葡萄VvERF655基因响应可可毛色二孢的表达模式及VvERF655与VvWRKY75的互作

可可毛色二孢Lasiodiplodia theobromae引起的葡萄溃疡病是葡萄上的主要枝干病害之一, 对葡萄生产造成巨大影响。课题组前期研究发现葡萄VvWRKY75负调控葡萄溃疡病的发生, 共表达网络分析表明, VvERF655基因与VvWRKY75共表达。为明确VvERF655是否参与葡萄对葡萄溃疡病的调控过程, 本研究克隆了VvERF655, 运用生物信息学对VvERF655的理化性质等进行分析, 并通过RT-qPCR对VvERF655基因的表达模式进行分析, 结果表明, VvERF655响应L. theobromae侵染并明显受脱落酸、水杨酸激素诱导。通过酵母双杂交技术分析发现, VvWRKY75与VvERF655在酵母中存在互作关系。以上结果表明, VvERF655可能参与寄主对可可毛色二孢的侵染以及激素调控过程。本研究为进一步揭示葡萄VvERF655基因的生物学功能奠定基础。     

可可毛色二孢β-1,4-葡聚糖内切酶LtEg1信号肽的鉴定及其酶活性分析

为明确可可毛色二孢β-1,4-葡聚糖内切酶LtEg1是否能够分泌至胞外及其活性,采用酵母互补试验分析了内切酶LtEg1的信号肽活性,利用3,5-二硝基水杨酸法检测了内切酶LtEg1活性,通过实时荧光定量PCR测定了LtEg1基因在不同侵染阶段的表达量。内切酶LtEg1的信号肽和酵母蔗糖酶的融合表达能够引起酵母蔗糖酶的外泌,使得酵母转化子能够在以棉子糖为唯一碳源的培养基上生长。内切酶LtEg1能够以羧甲基纤维素钠为底物,发挥其酶活功能。与营养菌丝阶段相比,LtEg1基因在病原菌侵染阶段的表达水平显著升高;且在接种后48 h,LtEg1基因的表达量达到高峰,约为营养菌丝阶段的12倍,表明内切酶LtEg1作为重要的外泌蛋白酶,在可可毛色二孢侵染致病过程发挥了重要作用。     

葡萄VvSUC27基因的克隆及病原响应表达分析

由葡萄座腔菌科Botryosphaeriaceae真菌引起的葡萄溃疡病(Botryosphaeria dieback)是葡萄上的主要枝干病害之一,严重影响葡萄产业的健康发展.其中,致病力最强的为可可毛色二孢Lasiodiplodia theobromae.糖转运蛋白在调节植物对病原菌的免疫过程中起着至关重要的作用,关于...     

海南芒果可可球二孢的致病力及其对多菌灵抗性

从海南5个芒果主产区采集分离芒果蒂腐病菌可可球二孢菌株,采用室内人工接种的方法对110个可可球二孢菌株进行致病力测定,采用区分剂量法和菌丝生长速率法测定其中106个菌株对多菌灵的抗性。结果表明:芒果蒂腐病自然发病率29.37%;人工接种的发病率100.00%,110株可可球二孢菌株中强致病力、中等致病力及弱致病力菌株分别占64.55%、25.45%和10.00%;在106株可可球二孢菌株中,抗性菌株发生频率为52.83%,其中高抗菌株可在含100 mg/L多菌灵培养基上生长,发生频率为45.28%,中抗菌株在含100 mg/L多菌灵培养基上不能正常生长,抗性频率为7.55%;没有发现低抗菌株。多菌灵对可可球二孢田间分离菌株EC₅₀的频率分布为不连续分布,研究结果表明海南地区危害芒果的可可球二孢对多菌灵抗性非常严重。     

海南省芒果可可球二孢对多菌灵的敏感性及菌株适合度研究

针对中国海南地区芒果蒂腐病致病菌可可球二孢对多菌灵的抗性水平及菌株的适合度进行了研究。采用区分剂量法,测定了2016年从海南省芒果园采集、分离的90株可可球二孢对多菌灵的敏感性,并对抗性及敏感菌株在菌丝生长、致病力、渗透压及相对渗率等方面进行了比较。结果表明:海南地区芒果可可球二孢对多菌灵的抗性频率为65.56%,且以高抗菌株为主（8个中抗菌株,51个高抗菌株）;该病菌对芒果危害甚大,其中具强致病力的菌株达65株,占72.22%。高抗菌株的菌丝生长优于敏感菌株,存在极显著差异（P0.01）;抗性菌株的致病力与敏感菌株无显著差异;敏感菌株的相对渗率高于抗性菌株,表明敏感菌株细胞膜的透性较高,胞内电解质渗出较多。研究表明,海南地区芒果可可球二孢对多菌灵的抗性水平较高,且抗性菌株适合度高于敏感菌株,易形成优势群体。     

球孢白僵菌体壁蛋白水解酶基因BbCDEP的克隆与分析

从玉米螟虫生真菌球孢白僵菌中分离到体壁蛋白水解酶基因片段BbCDEP,长度为1760bp,包含有3个内含子区段,分别位于331～405,586～646,1157～1223bp的位置,该基因编码了一个由377个氨基酸组成的蛋白,与蛋白数据库中同源蛋白AAL5578.1和AAK70804.1的最高同源性分别为98.9%和98.7%,为研究该基因在致病过程中的分子机理提供理论依据。     

水分胁迫下小麦TaWSI18基因的电子克隆及生物信息学分析

利用电子克隆和RT-PCR技术,从干旱胁迫的小麦叶片中分离出4条新的具有高度同源性的WSI18基因,即TaWSI18-1、TaWSI18-2、TaWSI18-3和TaWSI18-4(Gene Bank登陆号分别为:KP226849、KP226850、KP226851和KP226852);其开放阅读框均为678 bp,编码225个氨基酸。生物信息学分析表明,该类蛋白具有亲水性,存在多个磷酸化位点,无信号肽结构域及剪切位点,定位于细胞质中,无跨膜结构域;二级结构分析表明,该蛋白α螺旋和无规则卷曲的比例达到90%以上;同源性比较及聚类分析表明,小麦TaWSI18蛋白同无芒雀麦(Bromus inermis,BAN15016)WSI18蛋白和二穗短柄草(Brachypodium distachyon,XP 003569641)LEA3蛋白具有高度的同源性,相似性分别为94%和70%。小麦TaWSI18基因编码蛋白的氨基酸序列的N端存在与LEA蛋白的N端区域高度保守的序列。表明小麦TaWSI18蛋白的性质与LEA3蛋白的相似,说明了小麦的TaWSI18蛋白可能与LEA3蛋白存在相同或相似的功能,为进一步研究其在小麦水分胁迫下的功能奠定基础。     

球孢白僵菌SJ-05几丁质酶基因的克隆与序列分析

几丁质酶在昆虫真菌侵染寄主过程中起着重要的作用。本文对采自内蒙古准格尔旗沙棘木蠹蛾的球孢白僵菌菌株SJ-05进行了几丁质酶的基因分析,从中克隆了几丁质酶Bbchit基因,得到了重组质粒pMD19-T/chit。测序结果表明,Bbchit基因核苷酸序列阅读框架全长为1047 bp,编码348个氨基酸,推测分子量为36.924 ku,等电点为5.94。氨基酸序列比对结果表明,与球孢白僵菌Bb0062菌株（AAN41259.1）的氨基酸序列相似性最高,为99.71%。     

草莓胶孢炭疽菌CFEM候选效应子的生物信息学鉴定及其侵染过程中的转录分析

病原真菌通常分泌效应子到寄主组织中调控寄主的生理过程,从而有利于其侵染。CFEM(common in several fungal extracellular membrane)蛋白是真菌所独有的,且与致病性密切相关。本研究利用Pfam数据库对草莓胶孢炭疽菌全基因组进行搜索,鉴定获得22个CFEM蛋白。对CFEM蛋白的信号肽、跨膜结构域和亚细胞定位进行分析,结果表明仅有8个CFEM蛋白为分泌蛋白。对CFEM分泌蛋白在不同侵染阶段的转录情况进行转录组学及RT-PCR分析,结果显示8个CFEM蛋白在侵染后不同时期均有表达。其中,1个CFEM分泌蛋白于附着胞形成期特异表达,2个于活体寄生阶段特异表达,2个于死体寄生阶段特异表达。综合上述分析结果,预测这8个分泌蛋白可能为草莓胶孢炭疽菌的效应子。本研究为深入解析植物病原真菌CFEM效应子提供了理论依据。     

茶树病害病原菌Lasiodiplodia theobromae生物学特性研究

采用皿内测定方法对贵州省惠水县茶树叶斑病的病原菌Lasiodiplodia theobromae进行了病原生物特性研究。结果表明,其病原菌可在SM、PDA、OA和MEA培养基上生长,菌株在MEA培养基上的生长速率显著快于SM培养基、PDA培养基和OA培养基。病原菌适宜在OA培养基上产孢。碳素和氮素营养可影响菌株的生长及菌丝色素的形成。在PDA培养基上,菌株在33℃条件下的生长速度最快,可适应酸性和碱性条件。但在弱碱条件下生长最为迅速。菌株可在PDB培养基中生长,接种菌碟后生长88 h进入稳定期。     

Characterization of Lasiodiplodia species causing leaf blight,stem rot and fruit rot of fig (Ficus carica) in Malaysia

Fig (Ficus carica) is an exotic deciduous plant that is grown worldwide. Fungal diseases pose a major threat to fig plants, affecting their fruit quality and production. This study was conducted to characterize the fungal isolates associated with leaf blight, stem rot and fruit rot of F. carica in Malaysia through morphological analysis, DNA sequencing, multigene phylogenetic analysis and pathogenicity tests. From September 2018 to March 2019, 30 blighted leaves and 30 rotted stems and fruits of F. carica were collected from several nurseries in Malaysia. Thirty fungal isolates that belonged to Lasiodiplodia theobromae (27 isolates) and L. brasiliensis (three isolates) were identified based on morphological characteristics, comparison of DNA sequences and phylogenetic analysis of the internal transcribed spacer (ITS), elongation translation factor 1-α (tef1-α), β-tubulin (tub2) and DNA-directed RNA polymerase II subunit (rpb2). Among the 27 isolates of L. theobromae, nine isolates were obtained from leaves, eight isolates from stems and 10 isolates from fruits, whereas the three isolates of L. brasiliensis were obtained from stems (two isolates) and a leaf (one isolate). The results of pathogenicity tests revealed that L. theobromae and L. brasiliensis isolates were responsible for leaf blight and stem rot of F. carica, whereas fruit rot was caused by L. theobromae isolates. The present study highlighted two different species, L. theobromae and L. brasiliensis, as the causal agents of leaf blight and stem rot of F. carica. Additionally, L. theobromae caused fruit rot of F. carica in Malaysia.     

Relationship between incidence and severity of cashew gummosis in semiarid north-eastern Brazil

The incidence–severity relationship for cashew gummosis, caused by Lasiodiplodia theobromae , was studied to determine the feasibility of using disease incidence to estimate indirectly disease severity in order to establish the potential damage caused by this disease in semiarid north-eastern Brazil. Epidemics were monitored in two cashew orchards, from 1995 to 1998 in an experimental field composed of 28 dwarf clones, and from 2000 to 2002 in a commercial orchard of a single clone. The two sites were located 10 km from each other. Logarithmic transformation achieved the best linear adjustment of incidence and severity data as determined by coefficients of determination for place, age and pooled data. A very high correlation between incidence and severity was found in both fields, with different disease pressures, different cashew genotypes, different ages and at several epidemic stages. Thus, the easily assessed gummosis incidence could be used to estimate gummosis severity levels.     

RXLR类效应分子PITG_21645.2的基因序列分析及功能验证

 马铃薯晚疫病黑龙江菌株效应分子PITG_21645.2在本氏烟(Nicotiana benthamiana)上可以抑制由INF1引起的过敏性细胞坏死(Hypersensitive response, HR)。为验证其为致病疫霉致病的重要效应分子,克隆了10个致病疫霉菌株以及3个同属卵菌菌株中的PITG_21645.2同源基因,它们在氨基酸序列上相似度为93%~100%。与INF1共表达的结果显示,仅有PITG_21645.2^MP903丧失了抑制HR的功能。其中PITG_21645.2^MP903的第31位氨基酸存在特异性,编码丝氨酸,其余同源基因在该位点均编码天冬酰胺。PITG_21645.2^MP903回复突变体(S31N)能够恢复该基因抑制INF1引起HR的功能,说明第31位氨基酸是影响抑制功能的关键位点。另外,PITG_21645.2还能抑制BAX、大豆疫霉PsojNIP和效应分子Avh238在本氏烟上激发的HR,而PITG_21645.2^MP903都丧失抑制功能。本氏烟上表达PITG_21645.2能够促进致病疫霉在寄主上的定殖,从而提示PITG_21645.2在致病疫霉的侵染中发挥致病性功能。     

西藏一种芥菜型油菜PGIP基因的克隆与序列分析

防治油菜菌核病的有效方法是培育抗菌核病的油菜品种。多聚半乳糖醛酸酶阻遏蛋白(polygalacturonase inhibiting protein,PGIP),是植物体内一种重要的抗真菌蛋白。本研究根据NCBI中发表的PGIP基因序列设计引物,从芥菜型油菜‘藏油6号’中克隆得到全长1 048bp的PGIP基因序列。其核酸序列与NCBI数据库中登录的PGIP基因序列同源性为99%。该序列翻译后得到的氨基酸序列与NCBI上公布的油菜PGIP序列同源性为99%。该蛋白质序列中包含8个亮氨酸重复区(leucine-rich repeat,LRR)。过去研究结果显示芥菜型油菜比甘蓝型、白菜型油菜对菌核病的抗性普遍较高,但是未能搞清楚具体的原因,只是提出抗菌核病基因主要分布在芥菜型油菜中,其次是甘蓝型油菜中。通过研究证实了芥菜型油菜上PGIP基因具有保守性、疏水性,而且结构稳定,具有多个信号肽,能够高效地发挥其抗核盘菌的功能。今后在油菜育种上应利用芥菜型油菜的优势,发挥其抗病育种的潜力。     

马铃薯晚疫病菌效应子PITG_16427.2的基因序列分析及功能验证

 马铃薯晚疫病的病原是致病疫霉菌(Phytophthora infestans),该病原菌在侵染马铃薯过程中分泌大量RxLR型效应子,但目前绝大多数RxLR效应子的功能和作用机制尚不明确。本研究成功克隆了致病疫霉菌的一个RxLR效应子PITG_16427.2,在本氏烟中瞬时表达PITG_16427.2,发现该效应子能够抑制6种激发子(INF1、PsojNIP、BAX、SIF2、Avh238、Avh241)激发的植物免疫反应。进一步研究发现,效应子PITG_16427.2在晚疫病菌侵染马铃薯早期上调表达。在15个致病疫霉菌株和3个同属菌株中克隆该基因,克隆到氨基酸序列一致性超过93%的同源基因,这些同源基因均能在本氏烟中抑制INF1和Avh241引起的HR,揭示了该效应子在病原卵菌中序列和功能的高度保守性。在本氏烟和马铃薯感病品种Désirée中瞬时表达PITG_16427.2,发现该效应子能够显著促进晚疫病菌的侵染。通过qRT-PCR方法检测发现,乙烯信号的相关基因ERF1显著上调,而水杨酸信号相关基因PR1b显著下调,表明PITG_16427.2在晚疫病菌侵染过程中可抑制寄主的SA信号途径,促进晚疫病菌侵染。因此,RxLR型效应子PITG_16427.2是致病疫霉菌中一个重要的侵染致病因子。     

水稻中与白叶枯病菌hrf1基因同源序列克隆及功能分析

利用PCR技术从粳稻R109中克隆到与hrf1同源的序列,命名为hpfr1,与hrf1基因的同源性为92%。推导的氨基酸同源性为74.8%。hpfr1基因连接到含T7启动子的高表达载体pET30a（＋）上构建重组质粒pHOSJ4,并转化宿主菌BL21（DE3）产生表达菌株BL21（DE3）/pHOSJ4。hpfr1与hrf1基因在起始密码子ATG至225个碱基处完全相同。将ATG至225碱基的序列克隆,并构建表达载体pHOSJ4-225,表达产物注射烟草能引起过敏反应。BL21（DE3）/pHOSJ4和BL21（DE3）/pHOSJ4-225的蛋白抽提物诱导烟草抗烟草花叶病毒病均达极显著水平,浓度为60μl/ml时防效最显著,分别为96.35%、95.4%,与harpinXoo防效（95.9%）相近。     

Effects of pre‐ and post‐treatment with ethephon on gum formation of peach gummosis caused by Lasiodiplodia theobromae

Peach gummosis, caused by Botryosphaeria spp. fungi, is the process of gum accumulation and exudation in plants. Ethephon (2‐chloroethylphosphonic acid) has profound effects on plants, including enhanced production of secondary metabolites and regulation of plant diseases. This study investigates the effects of application of ethephon before and after inoculation with Lasiodiplodia theobromae on gum formation. Gum formation was promoted by ethephon treatment prior to pathogen inoculation, but inhibited by ethephon applied after the pathogen. The inhibitory effect was counteracted by 1‐methylcyclopropane, which is an ethylene signal inhibitor. 1‐methylcyclopropane also promoted gum formation. Exposure of three isolates of Botryosphaeria to ethephon inhibited mycelial growth. Both treatment methods increased the sugar content at 12 and 24 h post‐inoculation (hpi). However, the sucrose, glucose and fructose contents were significantly higher in shoots with ethephon post‐treatment (application of ethephon after the pathogen inoculation) than those in shoots with ethephon pre‐treatment (application of ethephon prior to pathogen inoculation) at 48 and 72 hpi. The expression of two putative senescence‐related genes, SEN2 and SEN4, were significantly enhanced in pre‐ and post‐treated shoots with ethephon at 24, 48 and 72 hpi. Ethephon application also up‐regulated expression of the pathogenesis‐related protein PR4 while down‐regulating PR1a and PR10. The results show that ethephon has a dual function in regulating gum formation by affecting both the peach shoots and the pathogen.