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.     

Cyclic AMP Restores Appressorium Formation Inhibited by Polyamines in Magnaporthe grisea

ABSTRACT Magnaporthe grisea, the causal agent of rice blast, forms a dome-shaped melanized infection structure, an appressorium, to infect its host. Environmental cues that induce appressorium formation in this fungus include the hydrophobicity and hardness of the contact surface and chemicals produced by the host. An elevated concentration of intracellular cyclic AMP (cAMP) has been implicated in appressorium differentiation in M. grisea. Polyamines (putrescine, spermidine, and sper-mine) are involved in cell growth and differentiation in a wide range of organisms. To understand the role of polyamines in appressorium differentiation in M. grisea, intracellular polyamines were quantified, and the effects of polyamines and polyamine biosynthesis inhibitors on conidial germination and appressorium formation were tested. High levels of polyamines were detected in freshly collected spores, but the levels decreased during conidial germination. Spermidine was found to be the major component. Polyamines and polyamine biosynthesis inhibitors did not affect conidial germination, but polyamines specifically impaired appressorium formation. Furthermore, exogenous addition of cAMP restored appressorium formation inhibited by poly-amines. These results suggest that polyamines may reduce intracellular cAMP levels in M. grisea, leading to the inhibition of appressorium formation.     

爵床提取物对辣椒炭疽病菌的抑制作用及其苗期防效

爵床(J usticia procumbens L.)是一种传统的中药材.为探明爵床提取物对辣椒炭疽病菌的抑制作用,采用生长速率法和孢子萌发法分别测定了爵床提取物对辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发的抑制作用,采用苗期盆栽试验观察爵床提取物对辣椒炭疽病的防治效果.结果表明,爵床甲醇提取物可抑制辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发,其活性随着浓度的提高而增强,抑制中浓度分别为4.23、2.16和2.98 mg/mL.通过对爵床提取物处理后病菌孢子萌发形态显微观察,发现病菌孢子芽管长度受到明显抑制甚至不能萌发.当提取物浓度为16mg/mL时对苗期辣椒炭疽病的防效达73.08％.     

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。     

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.     

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.     

一种优化的辣椒尖孢炭疽菌遗传转化体系

由辣椒尖孢炭疽菌(Colletotrichum acutatum)侵染引起的炭疽病是辣椒生产中最具有破坏性的真菌病害,严重影响辣椒的品质和产量。本研究以辣椒尖孢炭疽病菌HHDL02为对象,采用1 mol·L^-1 NH₄Cl 2%的酶裂解液裂解分生孢子萌发2 h的芽管,裂解2～3 h可以高效制备原生质体,结合PEG介导转化法成功将GFP基因导入,其转化菌株菌落形态、菌丝生长速率、分生孢子形态、孢子萌发、附着胞形成、产孢量和致病性与野生型菌株无明显差异,且后代荧光信号遗传稳定。PEG介导的原生质体转化适合辣椒尖孢炭疽菌遗传操作,有助于其致病机理的研究。     

Differential Interactions of aColletotrichum gloeosporioides isolate with green and red pepper fruits

Differential interactions ofColletotrichum gloeosporioides isolate KG 13 with green and red pepper fruits (Capsicum annuum were found when it was inoculated on unwounded and wounded fruits. The isolate produced the typically necrotic, sunken anthracnose symptom on unwounded and wounded green fruits, and wounded red ones, but not on unwounded red ones. Appressorial formation of the fungus on the surfaces of compatible green fruits was higher than on incompatible red ones up to 12 h after inoculation. More and longer infection pegs from appressoria were produced on green than on red fruits. When cuticular wax layers of green and red fruits were removed by dipping in chloroform, red ones only produced larger lesions and more conidia than water-dipped controls did. However, differences in lesion diameter and conidial production were not observed between green and red fruits wounded by pin-pricking. In addition, concentrations of wax extracted from the surface of green and red fruits affected conidial germination and appressorial formation of the fungus. These findings suggest that the isolate KG 13 ofC. gloeosporioides may react differentially to green and red pepper fruits, probably due to the physical and chemical differences in cuticular layers of the fruits.     

Effect of humidity and temperature on conidial germination and appressorium development of two Philippine isolates of the mango anthracnose pathogen Colletotrichum gloeosporioides

A comparison of rates of germination and appressorium formation by an isolate of Colletotrichum gloeosporioides on mango leaves, fruit surfaces and cellophane membranes showed that behaviour was broadly similar on all three substrates. Frequency of appressorium formation was slightly higher on cellophane membranes, and both hyaline and melanized appressoria were formed. Only melanized appressoria were formed on mango surfaces. In vitro experiments on membranes showed comparative differences in physiological behaviour with temperature for two Philippine isolates of C. gloeosporioides . The most stimulatory temperature for production of appressoria differed in isolates I-2 and I-4 (25 and 20°C, respectively). At 30°C more appressoria became melanized than at lower temperatures, but the frequency of formation of penetration pegs was highest at 25°C. Conidia of C. gloeosporioides germinated on cellophane membranes at relative humidities as low as 95%, but the percentage of conidia germinating and forming appressoria increased as the RH approached 100%. Approximately 18% of conidia of C. gloeosporioides I-2 held at 62 and 86% RH for 4 weeks retained viability, and some were capable of forming appressoria when placed at 100% RH. These results have implications for epidemiological models for disease control.     

8种杀菌剂对核桃炭疽病病原菌胶孢炭疽菌的室内毒力

由胶孢炭疽菌Colletotrichum gloeosporioides引起的核桃炭疽病是目前危害山东省核桃生产的主要病害。为筛选防治该病害的高效药剂,在确定了最适菌丝生长和分生孢子萌发温度的基础上,采用菌丝生长速率法和孢子萌发法,测定了8种常用杀菌剂对胶孢炭疽菌的毒力,评估了温度对咪鲜胺和戊唑醇毒力的影响,检测了17个胶孢炭疽菌菌株对咪鲜胺和戊唑醇的敏感性,同时评价了这2种杀菌剂的预防和治疗作用。结果显示:在胶孢炭疽菌菌丝生长和孢子萌发的最适温度28℃下,供试8种杀菌剂中,咪鲜胺、戊唑醇和三唑酮对供试菌株菌丝生长的抑制作用最强,平均EC50值分别为0.07、1.45和3.04 mg/L;异菌脲、代森锰锌和咪鲜胺对分生孢子萌发的抑制作用最强,平均EC50值分别为21.07、25.14和25.18 mg/L。咪鲜胺对菌丝生长的抑制作用受温度影响较小,而戊唑醇对菌丝生长的抑制作用随温度降低而增强;供试17个菌株对咪鲜胺和戊唑醇均有较高敏感性,平均EC50值分别为0.08 和 1.03 mg/L。建议在核桃炭疽病发病的不同时期,轮换使用咪鲜胺、戊唑醇、三唑酮、异菌脲和代森锰锌等杀菌剂,以有效控制该病害。     

Biocontrol activity and primed systemic resistance by compost water extracts against anthracnoses of pepper and cucumber

We investigated direct and indirect effects of compost water extracts (CWEs) from Iljuk-3, Iljuk-7, Shinong-8, and Shinong-9 for the control of anthracnoses caused by Colletotrichum coccodes on pepper and C. orbiculare on cucumber. All tested CWEs significantly (P < 0.05) inhibited in vitro conidial germination and appressorium formation of the fungal pathogens; however, DL-β-amino-n-butyric acid (BABA) failed to inhibit the conidial development of the pathogens. Direct treatments of the CWEs and BABA on pepper and cucumber leaves at 1 and 3 days before or after inoculation significantly (P < 0.05) reduced anthracnose severities; Iljuk-3, Shinong-9, and BABA for pepper and Iljuk-7 for cucumber had more protective activities than curative activities. In addition, root treatment of CWEs suppressed anthracnoses on the plants by the pathogens; however, CWE treatment on lower leaves failed to reduce the diseases on the upper leaves of the plants. The CWE root treatments enhanced not only the expression of the pathogenesis-related (PR) genes CABPR1, CABGLU, CAChi2, CaPR-4, CAPO1, and CaPR-10 in pepper and PR1-1a, PR-2, PR-3, and APOX in cucumber but also the activity of β-1,3-glucanase, chitinase, and peroxidase and the generation of hydrogen peroxide in pepper and cucumber under pathogen-inoculated conditions. However, the CWE treatments failed to induce the plant responses under pathogen-free conditions. These results indicated that the CWEs had direct effects, reducing anthracnoses by C. coccodes on pepper leaves and C. orbiculare on cucumber leaves through protective and curative effects. In addition, CWE root treatments could induce systemic resistance in the primed state against pathogens on plant leaves that enhanced PR gene expression, defense-related enzyme production, and hydrogen peroxide generation rapidly and effectively immediately after pathogen infection. Thus, the CWEs might suppress anthracnoses on leaves of both pepper and cucumber through primed (priming-mediated) systemic resistance.     

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

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

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。     

Ulvan effect on conidial germination and appressoria formation of Colletotrichum gloeosporioides

Ulvan is an algal polysaccharide known for its ability to induce resistance to plant diseases such as the Glomerella leaf spot of apple caused by Colletotrichum gloeosporiodes. This study was aimed at investigating microscopically, in tests in vitro and in vivo, whether ulvan interferes in the development of pre-infective structures of C. gloeosporioides. Conidial germination and appressoria formation were monitored hourly on agar and cellophane, and at 48 h on water- and ulvan-treated susceptible as well as resistant apple leaves. Amendment of agar with ulvan (10 mg ml^?1) enhanced the germination and resulted in longer germ tubes at 7 h of incubation. On cellophane it significantly delayed appressoria formation up to 8 h, but later after 14 h increased the number of appressoria per conidium. Spraying of susceptible leaves with ulvan 6 days before inoculation decreased disease severity by 50%. This was associated with inhibition of appressoria formation and stimulus in growth of germ tubes, without interfering with conidial germination, when compared with both water-treated control and resistant plants. Appressorium formation occurred preferentially on anticlinal walls of epidermal cells and its location was not influenced by host resistance or by ulvan treatment. This study suggests a new mode of action for ulvan interfering with appressorium formation that could protect apple plants against C. gloeosporioides infection.     

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.     

胶孢炭疽菌G蛋白信号调控因子CgRGS3的生物学功能

G蛋白信号调控因子(regulators of G-protein signaling,RGS)是G蛋白的一类负调控因子,在植物病原真菌生长发育及致病过程中起着重要作用。为探究胶孢炭疽病菌1个RGS基因CgRGS3的生物学功能,利用PCR技术扩增CgRGS3基因并进行生物信息学分析,通过同源重组的方法获得该基因的敲除突变体,并在突变体的基础上获得互补株,通过表型分析确定CgRGS3的生物学功能。结果表明:CgRGS3编码1个含有367个氨基酸的蛋白,包含1个RGS功能域和3个跨膜区域,表型分析发现与野生型菌株相比,敲除突变体营养生长缓慢,分生孢子产量附着胞形成率显著降低,对Cu~(2+)、Fe~(2+)、Zn~(2+)离子更加敏感,细胞壁完整性发生改变,黑色素产量降低及致病力减弱等。由此可见,CgRGS3参与调控胶孢炭疽菌的营养生长,分生孢子产生及附着胞的分化、细胞壁完整性、黑色素产生及致病性等。     

Histological characterization of the early-stage infection events of Setosphaeria turcica in maize

Northern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early-stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.     

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.