桑椹菌核病是由3种子囊菌真菌引起的相似症状病害的统称,为果桑生产中的毁灭性真菌病害,严重制约果桑产业的发展。这3种病原菌同为死体营养型病原菌,其侵染手段多样,侵染机制复杂。由于3种病原菌在人工培养上存在不同程度的困难(桑实杯盘菌和肉阜状杯盘菌难培养,核地杖菌在人工培养基上不能完成生活史),在一定程度上限制了病原菌的研究。本文综述了桑椹菌核病的侵染循环、病原菌、病害流行、病原菌与寄主互作等方面的研究进展,并对未来的研究进行了展望,以期对桑椹菌核病的深入研究提供参考。

马铃薯是重要的粮食和经济作物。马铃薯Y病毒(potato virus Y,PVY)是危害马铃薯安全生产的重要病毒。近年来,危害我国马铃薯的PVY株系组成发生了显著变化。PVY重组型株系尤其是PVY^NTN-NW SYRI和SYRII型成为优势株系,但与传统株系分离物相比,优势株系分离物在不同寄主上的侵染性及其致病力还不清楚。本研究分析了PVY^N株系代表性分离物PVY^N605和PVY^NTN-NWSYRI型分离物GZ在本氏烟、普通烟和辣椒上的侵染性,比较了二者在本氏烟和普通烟上的致病力。结果表明,PVY^N605和PVY^NTN-NWSYRI-GZ分离物均能侵染本氏烟和普通烟,并在普通烟上引起叶脉坏死;PVY^N605不能系统侵染辣椒品种‘特大牛角王’,而PVY^NTN-NWSYRI-GZ可系统侵染辣椒品种特大牛角王。PVY^NTN-NWSYRI-GZ在本氏烟细胞间的移动速度明显慢于PVY^N605,但引起本氏烟矮化程度强于PVY^N605。截至接种后第11天,PVY^NTN-NWSYRI-GZ在系统叶片的积累水平明显低于PVY^N605。研究结果对了解PVY种群变化及制定针对性防控策略具有重要意义。

以西番莲品种‘台农1号’为实验材料,通过电镜扫描技术比较观测TeMV和PWV侵染与健康西番莲的叶片、幼嫩果实外果皮组织形态结构,分析测定其叶片生理生化变化,研究西番莲病毒侵染对西番莲叶片和幼嫩果实外果皮组织结构和生理生化的影响。结果表明:TeMV与PWV侵染后,西番莲植株叶片的海绵组织细胞间隙缩小,发生成团堆积状排列,栅栏组织细胞萎缩,呈不规则长条形;幼嫩果实外表皮组织中石细胞层发生严重断裂,排列松散,海绵层细胞变得松散,且发生较严重的木质化现象;叶片与幼嫩果实外表皮表面纹理粗糙、褶皱,气孔保卫细胞萎缩,气孔周围组织皱缩、粗燥;感病果实外果皮表面上的腺毛凸起无序。病毒侵染后,感病植株第1~3叶的蔗糖含量较第4~6叶的高23.11%;健康叶片则相对平衡,降低了淀粉积累、PG酶、纤维素酶与PPO酶活性,提高了感病植株第1~3叶的α-淀粉酶、GS酶与POD酶活性。西番莲受TeMV与PWV侵染后,植株叶片第1~6叶营养物质分配不平衡,相关物质的合成分解与抗氧化防御酶系统受伤害,叶片与果实组织形态结构受到不同程度的损坏,植株正常吸收功能遭到破坏。

植物类甜味蛋白(Thaumatin like proteins, TLPs)是一类具有抗菌活性和抗非生物胁迫作用的病程相关蛋白。本研究克隆获得富士苹果(Malus domestica cv. Fuji)TLPs家族蛋白Mdtl2编码基因,该基因开放阅读框全长为912 bp,编码303个氨基酸。生物信息学分析显示Mdtl2含有多个保守的半胱氨酸及TLPs家族保守序列片段,且具有TLPs典型的三维结构,属于TLPs家族的糖苷水解酶64家族(GH64-TLP-SF Superfamily)。在烟草中过表达Mdtl2能够显著抑制疫霉(Phytophthora infestans)的侵染。在苹果中过表达Mdtl2显著抑制苹果树腐烂病菌(Valsa mali)的侵染,并且能够诱导胼胝质的积累。上述结果表明,Mdtl2作为糖苷水解酶家族蛋白,能够通过诱导胼胝质的积累,提高植物对病原菌的抗性。

香蕉枯萎病菌热带4号生理小种(Fusarium oxysporum f. sp. cubense tropical race 4, Foc TR4)严重威胁世界香蕉产业的可持续发展,一些特殊功能的蛋白在其侵染过程中起重要作用。我们分析Foc TR4接种巴西蕉后的转录组数据发现M35金属蛋白酶(metalloprotease 35)同源基因FocM35_2在病原菌侵染初期受诱导显著上调表达,经同源重组法敲除该基因,突变体致病力显著下降,菌丝生长速率降低,产孢量略微减少,回补突变体则可以恢复其致病力和生长发育;与野生型菌株相比较,突变体对外源氧胁迫、渗透压和细胞膜压力更敏感;在本氏烟草(Nicotiana benthamiana)中的瞬时表达结果显示该蛋白定位于质外体,并引起叶片产生超敏反应(HR,hypersensitive response);该蛋白的缺失还能够诱导寄主更高水平几丁质酶的活性。因此,FocM35_2是促进Foc TR4侵染寄主的的重要致病因子。

枝干病害葡萄溃疡病近年来严重限制了葡萄产业发展。研究表明葡萄蔗糖转运蛋白参与寄主植物和病原菌的互作过程。为解析蔗糖转运蛋白VvSUC12在葡萄免疫反应过程中的功能,本研究克隆了VvSUC12基因翻译起始位点上游1 500 bp的启动子区,通过生物信息学分析发现该区域包含4个Dof转录因子结合序列(A/TAAAG)及多种激素调节与防御相关的顺式元件。实时荧光定量分析显示,接种可可毛色二孢菌显著诱导VvSUC12和VvDof19基因的表达。通过酵母单杂交实验筛选得到与VvSUC12启动子互作的转录因子VvDof19。酵母双杂交实验证实VvDof19具有自激活活性;进一步通过烟草瞬时表达发现Dof19-GFP的相对GUS活性约为对照GFP的9倍,表明转录因子VvDof19能够激活VvSUC12基因的表达。研究结果为深入研究蔗糖转运蛋白在葡萄免疫反应中的功能奠定基础。

凝集素类受体激酶(lectin receptor-like kinase, LecRLKs)是一类在植物应答多种生物/非生物胁迫中发挥重要作用的类受体激酶。本研究在小麦与条锈菌互作的转录组中筛选到1个在小麦与条锈菌非亲和互作中显著上调表达的 LecRLKs 基因 TaLecRLK1。该基因全长 2 292 bp,编码蛋白含有1个胞外信号肽、B-lectin 结构域、PAN_AP结构域、跨膜域和胞内酪氨酸激酶域。qRT-PCR 分析表明: TaLecRLK1在小麦与条锈菌非亲和互作早期诱导表达,在烟草及小麦原生质体中瞬时表达,TaLecRLK1-GFP 定位在细胞膜上。利用大麦条纹花叶病毒介导的基因沉默技术(BSMV-VIGS)沉默 TaLecRLK1,接种无毒性条锈菌小种 CYR23 后,沉默叶片表面产生少量夏孢子堆,组织学观察发现沉默植株中条锈菌菌丝长度增长、侵染点附近活性氧积累面积减少;TaPR1、TaPR2、TaPR5 的表达受到抑制,TaCAT 和 TaSOD 则被迅速诱导表达。综上所述,TaLecRLK1对小麦抗条锈病起到正调控作用。

基因敲除技术日益成为基因功能研究的重要手段,为了深入研究甘蔗梢腐病菌致病基因的功能,构建了梢腐病菌YN41菌株的基因敲除体系。本研究构建了基于线性DNA的同源重组基因敲除技术,融合PCR构建敲除盒即带有潮霉素基因标记的线性DNA融合片段,配置0.05 g·mL^-1溶壁酶+0.01 g·mL^-1崩溃酶的复合酶液28 ℃酶解3 h获得了高产量的原生质体,利用聚乙二醇(PEG)介导的原生质体的转化,PCR鉴定技术和酶切鉴定技术对转化子进行鉴定,荧光定量PCR分析和Southern Blot方法进一步对基因缺失突变体进行验证,生物学表型(菌落形态、生长速率、产孢量、生物量和致病力)验证了PK基因敲除体系的成功构建。28 ℃酶解3 h获得了2×10⁷个· mL^-1原生质体;对编码丙酮酸激酶的PK基因进行了基因敲除验证,获得了纯合敲除突变体;荧光定量PCR检测转录水平无表达;Southern Blot结果显示使用PK基因探针杂交,基因缺失突变体无条带,野生型杂交到目的条带;生物学表型验证了PK基因敲除突变体对比野生型菌丝色素加深,生长速率先慢后快、产孢量增加,致病力增强,成功构建了PK基因敲除突变体。甘蔗梢腐病菌YN41基因敲除体系的成功构建,为甘蔗梢腐病菌致病基因的功能研究提供了技术平台。

由于缺少稳定的遗传转化体系,丝核菌致病相关基因的功能研究受到严重限制。本研究以防治丝核菌病害的常用杀菌剂靶标基因CYP51和Sdh为目标基因,体外合成禾谷丝核菌CYP51的3个同源基因以及Sdh的B、C、D亚基基因的dsRNA,采用喷雾诱导基因沉默(spray-induced gene silencing, SIGS)方法研究这6个基因产生的dsRNA对病菌生长和致病性的影响。结果表明,外源施加靶标基因dsRNA对禾谷丝核菌的生长和致病性均有抑制作用。在被侵染的大麦叶片上,外源dsRNA能够显著抑制病菌中对应基因的表达。100 ng·μL^-1 浓度的RcCYP51-3-dsRNA在大麦叶片上抑菌效果较为显著,且可以同时抑制3个RcCYP51同源基因的表达;RcSdhD-dsRNA同样可以抑制RcSdh三个亚基的基因表达,在50 ng·μL^-1浓度时抑菌效果最好。本研究探索了一种抑制禾谷丝核菌基因表达的方法,为使用SIGS方法研究丝核菌基因功能打下基础。

为了从内生菌资源中筛选出对向日葵菌核病有防效的生防菌株,本研究从12个向日葵品种籽粒中分离获得不同菌落形态的33株内生菌株,在此基础上,利用纸杯法进行初筛,从中选出两株对菌核病具有良好防效的菌株,进而采用离体叶片接种试验进行复筛,筛选出一株对菌核病离体防效达100%的内生菌菌株KB3(Bacillus subtilis)。温室盆栽条件下KB3能够显著降低向日葵苗期菌核病的病情指数,防治效果达79.47%。含菌培养基接菌试验表明菌株KB3发酵滤液对核盘菌的抑菌率为68.46%,分皿试验发现KB3能够抑制核盘菌黑色菌核的形成。通过全基因组测序,分析预测到KB3中有7个次级代谢产物合成基因簇。本研究所采取的纸杯法和离体叶片接种相结合的筛菌方法将对菌核病生防菌株的筛选具有参考意义,所获KB3菌株对于向日葵菌核病的防治具有一定的应用前景。

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。

为建立检测甜瓜黄斑病毒(melon yellow spot virus,MYSV)的SYBR Green I 实时荧光定量PCR(qPCR)方法。基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化。结果显示,优化后的qPCR方法最适退火温度为61.3 ℃,最适引物浓度为0.65 μmol·L^-1,特异性强,灵敏度高,比PCR高100倍。以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7。实验样品验证表明建立的qPCR方法可用于MYSV的定量检测。

Soybean root rot disease caused by the genus Fusarium was observed from 30 soybean fields during 2017 and 2018 in 12 counties or cities of Heilongjiang Province. A total of 250 Fusarium isolates were recovered from the root tissues of soybean seedling collected from those fields. Identification of the Fusarium species was performed by examining their morphological features and sequences analysis of internal transcribed spacers (ITS) of rDNA. Six species of Fusarium were identified, with F. oxysporum being the predominant species (72.8%), followed by F. solani (15.6%), F. tricinctum (7.2%), F. equiseti (2.8%), F. brachygibbosum (1.2%) and F. graminearum (0.4%). All Fusarium isolates were highly virulent on soybean (cv. Heinong 69), causing root rot disease symptoms, which were reisolated from diseased plants and identified, fulfilling the Koch’s postulates. The pathogenicity tests for representative strains of the six Fusarium species showed that at 14 days after inoculation by sorghum seeds (4 g) fully colonized by Fusarium, the disease indexes of F. oxysporum (m3-3xu), F. solani (p15-5zhu), F. tricinctum (pz2-2), F. equiseti (dy-mz2-1), F. brachygibbosum (p13-1), and F. graminearum (mx7-2) were 69.05, 42.86,78.57, 45.24, 58.96, and 35.94, respectively. Both conidia cultures and secretions of the six isolates could cause root rot on soybean at 14 days post inoculation. The rot was serious with increased concentration of conidial cultures and secretions. F. solani was the most virulent to soybean with a disease index of 56.2, followed by F. oxysporum (44.8), F. graminearum (39.1), F. equiseti (37.2), F. tricinctum (32.9) and F. brachygibbosum (16.2) at a concentration of 1×10⁸ conidia·mL^-1. The disease indexes of F. solani and F. equiseti were 41.0 and 27.7, respectively at concentration of 1×10⁵ conidia·mL^-1, which showed no significant difference related to the disease index at concentration of 1×10⁸ conidia·mL^-1. As for the virulence of fermentation filtrates, F. solani and F. oxysporum were the most virulent to soybean with the disease indexes of 48.6 and 48.6, respectively, followed by F. equiseti (44.8), F. tricinctum (41), F. graminearum (40.1) and F. brachygibbosum (27.2).

A new leaf spot disease causing reddish brown or black necrosis on Nandina domestica was identified in Jinhua city, Zhejiang province. Using tissue isolation, Koch’s postulation, morphological observation, and maximum likelihood estimate, we determined that the causative pathogen of N. domestica leaf spot was Diaporthe eres. To our knowledge, it is the first report to show that phytopathogenic fungus D. eres causes leaf spot disease of N. domestica in China. This study may provide important basis for rapid diagnosis and prevention of this new disease on N. domestica.

Common bean (Phaseolus vulgaris), also known as the Kidney bean, is widely grown in China for its fresh pod or edible dry seeds, which consumed as vegetable and cereal. In July 2021, severe outbreaks of common bean wilt were observed in several commercial fields in Inner Mongolia. A fungus was isolated from dark brown lesions on diseased stems and roots. The pathogenicity of the isolated fungus was confirmed from inoculation experiments. On the basis of morphological features and EF-1α and β-tubulin sequences, the pathogen was identified as Fusarium oxysporum. This is the first report of F. oxysporum causing Fusarium wilt on common bean in Inner Mongolia. As a destructive plant pathogen, F. oxysporum has a wide host range and is distributed all over China, control measures should be taken in advance.

From 2019 to 2021, the sample leaves with typical anthracnose disease symptoms were collected from tree peony (Paeonia suffruticosa Andr.) in Anhui of China. Thirty-one fungal isolates obtained from the diseased samples and the purified cultures exhibited the same morphological characteristics on potato sucrose agar. Pathogenicity tests for the representative AF614 revealed that that this fungus was the causal agent of the disease. Molecular analysis of the sequences for the internal transcribed spacer (ITS) regions of their rDNA and the partial DNA fragments of actin (ACT), β-tublin (TUB2), and chitin synthase (CHS) genes showed the orthologues with these of Colletotrichum siamense species. Combined with morphological and molecular characteristics, we named the fungal AF614 as Colletotrichum siamense. To the best of our knowledge, this is the first report of C. siamense causing anthracnose of tree peony in China.

In recent years, sugarcane brown stripe disease has spread across a large area in Yunnan province. To identify the pathogen causing sugarcane brown stripe, 58 diseased samples were collected from different areas and varieties. The pathogen causing sugarcane brown stripe was identified by morphological characteristics and sequences analysis of rDNA ITS region and GAPDH. The results showed that the pathogen was Bipolaris setariae, a new record species of sugarcane brown stripe in Yunnan, which provide scientific basis for epidemic prediction, disease-resistant breeding, and precise control of sugarcane brown stripe.

In order to deeply understand the variation and evolution of RBSDV S6 segment, we analyzed the genetic variation of RBSDV S6 sequence in 26 maize and rice samples from 13 locations. The results showed that there were a large number of base mutations in the coding region, indicating the presence of variation and recombination between S6 sequences. At the same time, by analyzing the amino acids and codon usage preferences encoded by S6, we found that S6 population has other codon usage preferences in addition to GC3s, and the codon usage of this population is under the pressure of natural selection. The genetic recombination analysis showed that there were 2 recombination events (S6-M-13-ⅤM-2 and S6-M-13-ⅤM-3) in 26 sequences. Further, based on phylogenetic analysis, S6 sequences can be divided into four groups. The above results show that S6 segment plays an important role in the genetic variation and evolution of RBSDV population, which provides a theoretical basis for further research on the pathogenic mechanism of RBSDV S6 in maize rough dwarf disease and rice black-streaked dwarf disease.

Passiflora edulis mosaic disease is a serious constraint to the production of Passiflora edulis. in China. During 2016-2019, 170 samples of P. edulis with typical mosaic symptoms were collected from 7 planting areas in Fujian, Guangxi and Guangdong. Cucumber mosaic virus (CMV), Potyvirus and Tobamovirus were detected using RT-PCR with degenerate primers and/or specific primers. The results showed that 90 out of 170 samples were detected positive. The detection rate of CMV was 51.18% while the detection rate of Potyvirus was 10.00 %, however, Tobamovirus was not found in this research. CMV and Potyvirus showed severe mixed infection which account for 15.56% of total infection rate. Further identification of viruses in the genus Potyvirus was performed and the amplified fragment shared 98.00%-100.00% identity with East Asian passiflora virus aviliable in the GenBank. Additionally, 5 isolates with polyprotein gene sequences were obtained and phylogenetic analysis of these 5 isolates indicated a high genetic relationship to the AO stain reported from Japan and China (Taiwan). These results will be helpful for the detection and effective control of Passiflora edulis mosaic disease.

柑橘叶斑驳病毒(citrus leaf blotch virus, CLBV)于20世纪70年代在美国加利福尼亚洲首次发现,之后许多国家陆续报道CLBV侵染不同寄主植物。2016年,我国在猕猴桃中首次检测到CLBV,随后在甜樱桃、柑橘、芍药、苹果、桑树和南天竹中发现CLBV,现已在多个地区普遍发生流行。本文系统梳理了CLBV的研究进展,重点综述了CLBV的发生分布、传播为害、分子变异、检测技术、基因组特征、基因功能等,以期为抗CLBV作物育种、病害综合防控及致病机理的研究提供基础支撑。

为了对马铃薯黄萎病菌的系统侵染进行研究,本研究首先建立了用于观察大丽轮枝菌侵染马铃薯过程的培养皿滤纸体系,在此基础上利用带有GFP标记的大丽轮枝菌对不同抗性水平马铃薯根系侵染过程进行了比较研究,结果表明:马铃薯根系在浓度为10⁷个·mL^-1的大丽轮枝菌分生孢子液中浸泡处理2 h后,分生孢子可附着在抗/感马铃薯根系的根毛上,单个视野中感病品种根系上分生孢子附着平均数量为65个,显著高于抗病品种根系上附着的分生孢子数量(23个);接种12 hpi,根系上附着的分生孢子开始萌发,但抗/感品种根系表面孢子的萌发率无显著差异;接种3 dpi后,菌丝体能够突破根系表皮,进入细胞间隙,但感病品种根系中菌丝的扩展速度更快,并在7 d后到达根系的维管束内部;而抗病品种则在接种9 dpi,在维管束内才能够观察到绿色荧光信号;接种10 dpi,感病品种根系中可见新的分生孢子梗和分生孢子形成,而抗病品种中未见有分生孢子的形成。接种30 dpi,感病品种地上部茎杆中定殖的病原菌量是抗病品种的5倍以上;同时,感病品种叶柄中的病原菌数量也显著高于抗病品种。

大丽轮枝菌是世界范围的植物病原真菌,能够侵染660多种植物,可造成严重的经济损失。大丽轮枝菌基因组分析发现不同菌株含有菌株特异基因,但是大部分基因的生物学功能还不清楚。本研究通过比较大丽轮枝菌VdLs17和JR2的基因组鉴定出1个JR2菌株特异性基因Chr6g02370,该基因编码乙醇脱氢酶,且在侵染过程中被诱导表达。敲除突变体的乙醇脱氢酶酶活性显著降低,但对乙醇等碳源的利用能力并未受到影响。敲除突变体对烟草和番茄的致病力显著降低。表型分析表明敲除该基因降低了黑色素合成能力,但不影响菌丝生长和产孢。研究结果丰富了人们对大丽轮枝菌菌株特异基因生物学功能的认识,为深入开展其功能基因组学研究提供了遗传材料。

由条形柄锈菌小麦专化型(Puccinia striiformis f. sp. tritici)引起的小麦条锈病严重危害我国小麦的生产。钙调素结合转录激活因子(calmodulin-binding transcription activator,CAMTA)是植物中最重要的受Ca²⁺/CaM调节的转录因子家族之一,在植物响应生物胁迫中发挥重要作用。本研究从小麦与条锈菌互作的转录组数据库中筛选出4个Pst处理下调表达的小麦CAMTA基因家族成员,通过实时定量PCR(qRT-PCR)以及病毒介导的基因沉默(virus-induced gene silencing,VIGS)技术对其在小麦与条锈菌互作中的功能进行了初步解析。结果表明,在亲和互作中,TaCAMTA1-A、TaCAMTA1-B和TaCAMTA3-D在不同时间点明显下调表达,而TaCAMTA2-B在条锈菌处理12 h显著上调表达。通过VIGS技术沉默TaCAMTAs基因后发现,沉默TaCAMTA2-B后与对照组相比,小麦叶片坏死斑和孢子数量均明显减少,抗病性增强。在此基础上,克隆了小麦TaCAMTA2-B基因,通过烟草亚细胞定位发现TaCAMTA2-B 定位于细胞核,此外,酵母自激活实验验证TaCAMTA2-B具有转录激活活性。外源激素SA、ABA和JA处理表明TaCAMTA2-B受ABA诱导上调表达,同时TaCAMTA2-B在小麦的根、茎、叶中均有表达,在根中的表达量最高。以上结果表明TaCAMTA2-B可能作为一个感病相关基因在小麦对条锈病互作过程中发挥功能,通过进一步解析小麦TaCAMTAs可能参与的感病机制,为创制广谱持久的小麦抗条锈菌品种提供理论依据。

苦瓜枯萎病是由尖孢镰刀菌苦瓜专化型(Fusarium oxysporum f. sp. momordicae Sun & Huang)侵染引起的一种严重制约苦瓜安全生产的土传病害。明确苦瓜枯萎病菌的致病机理对苦瓜枯萎病的防治具有重要的指导意义,但目前苦瓜枯萎病菌的致病机理尚不明确。前期分析苦瓜枯萎病菌强致病力菌株SD-1和弱致病力菌株SD-V(携带真菌病毒)转录组差异表达基因时,发现Ⅱ型卤酸脱卤酶(FoHAD-typeⅡ)基因在SD-V菌株中表达量显著下调,推测该基因与苦瓜枯萎病菌的致病性相关。本研究根据同源重组的原理,利用分割标记法(Split-Marker PCR)获得了FoHAD-typeⅡ基因的融合片段,分别通过PEG介导的原生质体转化和农杆菌介导的遗传转化获得该基因的敲除突变体和回补突变体,并对敲除和回补突变体的生物学特性和致病力进行了分析。结果表明,敲除突变体的生长速率和产孢量与野生型菌株相比没有显著差异,菌丝尖端形态和孢子形态也无明显差异,但气生菌丝减少,对渗透胁迫耐受性降低,致病力显著下降;回补突变体的生物学性状和致病力与野生型菌株一致。表明FoHAD-typeⅡ基因在苦瓜枯萎病菌的致病过程中发挥重要作用,但不参与苦瓜枯萎病菌生长和发育的调控。研究结果为后续探究苦瓜枯萎病菌的致病机制提供理论基础,同时,为研究真菌病毒的弱毒机理提供参考。

内吞作用在细胞的营养吸收和信号转导过程中起重要作用。在真核生物中Syp1和Ede1是网格蛋白介导内吞的早期蛋白成员。禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重影响小麦产量和品质。禾谷镰刀菌含有酿酒酵母SYP1的一个同源基因FgSYP1。酵母双杂交表明禾谷镰刀菌FgSyp1与FgEde1存在互作关系。利用同源重组的方法得到ΔSyp1突变体和ΔΔSyp1/Ede1双敲突变体,通过生长、产孢和致病性等实验测定禾谷镰刀菌FgSyp1功能。研究表明FgSyp1在禾谷镰刀菌菌丝生长、无性生殖和致病性发挥重要作用,ΔΔSyp1/Ede1双敲突变体在无性和有性生殖方面表现出比ΔSyp1突变体更显著的效果。

小麦赤霉病是以禾谷镰刀菌为优势种的镰刀菌复合种群引起的一种影响小麦产量和粮食安全的重大真菌病害。蔗糖转运蛋白在禾谷镰刀菌中的功能尚不明确。本研究通过与裂殖酵母蔗糖转运蛋白Sut1的氨基酸序列同源比对鉴定到禾谷镰刀菌蔗糖转运蛋白FgSut1和FgSut2,通过基因敲除获得禾谷镰刀菌的Fgsut1、Fgsut2突变体和Fgsut1Fgsut2双敲突变体。研究表明:Fgsut1、Fgsut2突变体和Fgsut1Fgsut2双敲突变体的营养生长、产孢、有性生殖和产毒过程均没有出现缺陷。在葡萄糖为单一碳源的培养基上Fgsut1突变体的生长速率减缓,Fgsut1Fgsut2双敲突变体生长速率下降更显著。Fgsut1Fgsut2双敲突变体在果糖和麦芽糖为碳源的平板上生长速率减缓,说明FgSut1和FgSut2对碳源的利用存在功能的分化和冗余。在小麦穗和小麦胚芽鞘接种实验中,Fgsut2突变体和Fgsut1Fgsut2双敲突变体的致病力降低,说明FgSut2可能在禾谷镰刀菌侵染过程中发挥主要作用, FgSut1和FgSut2在侵染过程存在功能冗余。本研究将禾谷镰刀菌在生长发育过程中对碳源吸收、毒素合成及致病建立联系,丰富了禾谷镰刀菌对碳源利用机制的研究。

黄单胞菌属病原菌能侵染约400种植物,造成严重的经济损失。黄单胞菌的一个重要特征是产生具有保护功能的菌黄素。菌黄素是一类含有两个溴修饰的芳香多烯类磷脂化合物,但负责溴修饰的基因及其溴化机理还不清楚。本研究以野油菜黄单胞菌XC1菌株和稻黄单胞菌PXO99^A菌株菌黄素生物合成基因簇中的xanJ为研究对象,开展了生物信息学、遗传学、生物化学和病理学等方面的探究,初步阐明了xanJ的生物学功能。XanJ含有一个FAD结合结构域或一个色氨酸卤化酶结构域,拥有高度保守基序GXGXXG和WXWXIP,属于一类FAD依赖型卤化酶;xanJ敲除突变体无法合成双溴菌黄素,但仍能合成少量无溴化修饰的菌黄素; xanJ突变体中菌黄素前体3-羟基苯甲酸和4-羟基苯甲酸无积累;在xanJ突变体中过表达已知的酚类卤化酶基因ab10、bmp5、aerJ和mcnD无法恢复双溴菌黄素的生物合成;敲除xanJ不影响病原菌的胞外纤维素酶﹑胞外蛋白酶和胞外多糖的生物合成,也不影响对甘蓝的致病性。因此,XanJ可能负责菌黄素多烯链上ß-碳原子的溴化,并参与菌黄素生物合成过程中的多烯链延伸。

WRKY转录因子是植物中特有的一类转录因子,已有研究表明WRKY group Ⅲ家族众多基因在抵御生物胁迫的过程中发挥着重要的作用。为了解WRKY group Ⅲ家族成员在芜菁根肿病抗性中的潜在功能,利用生物信息学分析方法,鉴定到21个候选WRKY group Ⅲ家族基因,其不均匀分布于芜菁的9条染色体,编码蛋白包含258~916个不等的氨基酸残基,含0~8个不等的motif结构,大部分成员包含按顺序排列的motif 4-5-7/(8-2)-1-3。WRKY group Ⅲ家族各成员启动子区域含有大量光、激素和逆境胁迫响应元件,在激素响应元件中以Me-JA最多。qRT-PCR结果显示WRKY group Ⅲ家族基因可响应根肿病菌诱导,其中BrWRKY46-2、BrWRKY70-2在抗病材料根部表达量显著上调,感病材料中降低;经外源SA诱导后表达量上调,Me-JA诱导后下调,表现出相反的表达模式。亚细胞定位结果显示,BrWRKY46-2和BrWRKY70-2均定位于细胞核中。推测芜菁WRKY group Ⅲ家族基因广泛参与了根肿菌响应过程,且BrWRKY46-2和BrWRKY70-2与芜菁品种抗病和感病密切相关并受SA和JA的强烈诱导,暗示其对根肿病具有重要调控作用,可作为进一步抗病研究和功能分析的候选基因。

MYB基因家族作为植物体内最大的转录因子家族之一,在调节植物生长发育以及抗逆境胁迫方面发挥着至关重要的作用。然而,OsMYBs在水稻抗病毒通路中的作用还尚不清楚。为了探究OsMYBs家族在水稻抗病毒通路中所扮演的角色,对其结构、特征,以及对已报道的单链RNA病毒水稻草状矮化病毒(rice grassy stunt virus, RGSV)和双链RNA病毒水稻锯齿叶矮缩病毒(rice ragged stunt virus, RRSV)侵染后的表达谱进行分析,发现51个响应RGSV侵染,50个响应RRSV侵染。进一步分析发现,26个OsMYBs可同时响应两种不同类型病毒。从中筛选出10个表达量相对较高且差异倍数较大的基因进行实时荧光定量PCR(Real-time Quantitative PCR, qRT-PCR )验证,结果表明,OsMYB30、LOC_Os02g42850、LOC_Os02g47744、LOC_Os05g37050、LOC_Os06g43090和LOC_Os08g33150在响应两种病毒侵染后表达量下调;而LOC_Os05g38460在响应两种病毒侵染后表达量上调。LOC_Os05g51160和LOC_Os09g26170在RGSV侵染后表达量上调,在RRSV侵染后表达量下调;而LOC_Os08g05520在RGSV侵染后表达量下调,在RRSV侵染后表达量上调。这10个基因中有7个在两种病毒侵染后表达量变化趋势相同,表明这些基因可能对病毒响应具有广谱性。这为深入解析OsMYBs在水稻与病毒互作中的功能提供了有用的信息。

“发育快,交配效率高”是松材线虫种群扩张和病害流行的基础。通过生物信息学分析、原位杂交和RNAi等技术,本研究明确了Bxy-egl-30在松材线虫(Bursaphelenchus xylophilus)个体生长发育过程中的功能。生物信息学分析结果表明Bxy-egl-30编码区长度为1 128 bp,编码375个氨基酸,属于EGL-30蛋白亚基家族。原位杂交结果显示Bxy-egl-30基因在松材线虫不同发育阶段的表达具有特异性,在胚胎期与幼虫期该基因广泛表达于性腺、肠道和附近的体壁细胞等部位;在成虫期,该基因在雌虫的阴户肌肉和雄虫的性腺部位集中表达。RT-qPCR结果显示Bxy-egl-30在二龄期表达量最高,胚胎期次之,三龄期、四龄期和成虫期表达量依次降低。浸泡Bxy-egl-30双链 RNA 后,松材线虫胚胎的孵化率下降了10.88%,二龄期、三龄期幼虫活力明显下降,每分钟头摆频率约下降4次。致病性接种实验结果显示松材线虫被Bxy-egl-30 双链RNA干扰后致病性减弱,减缓了黑松的死亡时间,干扰组黑松的死亡时间比对照组晚9 d。本研究明确了Bxy-egl-30基因在松材线虫不同发育阶段的时空动态表达特性和生物学功能,揭示了该基因调控松材线虫早期个体发育和运动等方面的重要作用。

miRNAs对油菜抗核盘菌(Sclerotinia sclerotiorum)的调控作用尚不明确。本研究分析了油菜miR6030在菌核病抗性调控中的功能和机制。结果表明,miR6030在油菜不同组织中表达水平有差异,以根和茎中较高,花中最低。miR6030瞬时超表达和沉默分析以及基因沉默转基因油菜植株分析结果均显示,miR6030负调控油菜对核盘菌的抗性。降解组分析和GUS染色分析显示,miR6030靶标2个产物具有CC-NBS-LRR(CNL)结构的基因BnaCnng64090D和BnaA09g10520D。这2个基因均正调控油菜对核盘菌的抗性。综上,油菜miR6030降解编码CNL蛋白的BnaCnng64090D和BnaA09g10520D基因转录本,从而负调控对菌核病的抗性。该研究结果增进了对miR6030生物学功能以及油菜对核盘菌抗性分子机制的理解,并为油菜抗菌核病分子育种提供新思路。

为了明确抗性基因对番茄黄化曲叶病毒的抗性水平及田间应用效果,本研究利用已报道的番茄黄化曲叶病毒抗性基因Ty-1、Ty-2、Ty-3及Ty-3a的分子标记对43个番茄品种(含供试品种37个,对照品种6个)进行抗性基因型分析,并采用田间自然鉴定方法验证不同抗性基因型的抗性水平。结果表明:43份参试番茄品种中,31个品种携带抗病基因,占全部参试品种的72.09%,其中Ty-1检出率为65.12%,在番茄抗病毒病育种中应用最为广泛;共检出10种抗性基因型,分别包括1~3种抗性基因,对番茄黄化曲叶病毒的抗性由高到低依次为:Ty-1/ty-1+Ty-2/ty-2+Ty-3a/ty-3a、Ty-1/Ty-1+Ty-2/ty-2+Ty-3a/ty-3a、Ty-1/Ty-1、Ty-1/ty-1+Ty-3/ty-3、Ty-1/ty-1+Ty-3a/ty-3a、Ty-1/Ty-1+Ty-3a/ty-3a、Ty-3a/ty-3a、Ty-1/ty-1+Ty-2/ty-2、Ty-1/ty-1、Ty-2/ty-2,其中含有单基因杂合抗性的番茄品种,抗性表现为中抗或感病;含有2个及2个以上抗性基因的品种表现为中抗或以上水平抗性;方差分析显示,携带3个抗性基因位点表现出的聚合抗性显著高于Ty-1、Ty-2单基因杂合抗性,说明抗性基因具有累加效应;抗性分析结果显示:37个供试番茄品种中,筛选出6个高抗品种、9个抗病品种、9个中抗品种、13个感病品种,鉴定圃中TYLCV发病率为58.03%,ToCV发病率为4.67%,ToCV与TYLCV复合侵染后可显著降低品种抗性,暗示TYLCV及ToCV复合侵染对抗性基因有潜在不利影响。本研究结果为抗病基因的应用提供了理论依据。

为进一步了解人参交链孢菌(Alternaria alternata)对氯氟醚菌唑的抗药性风险,本研究通过药剂驯化法获得4株不同抗性水平的突变菌株,通过检测其遗传稳定性、生物学性状以及与其他杀菌剂间的交互抗性来进行A. alternata的抗性风险评估。结果表明高抗突变体适合度较高且能稳定遗传,一旦产生,有成为田间优势菌株的风险。氯氟醚菌唑抗药性突变体对氯氟醚菌唑的抗药性与对嘧菌酯、醚菌酯、苯醚甲环唑、吡唑醚菌酯和己唑醇5种药剂间不存在正交互抗性。A. alternata在药剂选择压力下可形成抗氯氟醚菌唑突变体,存在抗药性风险。本研究结果为吉林省人参黑斑病的防治及氯氟醚菌唑的合理使用提供科学依据。

为了解河南省假禾谷镰孢菌对氟环唑的敏感性,本文采用菌丝生长速率法测定了氟环唑对2019年从河南省16个地市分离的100株假禾谷镰孢菌的毒力。结果表明:氟环唑对供试菌株菌丝生长的EC₅₀值为0.002 4~0.505 4 μg·mL^-1,EC₅₀平均值为(0.109 3±0.095 7)μg·mL^-1;敏感性频率分布图显示,89株供试菌株位于相应的主峰范围内,敏感性频率分布为连续单峰曲线,将其EC₅₀平均值为(0.082 1±0.046 8)μg·mL^-1作为假禾谷镰孢菌对氟环唑的敏感性基线。方差分析显示:不同地市菌株对氟环唑的敏感性差异较小,各地市菌株EC₅₀平均值变化范围为0.050 8~0.296 8 μg·mL^-1;同一地市菌株对氟环唑的敏感性差异较大,商丘虞城最不敏感菌株的EC₅₀值是最敏感菌株的210.58倍。聚类分析表明:河南省假禾谷镰孢菌对氟环唑的敏感性差异与菌株的地理来源无明显关联性。氟环唑对假禾谷镰孢菌的毒力与吡唑醚菌酯、咯菌腈、苯醚甲环唑、多菌灵、氰烯菌酯及戊唑醇毒力之间无明显相关性。氟环唑拌种阻碍小麦生长,对其根长、株高及鲜质量均有抑制作用。温室试验中,12.5%氟环唑悬浮剂有效浓度为150 μL·mL^-1时喷雾防效最高,达到60.18%;离体试验中,治疗性喷雾,不仅有较好的防治效果,且对小麦生长无影响,12.5%氟环唑悬浮剂有效浓度为150 μL·mL^-1时防效最高,达到79.78%。本研究结果为氟环唑应用于小麦茎基腐病防治提供理论依据,为病原菌对药剂的敏感性监测奠定基础。

From 2019 to 2021, a new leaf spot disease was detected on Salvia miltiorrhiza Bge. in Henan province. The leaf spot disease could be found in all 12 investigated fields, and the rate of diseased plants was 20% to 70%. The disease spot was round or oval, gray white in the center, and appeared perforation at later stages which eventually led to leaf wilt by meeting together with multiple spots. The pathogen was isolated and purified by conventional tissue isolation, and was verified according to Koch′s rule. Based on morphological observation and rDNA-ITS, EF-1α and β-tubulin gene sequence analysis, isolate DS1 was identified as Corynespora cassiicola, the causal agent of the new leaf spot disease. This is the first report of leaf spot disease on S. miltiorrhiza caused by C. cassiicola in China, which will provide theoretical basis and technical support for the effective control of the disease in the planting and production of S. miltiorrhiza in the future.

Recently, the needles of Cryptomeria japonica in parks at Fuzhou have suffered from red blight seriously, and this situation is even getting worse. Four diseased needle samples of C. japonica were collected from different parks around. Four Pestalotiopsis strains were obtained after isolation and purification. Based on morphological characters in couple with phylogenetic analyses of sequences of ITS, β-tubulin and tef1, all the strains were identified as Pestalotiopsis hispanica. The results of Koch′s postulates for the represent isolate LS-1 on the needles of healthy C. japonica confirmed P. hispanica as the pathogen of the disease. This is the first report of P. hispanica on C. japonica, even in China.

As a famous medicinal plant, the growth and quality of Curcuma wenyujin can be affected by leaf sheath rot disease, which severely occurs in Dongshan plantations, Haikou city, Hainan province of China in 2017. Based on morphological traits and phylogenetic analyses of multi-loci (ITS, Cox I and Cox II), the pathogen isolated from the diseased plants was identified as Pythium myriotylum. The results provide a theoretical basis for the field control of leaf sheath rot disease on C. wenyujin.

A Ganoderma stem rot disease was found to infect Dimocarpus longana in 2021 in Binqiao town of Guangxi province. In order to clarify the pathogen of this disease, ten isolates with the same morphology were isolated from basidiocarps by using aconventional tissue separation method. LYJF001, a representative strain with excellent growth, was selected for pathogenicity test,morphological identification, sequence analysis of rDNA-ITS and SSU, and phylogenetic tree construction. The results showed that the pathogen causing the stem rot of D. longana was Ganoderma australe.

Bacterial leaf spot of tomato is a worldwide disease, which seriously affects the quality and yield of tomato because of its diverse routes of infestation, rapid spread and difficulty in control. To identify the pathogen of the disease, samples of tomato with leaf spots were collected and prepared for pathogen analysis. Five representative bacterial strains FQ21051401-FQ21051405 strains were isolated and purified from diseased tomato plants. Pathogenicity tests and re-isolation and re-identification of the bacteria were performed to confirm the isolate and fulfill the Koch′ postulates. Based on morphological and biochemical characteristics, phylogenetic analysis, and Koch′s postulates, the bacterial isolates were identified as Erwinia persicina. The aim of this study was to isolate and identify the causal agent of bacterial leaf spot disease of tomato, which will provide a better understanding of pathogenesis of the E. persicina and provided a scientific basis for its control.

In order to clarify the occurrence and distribution of garlic virus in Shandong Province, 382 garlic samples were collected in 2019. PCR was used for virus detection with specific primers. Eleven viruses were detected: leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), garlic common latent virus (GCLV), shallot latent virus (SLV), garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus D (GarV-D), garlic virus E (GarV-E), garlic virus X (GarV-X), milk vetch dwarf virus (MDV)and tobacco mosaic virus (TMV). The co-infection rate of the viruses was 44.24%, and there was co-infection of six kinds of viruses at most. Phylogenetic trees were constructed with CP of LYSV and OYDV. LYSV isolates were closely related to the Chinese, Turkish and Mexican isolates. OYDV isolates were closely related to the Chinese, Indian and Polish isolates.