA play an important, but complex, role during early B. graminis conidial development. Here, we demonstrate that a rise in cAMP levels correlates with conidial differentiation on the barley leaf surface. No change in cAMP levels is observed when conidia fail to differentiate on a non-inductive surface. Moreover, the cAMP levels appear to both increase and decrease during conidial development on barley leaves, suggesting that appressorial differentiation requires differential activation of the cAMP-signalling pathway. In addition, we have dissected the time periods over which cAMP, the cAMP analogue 8-Br-cAMP, and the PKA inhibitor H89, are able to affect conidial differentiation. This reveals that H89 is only active prior to 4 h post-inoculation, corresponding to the initiation of appressorial germ tube development, whereas only cAMP and 8-Br-cAMP inhibit the later process of appressorial hooking, at 4–8 h post-inoculation. Thus, we provide data that supports a model in which cAMP signalling is required to be active to trigger initiation of appressorial germ tube development and inactive to allow subsequent appressorial germ tube hooking.     

Rice Defense Mechanisms Against Cochliobolus miyabeanus and Magnaporthe grisea Are Distinct

ABSTRACT Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.     

水稻内生枯草芽孢杆菌对稻瘟病菌和稻恶苗病菌的抑制作用

自水稻茎和根内分离获得的枯草芽孢杆菌J215和G87菌株培养菌液和滤液对稻瘟病菌和稻恶苗病菌具有较强的抑制作用.其培养菌液(109cfu/ml)稀释2～100倍时,对稻瘟病菌和稻恶苗病菌菌丝生长抑制分别为83.6%～91.0%和60%左右;培养滤液稀释2倍时抑制率达52.4%～75.4%.细菌培养滤液处理能破坏病菌菌丝形态,使稻瘟病菌菌丝细胞膨大、细胞壁破损、原生质外渗、菌体崩溃;但对稻恶苗病菌菌丝损坏较轻,主要使菌丝细胞膨大、生长缓慢.另外,细菌培养滤液稀释10倍时对稻瘟病菌分生孢子形成和萌发的抑制分别达85%和95%以上;对稻恶苗病菌分生孢子形成和萌发的抑制率分别为90%和60%以上.