Genome-wide identification and characterization of the PbrATG family in Pyrus bretschneideri and functional analysis of PbrATG1a in response to Botryosphaeria dothidea

The pear is an economic fruit that is widely planted around the world and is loved by people for its rich nutritional value. Autophagy is a self-protection mechanism in eukaryotes, and its occurrence often accompanied by the degradation of damaged substances in cells and the recycling of nutrients. Autophagy is one of the mechanisms through which plants respond to environmental stress and plays an important role in plant development and stress resistance. Functional studies of autophagy-related genes (ATGs) have been performed on a variety of plant species, but little information is available on the ATG family in pear (Pyrus bretschneideri Rehd). Therefore, we analyzed the evolutionary dynamics and performed a genome-wide characterization of the PbrATG gene family. A total of 28 PbrATG members were identified.Phylogenetic analysis showed that PbrATGs were more closely related to ATGs of European pear and apple. Evolutionary analysis revealed that whole-genome duplication (WGD) and dispersed duplication events were the main driving forces of PbrATG family expansion.Expression analysis of different pear tissues showed that all the genes were expressed in different pear tissues, and different PbrATGs are expressed at different times and in different locations. Moreover, all PbrATGs also responded to different abiotic stresses, especially salt and drought stress, which elicited the highest expression levels. Pear seedlings were subsequently infected with Botryosphaeria dothidea (B.dothidea). The results showed that different PbrATGs had different expression patterns at different infection stages. According to the gene expression data, PbrATG1a was selected as a key autophagy gene for further analysis. Silencing of PbrATG1a reduced the resistance of pear to B. dothidea, which resulted in increased lesions, reactive oxygen species (ROS) contents, antioxidant enzyme activity, and gene expression levels in the silenced pear seedlings after B. dothidea inoculation. In this study, a comprehensive bioinformatic analysis of ATGs was conducted, and the functions of PbrATGs in pear development and in response to stress were elucidated, which laid a foundation for further study of the molecular mechanism of autophagy and a new strategy for pear resistance breeding.

茶树病害病原菌Botryosphaeria dothidea生物学特性研究

采用皿内测定方法,研究了河南省信阳市茶树叶部病害病原菌Botryosphaeria dothidea生物学特性。实验结果表明:Botryosphaeria dothidea能正常生长于PDA、OA和MEA培养基上,在MEA培养基上的生长速率较前两者略慢,产孢情况于OA培养基上相对较好。在不同营养因子影响下,病原菌的生长速度不相同,在以葡萄糖为碳素的培养基上菌株生长速度最快,以蛋氨酸作为氮素的培养基中生长速度最快。菌株生长的最适温度为25℃。菌株在液体培养基中可正常生长,菌落呈球形,并伴随新菌落的产生。最适pH值为6,能够适宜微酸环境,当pH值为8时菌株几近停止生长。

Extracellular and intracellular infection of Botryosphaeria dothidea and resistance mechanism in apple cells

Apple ring rot,which is caused by Botryosphaeria dothidea,severely affects apple production.The mechanisms employed in apple cells against B.dothidea remain unknown.In this research,the pathogen infection mode and the relationship between cell death and disease resistance in‘Fuji’/B.dothidea interaction pathosystem were investigated.By using transmission electron microscopy(TEM),our research showed that the pathogen infects apple cells both intracellularly and extracellularly.However,compared with that in immature fruit,the incidence of hyphae in the interior of mature apple fruit cells increased dramatically,suggesting that cell wall-mediated penetration resistance could be important in apple resistance against B.dothidea.TEM ultrastructural characterization identified the nuclear morphology of programmed cell death induction in both apple fruit and callus cells under B.dothidea infection.Overexpression of MdVDAC2(MDP0000271281),which encodes an outer-membrane localized anion channel protein in mitochondria,significantly promoted cell death under B.dothidea infection and simultaneously inhibited pathogen infection,suggesting that cell death represents a disease resistance mechanism in apple against B.dothidea infection.Furthermore,BdCatalase(KAF4307763),a cytochromeP450 family protein BdCYP52A4(KAF4300696),and subtilisin-domain containing proteinswere identified fromB.dothidea-secreted proteins,which suggested the potential involvement of active oxygen species and phytoalexins in combating B.dothidea infection and triggering or dampening apple resistance.Collectively,our research suggested that cell wall-mediated penetration resistance,programmed cell death machinery and microbial effector-interrelated signaling were among strategies recruited in apple to combat B.dothidea.The current research laid the foundation for further investigations into resistance mechanisms in apple.

葡萄座腔菌Botryosphaeria dothidea胞内miRNA及siRNA的鉴定与分析

通过ITS-rDNA检测和形态学典型特征观察确定葡萄座腔菌Botryosphaeria dothidea的遗传背景,并对其胞内小RNA测序.新预测10个miRNAs和298个siRNAs,并分析了预测的miRNAs和siRNAs的序列信息、结构、长度分布、碱基偏好性、表达情况,为在分子水平了解葡萄座腔菌发育和致病机制提供了理论基础.

Botryosphaeria dothidea在苹果果实上的侵染过程(英文)

对Botryosphaeria dothidea通过皮孔侵入果实的过程进行了光学显微镜和扫描电镜观察,发现接种后2d分生孢子完成萌发和附着胞形成过程。接种后10d,菌丝体在皮孔表面缓慢生长。从20￣30d菌丝体加速扩展并形成几个分枝,通常从皮孔外围侵入果实,但仅限于皮孔表面组织。从40～50d,菌丝体扩展更为繁茂,一些菌丝开始突入皮孔的第2层组织。接种皮孔中可检测到多聚半乳糖醛酸酶(PG),但活性比对照高。皮孔有3层,第1层为含果胶的死组织,易被病菌侵染降解,底部的第3层非常坚固,是阻止病菌侵染的有效屏障。随果实发育,皮孔大小、数目和裂口均有规律地增加。我们的研究结果进一步证实了苹果轮纹病的潜伏侵染特性。

杨树接种溃疡病菌Botryosphaeria dothidea后蛋白质表达差异分析

应用双向凝胶电泳技术,分析了抗病毛白杨和感病北京杨接种溃疡病菌Botryosphaeria do-thidea前后的蛋白质表达差异。通过比较两个树种接菌前后的双向凝胶电泳图谱,结果得到在两树种之间共有35个3倍蛋白质差异点,其中接种前毛白杨与北京杨相比上调蛋白4个,下调15个;接种后相比则上调蛋白7个,下调9个。同一树种内,毛白杨接种后与对照相比检测到8个上调蛋白,6个下调蛋白;北京杨接种后与对照相比有8个上调蛋白、10个下调蛋白。对这些差异蛋白产生条件及含量进行分析,笔者认为接菌前抗病毛白杨与感病北京杨相比上调蛋白可能与杨树抗溃疡病的组成型抗病机制有关,接菌后抗病毛白杨与感病北京杨相比上调蛋白可能与杨树对溃疡病的诱导型抗病机制密切相关。最后利用质谱技术鉴定出其中的4个差异蛋白分别是核酮糖二磷酸羧化酶小链、预测蛋白、过氧化物歧化酶、异戊烯二磷酸-异构酶,这些差异蛋白可能与杨树对溃疡病的抗性有一定关系。

葡萄座腔菌(Botryosphaeria dothidea)的有性阶段在我国苹果主产区的发生

【目的】为了明确苹果轮纹病的病原葡萄座腔菌(Botryosphaeria dothidea(Moug.)Ces.et De Not.)的有性生殖阶段在我国是否发生及在我国苹果主产区的发生情况,【方法】2011年10月至2012年11月,对我国山东等8个苹果主产省市的38个果园进行了调查与采样,对田间发现的子囊孢子通过形态观察、ITS序列的比对进行种类鉴定,并对子囊孢子进行了致病性测定。【结果】在北京、山东、河南、河北、陕西、山西和辽宁7个省市的20个果园的干腐型枯枝上发现了葡萄座腔菌的有性阶段,占调查省份的87.5%,占调查果园的52.6%;致病性测定结果显示,枯枝上形成的葡萄座腔菌的子囊孢子不仅可侵染苹果果实引起果腐,而且可侵染苹果枝条引起枝干溃疡。【结论】葡萄座腔菌的有性生殖在我国苹果主产区果园中发生普遍,子囊孢子不仅是葡萄座腔菌的一种越冬方式,也可以成为引起苹果轮纹病发生的初侵染源,在今后的轮纹病防治中,加强对苹果轮纹病田间干腐型枯枝的处理,将有助于提高病害防治效果。

铁载体产生菌筛选、鉴定及防治Botryosphaeria dothidea

葡萄座腔菌属(Botryosphaeria spp.)真菌在全球范围广泛引起多种果树发生溃疡病和流胶病等病症,严重制约种植业的健康发展。作者从蜂蜜中分离出产儿茶酚型铁载体的H47菌株和产异羟肟酸型铁载体的H114菌株,经16S rRNA基因分析和生理生化鉴定,两菌株均为解淀粉芽孢杆菌。菌株用MM9培养基发酵,上清液经有机溶剂萃取、真空干燥获得铁载体的粗品。铁载体粗品对B. dothidea Ces.&De Not ACCC 38026进行平板抑菌实验,结果表明:H47和H114菌株产生的不同类型铁载体粗品对葡萄座腔菌均有抑菌作用,毒力回归方程分别为y=1.432x+0.578和y=1.744x+0.063。由此可见,H47菌株和H114菌株有成为生物防治剂的潜力。

树木溃疡病菌Botryosphaeria dothidea不同菌株致病力分化与产毒强弱的关系

为了研究溃疡病菌致病力分化与其产毒强弱间的关系,以来源于不同地区、不同寄主上的13个葡萄座腔菌Botryosphaeria dothidea菌株及其毒素原液(培养滤液)对北京杨Populus×beijingensis的30cm长枝条的致病程度及毒害程度为评价指标,对供试13个溃疡病菌菌株致病力分化、产毒强弱以及致病力分化与产毒强弱间的关系进行了研究。结果表明:供试13个溃疡病菌菌株致病力分化和其产毒强弱差异均极显著,达到0.01显著水平。聚类分析将13个菌株分成强、中、弱等3种不同的致病类群和强、中、弱等3种不同的产毒类群。不同溃疡病菌菌株产毒强弱与其致病力强弱有明显的正相关性,相关系数为0.854。

First Report of Botryosphaeria dothidea Causing Sweet Osmanthus Leaf Dieback in China

Sweet osmanthus is one of the ten traditional famous flowers in China.The occurrence of the diseases caused by fungi other than Botryosphaeria spp.has been reported mainly from China on sweet osmanthus.A leaf dieback of sweet osmanthus caused by Botryosphaeria sp.was found for the first time in 2007 in Nanning City,Guangxi,China.The objectives of the present study were to isolate and characterize the causal organism of sweet osmanthus leaf dieback.The fungus was isolated from the lesions of affected sweet osmanthus leaves and its pathogenicity to sweet osmanthus was confirmed using a detached-leaf-inoculation method.The identification of the pathogen was carried out mainly based on the morphological characters and molecular analysis of internal transcribed spacer （ITS） sequences.The morphological characters of the pathogenic isolate GHX6 were agreed with that of Botryosphaeria dothidea.The ITS sequence of the isolate was amplified with primers ITS1 and ITS4,and submitted to GenBank （accession no.GQ368251）.Molecular analysis based on ITS sequence comparison between the isolate GHX6 and the other related fungi derived from GenBank supported that the causal agent of the sweet osmanthus leaf dieback belonged to Botryosphaeria dothidea.This is the first report of Botryosphaeria dothidea causing leaf dieback on sweet osmanthus in China.

Involvement of the autophagy-related gene BdATG8 in development and pathogenicity in Botryosphaeria dothidea

Botryosphaeria dothidea is a destructive fungal pathogen that causes Botryosphaeria canker and fruit ring rot on apple worldwide.Autophagy is a process of self-degradation that maintains intracellular homeostasis via lysosomal pathway.To date,the biological role of autophagy in B.dothidea remains unknown.In this study,we identified and characterized the autophagy-related gene BdATG8 in B.dothidea.BdATG8 was able to restore the defect in nitrogen starvation tolerance of Saccharomyces cerevisiae ATG8 deletion mutant.GFP-BdAtg8 was shown to be a useful marker for monitoring autophagy in B.dothidea.Target deletion of Bd ATG8(ΔBd Atg8)blocked autophagy and significantly impaired mycelial growth,conidiation and perithecium formation.In addition,ΔBdAtg8 showed significantly increased sensitivity to phytoalexin and oxidative stress,suggesting that BdATG8 plays critical roles in overcoming phytoalexin and reactive oxygen species(ROS)-mediated plant immunity.Pathogenicity assays revealed thatΔBdAtg8 almost lost ability to infect hosts.Overall,our results indicate that BdATG8 plays an important role in fungal development,stress responses and pathogenesis in B.dothidea.

DES Mutagenesis of Antagonistic Streptomyces of Botryosphaeria dothidea and Optimization of Its Fermentation Conditions

[ Objective] The paper wag to improve the antimicrobial effect of antagonistic Streptomyces on Botryosphaeria dothidea. [ Method ] Different concentrations of DES were adopted for mutagenic treatment of antagonistic Streptomyces F-58. After fermentation, the mutant strain numbered F-58-06 with genetic stability and good antimicrobial effect was selected among 200 mutant strains; the biological activity of the strain was determined with B. dothidea as the indicator fungus, and the best concentration of DES wag 2%. The fermentation conditions of antagonistic substances of Streptomyces strain F-58-06 produced in shake flasks were opti- mized using the method of single factor and orthogonal experimental design. [ Result] The optimal fermentation conditions were carbon source corn flour, nitrogen source KNO3, initial pH 7, loading volume 30 mL in 250 mL shake flask, inoculation volume 3%, incubation time 132 h. Based on orthogonal experiments, the best cultured medium ratio was corn flour 4%, KNO3 0.5%, CaCO3 0.2%, NaCI 0.35%, MgSO4 · 7H2O 0. 15%. [ Conclusion ] After mutation and optimization of culture medium, the antimicrobial activity of Streptomyces F-58 was greatly improved, and the average inhibition zone diameter was 1.35 times of that of the original strain.

Botryosphaeria dothidea营养体亲和性 (VCG_s)的研究(英文)

Botryosphaeriadothidea是重要的树木溃疡病菌。为揭示该菌种内群体遗传分化 ,本研究采用平板对峙培养法和非配对加权算术平均法 ,将我国北方Botryosphaeriadothidea分为 5个VCGs。经Shannon指数计算和T检验 ,表明Botryosphareiadothidea种群为一渐变种群 ,VCGs,VCGs 多样性指数由北向南递减。

不同地区苹果轮纹病菌(Botryosphaeria dothidea)菌株对杀菌剂敏感性的研究

从不同地区采集的苹果轮纹病病果以及大樱桃流胶病枝条木质部中分离纯化得到7株苹果轮纹病菌,用菌丝生长速率法测定了6个不同结构类型的杀菌剂对7株轮纹病菌的毒力。结果显示,所试杀菌剂对7个菌株的毒力由大到小依次是咯菌腈、戊唑醇、甲基硫菌灵、苯醚甲环唑、嘧霉胺和嘧菌酯。菌株不同,对杀菌剂的敏感性不同。所有菌株对咯菌腈、戊唑醇、甲基硫菌灵相对都比较敏感,EC50值均小于1μg/mL,最敏感菌株与耐药性最强的菌株间EC50值分别相差7.5倍、13.67倍、9.14倍。所试菌株对苯醚甲环唑的敏感性稍差,EC50值介于0.51-2.27μg/mL。嘧霉胺和嘧菌酯对所试的7个菌株均表现较低的毒力,不能作为轮纹病菌的防治药剂。仅菌株1010对嘧霉胺的EC50值=0.02μg/mL〈1μg/mL,但其回归方程的b值很小,EC95值达到2.95×10^4μg/mL,也失去实际应用价值。

苹果轮纹病菌(Botryosphaeria dothidea)拮抗细菌的筛选与鉴定

苹果轮纹病是苹果的主要病害之一。为了获得防治苹果轮纹病菌(Botryosphaeria dothidea)的生防细菌,该研究采用平板对峙法对供试的70株细菌进行筛选,初筛结果表明:共有18株供试菌株对苹果轮纹病有不同程度的抑制作用。通过计算抑菌率发现,4株细菌BS-1、B16、B169和1301的抑菌率在50.00%以上,其中B16的抑菌率最高,达到59.30%。经鉴定,BS-1为蜡样芽孢杆菌(Bacillus cereus)、B16、B169为莫海威芽孢杆菌(Bacillus mojavensis)、1301为多黏类芽孢杆菌(Paenibacillus polymyxa)。该研究表明,BS-1、B16、B169和1301对苹果轮纹病的防治具有一定的应用前景。

Botryosphaeria dothidea突变体库的构建及分析

Botryosphaeria dothidea是重要的林果溃疡病害病原,分布广,危害重。本研究应用ATMT(Agrobacterium tumefaciens-mediated transformation)介导的B.dothidea遗传转化体系成功构建了1053个转化子的突变体库,并且通过继代培养、PCR验证和Southern blot证明潮霉素B抗性基因整合到B.dothidea基因组中且可稳定遗传。以B.dothidea sdau11-126为对照,对526株转化子的菌落形态、生长速率和致病性进行分析,筛选获得了8个变异明显且稳定的突变体,以期为B.dothidea致病基因的分离、克隆和功能鉴定奠定基础。

Matrine inhibits mycelia growth of Botryosphaeria dothidea by affecting membrane permeability

Matrine is a promising botanical antifungal;however, the mechanism underlying the antifungal activity is yet limited. We studied the antifungal activity of matrine and the underlying mechanism in Botryosphaeria dothidea as a model strain. Matrine strongly inhibited mycelial growth of B. dothidea in a dose-dependent manner. Matrine-treated B. dothidea showed morphological and ultrastructural alterations, including shriveled hyphae, plasmolysis, and leakage of cytoplasm related to cell membrane deterioration. In addition, matrine caused significantly high conductivity and absorbance (260 nm) in extracellular matrices and low lipid contents in B. dothidea, indicating increased membrane permeability. Lipid peroxidation showed that matrine resulted in increased malondialdehyde content while enhancing the generation of reactive oxygen species and the activities of superoxide dismutase, catalase, and peroxidase. These results showed that matrine inhibited the mycelial growth of B. dothidea by enhancing cell membrane permeability via membrane lipid peroxidation.

不同杀菌剂对猕猴桃软腐病菌(Botryosphaeria dothidea)的防治效果评价

[目的]筛选对猕猴桃软腐病的最佳药剂,探明猕猴桃软腐病的化学防治方法,为猕猴桃软腐病的科学防治提供依据。[方法]通过菌丝生长抑制法测定了8种杀菌剂对猕猴桃软腐病病原菌葡萄座腔菌(Botryosphaeria dothidea)的室内毒力,开展了6种杀菌剂的离体防效评价,选用毒力较强的杀菌剂评价田间和贮藏期的防治效果,并对贮藏期杀菌剂的残留量进行了安全性评价。[结果]室内毒力测定结果表明,97%咪鲜胺对病菌菌丝生长的抑制活性最强,97.5%吡唑醚菌酯最弱,EC_(50)分别为0.1349μg·m L^(-1)、9.9987μg·m L^(-1);在室内离体防效测定中,75%肟菌酯·戊唑醇水分散粒剂WG表现出较好的预防效果,防效达78.15%,而25%咪鲜胺乳油EC却表现出较好的治疗效果,防效达89.38%。田间试验中,75%肟菌酯·戊唑醇WG和42.4%唑醚·氟酰胺悬浮剂SC在田间喷施对软腐病的防治效果较好,防效分别为92.30%和80.90%;贮藏期防效测定中,25%咪鲜胺EC在贮藏前浸果对软腐病的防治效果较好,贮藏90 d后,防效达95.98%,并且农药残留低于最大残留限量(MRL),未出现超标。[结论]综合室内、生长期及贮藏期药效试验结果,在猕猴桃生长期田间防治中可选用肟菌酯·戊唑醇、唑醚·氟酰胺交替使用,此外在贮藏期选用咪鲜胺对果实进行浸果处理有较好的防治效果。

葡萄座腔菌Botryosphaeria dothidea侵染及其对苹果果实影响的组织细胞学研究

利用光镜和电镜技术系统研究了苹果轮纹病菌葡萄座腔菌在成熟果实上的侵染扩展过程及其细胞学特征。扫描电镜观察发现,接种后3h位于皮孔处的分生孢子开始萌发,萌发后的孢子从一端或两端产生芽管直接侵入皮孔细胞,接种后9h完成侵入。30d后果面接种部位表现症状,45d后产生子实体。对接种部位取样进行光镜和透射电镜观察发现,病菌菌丝主要存在于寄主细胞壁、细胞内、细胞间隙及细胞壁与细胞膜之间。菌丝呈丝状,分枝,具隔膜。菌丝细胞内含有细胞核、线粒体、液泡等细胞器;菌丝外散发出一些高电子密度的颗粒物质,这些物质以菌丝为中心,呈放射状分布。病菌在果肉细胞生长扩展过程中,果肉细胞发生一系列变化。果肉细胞壁膨胀、变形,胞间层分离、破裂。与菌丝接触或相邻的果肉细胞细胞壁电子致密度降低,被降解成为如散发状的胞壁纤维束丝。果肉细胞的液泡破裂,质壁分离,细胞质凝结坏死并沉积于细胞壁周围,或通过受损的细胞壁胞间连丝从一个细胞转移到另一个细胞。后期菌丝在表皮下聚集生长、发育成分生孢子器。分生孢子器内壁细胞排列紧密,细胞中含有由数条丝状物平行排列而成的细胞器。该细胞器形状多样,周围总是分布着丰富的脂肪粒,推测可能与营养的运输与积累有关。

葡萄座腔菌(Botryosphaeria dothidea)全基因组分泌蛋白的预测及功能分析

葡萄座腔菌(Botryosphaeria dothidea)是引起多种果树和林木枝干溃疡病和果实腐烂病的重要病原菌。为明确葡萄座腔菌的致病机理,采用SignalP、WoLF PSORT、TMHMM、GPI-SOM等软件,对葡萄座腔菌全基因组14 998条蛋白序列进行了分泌蛋白预测和功能分析。结果表明B.dothidea全基因组编码蛋白中有851条序列具有典型的分泌蛋白特征,占蛋白总数的5.67%,其长度主要分布在100~700个氨基酸范围内。信号肽的分析结果表明,以19个氨基酸为信号肽的分泌蛋白数目最多,非极性氨基酸丙氨酸(A)在信号肽中的使用频率最高,而有带电侧链的天冬氨酸(D)和谷氨酸(E)使用频率最低,信号肽-3和-1位置的氨基酸相对保守,其切割位点属于典型的A-X-A型。对分泌蛋白功能预测结果表明,578个分泌蛋白获得了功能注释,其功能主要涉及碳水化合物的运输、代谢过程,蛋白翻译后修饰和氨基酸代谢、运输过程。分泌蛋白中效应蛋白(effector)预测结果表明,B.dothidea分泌蛋白中共有119个潜在的效应蛋白,其中11个可被PHI数据库注释到,其与引起其他植物病原菌致病力变化的致病效应蛋白具有较高的相似性。碳水化合物活性酶类(CAZymes)的预测结果表明,B.dothidea分泌蛋白组中共有279个CAZymes,其中GHs家族最多。这些结果的获得为今后进一步筛选B.dothidea的效应蛋白,明确B.dothidea的致病机理,以及筛选寄主新的抗性基因提供了必要的基础。