DHN黑色素与玉米大斑病菌附着胞膨压形成的关系

 【目的】探讨玉米大斑病菌胞内DHN黑色素在附着胞膨压产生过程中的作用,明确玉米大斑病菌的侵入机理。【方法】通过诱导玉米大斑病菌附着胞产生,确定附着胞形成的最佳条件,利用溶质排斥技术及incipient-cytorrhysis技术对玉米大斑病菌野生型菌株01-23和黑色素缺失突变体△St3hnr附着胞细胞壁孔径大小和膨压进行测定。【结果】野生型菌株01-23细胞壁孔径范围是2.1—2.7 nm,膨压在5.4 MPa左右;黑色素缺失突变体△St3hnr细胞壁孔径范围是2.7—3.3 nm,膨压在4.1 MPa左右;缺乏黑色素的附着胞不能形成高膨压,丧失了穿透能力。【结论】黑色素层对溶质分子外渗的阻挡作用导致了附着胞高膨压的产生,膨压产生的机械穿透力在玉米大斑病菌穿透基质平面的过程中发挥了重要作用。     

玉米大斑病菌小柱孢酮脱水酶StSCD家族鉴定及其功能分析

【目的】小柱孢酮脱水酶（scytalone dehydratase,SCD）是黑色素合成过程中的关键酶。本文旨在鉴定玉米大斑病菌（Setosphaeria turcica）小柱孢酮脱水酶基因（StSCD）家族,并分析玉米大斑病菌附着胞发育过程中StSCD基因家族表达量差异及SCD抑制剂对黑色素合成的影响,为进一步研究StSCD基因家族在黑色素合成和附着胞发育中的作用打下基础。【方法】利用玉米大斑病菌野生型菌株01-23的全基因组数据,获得StSCD基因家族的全序列,并与玉米小斑病菌（Cochlibolus heterostrophus）、稻瘟病菌（Pyricularia oryzae）、瓜类炭疽病菌（Colletotrichum lagenaria）等真菌的SCD进行序列比对;收集玉米大斑病菌附着胞不同发育时期的材料进行实时荧光定量PCR（qRT-PCR）分析,获得不同时期、不同StSCD的表达量,从而确定与病菌侵染和附着胞黑色素化密切相关的脱水酶基因;使用SCD抑制剂环丙酰菌胺处理玉米大斑病菌,测定菌落生长速度、黑色素合成量、附着胞膨压等,确定StSCD在附着胞发育过程中的作用。【结果】玉米大斑病菌全基因组中共鉴定到4个StSCD,其编码蛋白具有SCD保守结构域及保守的催化及底物结合氨基酸残基。StSCD3与灰葡萄孢（Botrytis cinerea）中功能冗余的SCD2蛋白具有较高同源性,StSCD4与多主棒孢（Corynespora cassiicola）中参与黑色素合成的SCD蛋白具有较高同源性。通过分析玉米大斑病菌生长发育过程中不同时期StSCD的表达量发现,在附着胞时期4个StSCD表达量均上调,其中StSCD3、StSCD4表达量上调尤为显著,附着胞再生菌丝时期StSCD3、StSCD4的表达量均显著下降,并且在附着胞诱导整个时期StSCD4的表达量较高。环丙酰菌胺处理玉米大斑病菌后,黑色素合成受阻,附着胞膨压显著降低。【结论】玉米大斑病菌含有4个StSCD,推测StSCD4参与DHN（1,8-间苯二酚）黑色素的合成,并进而影响附着胞膨压积累。     

Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeriaturcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB), which leads to considerable crop losses. Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection. However, how the regulatory mechanism works was still largel...     

玫烟色拟青霉孢子萌发和附着胞形成的影响因子研究

用不同种类、不同浓度的培养液离体培养玫烟色拟青霉孢子,观察其萌发过程,结果显示,玫烟色拟青霉孢子萌发可以产生单极芽管和双极芽管,而且芽管顶端膨大可形成附着胞,附着胞形态以球形和棍棒状为主;不同培养液中,孢子萌发和附着胞形成率不同,其中0.1%的菜青虫体壁浸出液(CL)培养液中孢子萌发率和附着胞形成率最高,分别达到79.01%和18.69%。不同基质对玫烟色拟青霉孢子萌发和附着胞形成的影响也不同,蝉翅较适合玫烟色拟青霉孢子萌发和附着胞形成。     

胶孢炭疽菌侵染杨树叶片的组织病理学研究

目的明确胶孢炭疽菌在杨树叶片上的侵染过程,为进一步从分子水平研究该菌的致病机制和杨树抗病分子育种奠定基础。方法用绿色荧光蛋白标记的胶孢炭疽菌菌株BH12-2的分生孢子悬浮液接种健康杨树叶片,采用光学显微镜和电子显微镜观察病原菌的侵染过程和杨树叶片的防卫反应。结果接种4h后,孢子开始萌发产生芽管;8h时芽管顶端形成附着胞;12h时成熟的附着胞中央形成侵染钉;24h时,孢子另一端萌发形成芽管和附着胞;48h后芽管不断分枝异化成菌丝并产生次级分生孢子;接种3d时,附着胞基部的侵染钉穿透寄主角质层和表皮细胞壁膨大形成侵染泡囊,侵染泡囊初始生长在寄主细胞壁和细胞膜之间,不穿透寄主的原生质体,随后产生初生菌丝和次生菌丝的分化;接种4~5d后,次生菌丝在寄主表皮和叶肉组织内大量扩展;第6d时,菌丝聚集在角质层下形成子座组织,并产生分生孢子梗和分生孢子。随着菌丝的扩展,叶片组织发生一系列的病理变化,在侵入点周围的叶肉细胞壁附近产生胼胝质,细胞壁向内凹陷并发生溶解,细胞质消解,叶绿体等细胞器解体以及寄主细胞坏死塌陷,最终在叶表面产生典型的褐色坏死病斑。结论胶孢炭疽菌在侵染过程中,一个分生孢子可萌发形成多个芽管和附着胞,提高其成功侵染的几率;胶孢炭疽菌对杨树叶片的侵染类型为细胞内半活体营养侵染型。      

Characterization of laccase gene StLAC6 and its involvement in the pathogenicity and peroxisome function in Setosphaeria turcica

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. tur...     

Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

Northern corn leaf blight （NCLB）, caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism （46%）, protein destination and storage （12%）, and disease defense （18%）. Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.     

MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize

Mitogen-activated protein kinase (MAPK) cascades play an important role in extracellular signal transduction and are involved in the pathogenicity of fungal pathogens to host plants. In Curvularia lunata, the roles of two MAPK genes, Clk1 and Clm1, have already been studied. Clk1 is involved in conidia formation and pathogenicity, and Clm1 is closely related to pathogen cell wall formation and pathogenicity to maize leaves. In this study, a third C. lunata MAPK gene, Clh1, which is homologous to hog1, was successfully cloned. We found that a Clh1 deletion mutant had lower intracellular glycerol accumulation than the wild-type stain and was unable to grow normally under osmotic stress conditions. Furthermore, the deletion mutants of three C. lunata MAPK genes (Clk1, Clm1 and Clh1) had lower levels of acetyl-CoA, which is an important intermediate product in the synthesis of melanin and furan toxin, and down-regulated expression of pathogenicity-associated genes. Furthermore, pathogenicity and the ability to produce toxin were restored after adding acetyl-CoA to the culture medium, suggesting that acetyl-CoA is closely involved in the pathogen MAPK signaling pathway.     

温湿度对草莓炭疽菌侵染草莓叶片的影响

为探索草莓炭疽病潜伏侵染条件提供理论依据。本研究在离体条件下通过调控室内温湿度对草莓炭疽病的发病情况及侵染特性差异性进行研究。结果表明：温湿度组合处理25℃/85%、20℃/80%、15℃/80%、10℃/80%、15℃/75%、10℃/75%在接种7天后均能发病,处理10℃/70%接种后28天才轻微发病。侵染特性差异性研究表明,处理25℃/85%,接种6 h,25%的分生孢子开始萌发;接种12 h,附着孢开始形成,形成率为16%,接种48 h,叶片开始出现零星病斑;接种7天,叶片组织细孢大量腐烂崩解,病指为39.07。处理10℃/70%,接种1天,2.88%分生孢子开始萌发;直至接种24天,分生孢子萌发率达到100%,附着孢开始形成,形成率为8.83%;接种28天,叶片开始出现零星病斑,病指仅为1.85;接种42天,叶片组织大面积腐烂,形成典型的炭疽病斑。得出处理10℃/70%接近该病害发病的底线温湿度条件,此条件下的潜伏期能够达到28天。     

Germination and appressorium formation of Pyricularia oryzae Cavara can be inhibited by reduced concentration of Blasin®Flowable with carbon dioxide microbubbles

We investigated the possibility to reduce the usage of Blasin^®Flowable (BF), a disinfectant inhibiting the germination and appressorium formation of Pyricularia oryzae Cavara conidia, by using carbon dioxide microbubbles (CO₂MB). Germination was significantly inhibited by 10 000-fold diluted BF solution containing CO₂MB generated by the decompression-type generator compared to CO₂ millibubbles (CO₂MMB) and CO₂MB generated by the gas-water circulating-type generator. Appressorium formation in the 10 000-fold diluted BF solution containing both CO₂MBs was less than that in CO₂MMB. Scanning electron microscopy showed wrinkles and dents on the surface of conidia treated with 5 000-fold diluted BF solution containing both CO₂MBs. Via transmission electron microscopy, we observed the expansion of the vacuole and the intracellular space and bloated or absent lipid granules in the conidia treated with BF solution containing both CO₂MBs. Our results show that inhibition of the conidium germination and appressorium formation of P. oryzae Cavara by 10 000-fold diluted BF solution could be achieved by using the decompression-type CO₂MB.     

甜瓜与单囊壳白粉菌亲和互作组织病理学及超微结构研究

利用光学显微镜与透射电镜技术,观察了单囊壳自粉菌(Podosphaera xanthii)1号生理小种侵染感病甜瓜叶片组织病理学和超微结构特征.结果表明:分生孢子接种后4 h开始萌发产生管状芽管,生出的第1个芽管顶端膨大形成附着胞,随后附着胞中部产生吸器侵入寄主表皮细胞,其余芽管也不断分化出菌丝并产生吸器,接种后120...     

Stk2, a Mitogen-Activated Protein Kinase from Setosphaeria turcica,Specifically Complements the Functions of the Fus3 and Kss1 of Saccharomyces cerevisiae in Filamentation,Invasive Growth,and Mating Behavior

Setosphaeria turcica, an essential phytopathogenic fungus, is the primary cause of serious yield losses in corn; however, its pathogenic mechanism is poorly understood. We cloned STK2, a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum, 56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae. To deduce Stk2 function in S. turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S. cerevisiae, a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S. cerevisiae. The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores, and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain. Moreover, STK2 complemented the function of KSS1 in filamentation and invasive growth, as well as the mating behavior of FUS3 in S. cerevisiae, however, its exact functions in S. turcica will be studied in the future research.     

橡胶树白粉病菌在疏水介质表面的侵染特征和RNA提取

将白粉菌孢子接种到石蜡膜(parafilm)介质表面,观察不同温度条件下橡胶树白粉菌(Oidium heveae B.A.Steinmann)菌株孢子在疏水介质上的侵染过程,确定0~24 hpi孢子侵染结构发育特征,提取侵染发育过程白粉菌的总RNA,采用RT-PCK扩增分析18S保守基因并评价其质量。结果表明:橡胶树白粉菌孢子在15~34℃温度条件下接种于parafilm介质表面能萌发并形成附着胞,萌发和附着胞形成的适温范围22~28℃,最适离体诱导温度22℃。孢子在parafilm介质表面0~24 hpi(hour Post moculation)侵染发育过程可分为未萌发(0hpi)、萌发(4 hpi)、附着胞形成(6,8 hpi)和次生附着胞形成(16,24 hpi)等4个阶段;采用改良Trizol方法提取白粉菌孢子侵染发育过程4个阶段总RNA,可满足转录组文库要求,并扩增18S序列,进化分析显示其与白粉菌科物种为同一分支。结果表明,病原菌孢子在22~28℃温度条件下能够在介质表面形成侵染结构,0,4,6,8,16,24 hpi侵染发育阶段总RNA可用于转录组测序分析研究。     

种子带菌作为玉米弯孢菌叶斑病初侵染源的研究

[目的]对种子带菌作为玉米弯孢菌叶斑病的初侵染源进行研究,为生产上防治该病提供理论依据。[方法]采用玻片萌发法和组织分离法测定种子外部带菌、内部带菌的存活力;采用沙培法在室内检测种子带菌对幼苗的侵染;应用田间培养法测定种子带菌对玉米的侵染。[结果]玉米种子所带弯孢菌孢子和菌丝可以越冬成活,种子内部带菌影响种子萌发,种子外部带菌不影响种子萌发;种子内外带菌均能侵染玉米幼苗的胚根和胚芽。[结论]玉米种子带弯孢菌孢子和菌丝可以越冬并侵染幼苗,使幼苗长势衰弱。     

A new Curvularia lunata variety discovered in Huanghuaihai Region in China

The purpose of this study was to identify the dominant pathogens of Curvularia leaf spot and their pathogenicity variation in Huanghuaihai Region of China in recent years. In 2013 and 2016–2017, the occurrences of Curvularia leaf spots on maize were investigated in fields located in Henan, Hebei, Shandong, and Anhui provinces, and 292 fungi were isolated from diseased leaves. These fungal isolates were subjected to morphological identification, and 232 isolates were found to have about 70% uncurved conidia and were identified as Curvularia lunata var. Most of the conidia of 2 representative isolates, namely, HNWB-131 and HNWB-185, were oblong with parallel septations and were distinctly different from a reference isolate CX-3. For further determination, the internal transcribed spacer(ITS), glyceraldehyde 3-phosphate dehydrogenase(GPDH), the large subunit(LSU), and translation elongation factor 1-alpha(EF1-α) sequences of HNWB-131, HNWB-185, and CX-3 were amplified and sequenced. The results of sequence analysis showed that the 4 gene sequences from the 3 isolates had a similarity of more than 99% to C. lunata. Based on the sequences of ITS and the combined data of the 4 genes, neighbor-joining trees were constructed for phylogenetic analysis. The results indicated that these 3 isolates were clustered together with C. lunata. The expression of Clg2 p and ClUrase genes in mycelia and conidia was significantly(P0.05) higher in CX-3 than in HNWB-131 and HNWB-185. This study found that the dominant pathogen of Curvularia leaf spot was a new variety of C. lunata with morphological variations in Huanghuaihai Region from 2013 to 2017. The pathogenicity of the C. lunata var. was not significantly enhanced, and the expression of Clg2 p and ClUrase genes of C. lunata var. was decreased.     

Intelligent diagnosis of northern corn leaf blight with deep learning model

Maize (Zea mays L.), also known as corn, is the third most cultivated crop in the world. Northern corn leaf blight (NCLB) is a globally devastating maize foliar disease caused by Setosphaeria turcica (Luttrell) Leonard and Suggs. Early intelligent diagnosis and warning is an effective and economical strategy to control this disease. Today, deep learning is beginning to play an essential role in agriculture. Notably, deep convolutional neural networks (DCNN) are amongst the most successful machine learning techniques in plant disease detection and diagnosis. Our study aims to identify NCLB in the maize-producing area in Jilin Province based on several DCNN models. We established a database of 985 leaf images of healthy and infected maize and applied data augmentation techniques including image segmentation, image resizing, image cropping, and image transformation, to expand to 30 655 images. Several proven convolutional neural networks, such as AlexNet, GoogleNet, VGG16, and VGG19, were then used to identify diseases. Based on the best performance of the DCNN pre-trained model GoogleNet, some of the recent loss functions developed for deep facial recognition tasks such as ArcFace, CosFace, and A-Softmax were applied to detect NCLB. We found that a pre-trained GoogleNet architecture with the Softmax loss function can achieve an excellent accuracy of 99.94% on NCLB diagnosis. The analysis was implemented in Python with two deep learning frameworks, Pytorch and Keras. The techniques, training, validation, and test results are presented in this paper. Overall, our study explores intelligent identification technology for NCLB and effectively diagnoses NCLB from images of maize.     

大蒜提取物防治黄瓜黑星病的初步研究

测定了大蒜提取物对黄瓜黑星病菌(C lad osp orium cucum erinum)的抑制作用和对苗期黄瓜黑星病的防治效果,并对黑星病菌在经大蒜提取物处理后的黄瓜叶片上的侵染过程进行了组织病理学观察。结果表明,0.3 g/mL的大蒜提取物可以完全抑制病菌菌丝生长和分生孢子萌发,该浓度大蒜提取物能有效预防病菌的侵染,作用效果优于多菌灵,但其治疗作用较差。组织病理学研究表明,大蒜提取物可以有效阻止病菌在叶片上的萌发和附着孢的形成,从而阻断病菌的侵入过程。     

玉米弯孢菌黑色素性质及其在致病性中的作用研究

为了探明玉米弯孢菌黑色素性质及其在致病性中的作用。利用酸碱沉淀法提取了玉米弯孢菌胞内及胞外黑色素,并分别采用紫外光谱、红外光谱研究了黑色素的性质;采用黑色素抑制实验研究了黑色素在玉米弯孢菌致病性中的作用。紫外光谱分析表明,黑色素在紫外及可见光区光谱扫描没有极值,吸光值取对数后对波长作图可以得到一条斜率为负值的直线,说明该实验提取的物质确实是一类黑色素类物质。红外光谱分析表明,玉米弯孢菌叶斑病菌菌株CX-3胞内与胞外黑色素红外光谱图有一定差异。玉米弯孢菌胞内黑色素以DHN为主,胞外是DOPA;初步证明DHN黑色素在病菌侵染感病玉米叶片过程中有一定作用。     

基于高光谱优化NDVI值反演玉米叶片氮含量

为研究基于高光谱优化NDVI值在反演喇叭口期玉米叶片氮含量的可行性,使用Fiedspec3.0光谱仪和Greenseeker分别对不同施氮量下玉米叶片的光谱反射率和NDVI进行了测定,并对NDVI值进行了提取和优化,建立玉米叶片氮含量优化NDVI反演模型,对其反演精度进行验证。结果显示:优化NDVI值与NDVI测定值、玉米叶片氮含量均呈显著正相关(r=0.913 2,0.949 8),优化NDVI值对玉米叶片氮含量反演精度达到显著水平(r=0.908 1),反演精度高于NDVI测定值。优化NDVI值能通过群体的速测来决策玉米叶片的氮含量,为玉米进行氮营养诊断提供新的技术支持,具有重要的现实意义。     

Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination, Appressorium Production, and Pathogenicity of Setosphaeria turcica

Systemic studies on the effects of mitogen-activated protein kinase （MAPK） signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126.