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。     

苹果盘二孢Marssonina coronaria的分离培养研究

苹果盘二孢Marssonina coronaria引起的褐斑病是造成我国苹果树早期落叶的主要原因,由于该病菌分离培养困难,阻碍了对其生物学特性的研究,进而影响了对其防治机理和流行规律的研究.本研究应用4种培养基质,探索了3种方法对苹果盘二孢的分离效果.结果表明,3种方法均可分离到病原菌,但组织块分离法和分生孢子团分离法成功率仅有10%左右,而单孢分离法污染少,成功率高达到90%以上,明显优于其他两种方法.不同培养基上菌落形态、大小和产孢情况差异也很大,培养1个月(25℃)后PDA上菌落黑褐色隆起,表面蚯蚓粪状,无气生菌丝,无子实体和基内菌丝;10%V8培养基上菌落中央隆起,黑褐色,表面生少量气生菌丝,边缘放射状,基内菌丝深褐色,有子实体;苹果叶片葡萄糖琼脂培养基(LDA)上菌落平坦,黄褐色,表面生茂密的金黄色气生菌丝,基内菌丝深褐色,有子实体;苹果叶片煎汁葡萄糖琼脂培养基(LEDA)上菌落有明显的不规则隆起,黄褐色至黑褐色,表面生少许气生菌丝,菌落生长缓慢,无基内菌丝,分生孢子盘菌落表面生,菌落直径仅2mm左右,而在其他培养基上的菌落直径可达6-8mm,说明培养基质、分离方法均对苹果盘二孢的分离培养和生长发育有明显的影响.     

指状青霉Penicillium digitatum侵染柑橘果实的细胞学研究

采用电子显微镜技术系统研究了指状青霉Penicillium digitatum对柑橘果实的侵染过程及超微结构特征。结果表明室温条件下,接种12h后,伤口附近的分生孢子开始萌发产生芽管;然后从伤口处直接侵入果实表皮细胞内;接种24h后,受侵染果实细胞中的菌丝向相邻细胞扩展蔓延,寄主细胞壁开始消解,质壁分离,细胞内含物及各类细胞器凝集,颜色加深,最后完全消解,伤口部位的果皮开始变软,伤口处的菌丝向外生长;84h后伤口处病斑软化,部分长出白色的霉层;96h后病斑软化面积直径达3cm,白色霉层面积逐渐扩大;120h后白色霉层中间伤口处霉层颜色加深变为灰绿色;144h后整个果实变软腐烂。果胶质标记结果表明,菌丝侵入果实后产生果胶酶并降解柑橘细胞壁中的果胶,使得细胞壁松弛,软化腐烂。     

白粉菌BGP(TG)在不同抗性草地早熟禾叶片上的侵染过程观察

【背景】草地早熟禾(Poa pratensis)白粉病是气传性病害,种植抗病品种是防治白粉病最经济、环保和有效的方法。【目的】明确禾布氏白粉菌BGP(TG)在3个不同抗性草地早熟禾叶片上的侵染过程和乳突在草地早熟禾抗白粉病中起的重要角色。【方法】利用考马斯亮蓝染色结合显微镜观察的方法,对不同抗性的3个草地早熟禾品种进行白粉菌侵染过程观察,统计分生孢子形成次生芽管的速度、吸器和乳突的形成情况,分析白粉菌对不同抗性草地早熟禾侵染过程的差异。【结果】‘探险家’草地早熟禾病症较其他2个品种更为严重,禾布氏白粉菌BGP(TG)侵染不同抗性品种的侵染过程基本一致,但‘探险家’叶片上初生吸器形成早于其他2个品种;接菌1-2 d后,在‘太行’叶表面分生孢子形成5级芽管的速度显著低于‘探险家’和‘黑杰克’;接种1d后,‘太行’草地早熟禾有效乳突形成率显著高于‘黑杰克’(P<0.05),‘黑杰克’有效乳突形成率显著高于‘探险家’(P<0.05)。【结论】明确了白粉菌在草地早熟禾叶片上的侵染过程和乳突在草地早熟禾抵御白粉菌侵入过程中起的重要作用,为草地早熟禾白粉病的预防和防治提供了理论基础和参考...     

小麦雪霉叶枯病菌侵染过程的细胞学研究

采用电镜技术研究了小麦雪霉叶枯病菌(Gerlachia nivalis)侵染过程的细胞学特征。电镜观察发现,分生孢子萌发产生的芽管由孢子细胞壁内层延伸而成;病菌侵入寄主体内后,胞间菌丝先在寄主细胞间扩展,随后胞间菌丝侵入坏死的寄主细胞,形成胞内菌丝;胞间菌丝和胞内菌丝在形态结构上无明显差异。在病菌扩展过程中,寄主细胞发生了一系列的病理变化,并最终坏死消解,寄主细胞的变化可能与病菌分泌的毒素有关。     

不同苹果品种对褐斑病抗性的鉴定及抗性生理研究

对42个苹果栽培品种叶片褐斑病进行了田间发病状况调查与抗性鉴定,分析了叶片气孔密度和大小与抗病性之间的关系,同时研究了不同抗性品种离体叶片接种病原菌后超氧化物歧化酶（SOD）、多酚氧化酶（vPo）、过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）活性和木质素含量的差异变化。依照抗性分级标准,供试材料中抗病品种有14个（其中高抗品种2个）,感病品种有28个（其中高感品种7个）;叶片气孔密度与病情指数之间存在显著正相关,相关系数r=0．683;叶片接种后,诱导了4种酶活性和木质素含量的升高,抗病和感病品种的SOD和PP0活性无显著差异,而抗病品种的POD和PAL活性以及木质素含量显著高于感病品种。苹果叶片的气孔密度、POD和PAL的活性以及木质素含量与褐斑病抗性有关。     

球孢白僵菌不同感染方式侵染棉铃虫幼虫的毒性比较及组织病理变化

为明确球孢白僵菌在不同感染方式下对棉铃虫的侵染能力, 采用饲喂法和浸渍法测定了球孢白僵菌HFW-05对棉铃虫Helicoverpa armigera (Hübner) 2龄幼虫的致病力, 并通过组织切片显微技术、 扫描电镜技术观察了球孢白僵菌HFW-05对棉铃虫的致病方式。结果表明: 白僵菌HFW-05可通过消化道（饲喂法）成功侵染2龄棉铃虫, 接种感染6 d后的校正死亡率为75.8%。经由体表（浸渍法）接种白僵菌HFW-05的试虫, 试验中体重的变化和取食量与对照相近（6 d校正死亡率仅为17.3%, 不能通过体表达到致病效果）。组织病理学变化表明: 26±1℃条件下, HFW-05菌株对棉铃虫以消化道侵染为主, 侵染后可导致寄主中肠微绒毛脱落严重, 肠壁组织溶解并最终只剩余底膜; 马氏管变形萎缩, 边缘向外突出隆起, 管径变大; 脂肪体萎缩解体, 结构松散; 表皮下的细胞被菌丝侵染破坏。浸渍法接种的试虫, 切片观察处理6 d后试虫, 体内未发现菌丝, 肠壁组织正常完整。扫描电镜观察, 浸渍法接种的分生孢子未能穿透棉铃虫表皮, 而是贴于寄主表皮表面生长, 在湿度合适的条件下, 菌丝生长到一定时间后断裂成为芽生孢子。白僵菌HFW-05可经由消化道对棉铃虫达到较高的致病效果, 在一定程度上弥补外界环境对白僵菌侵染的不利影响, 对今后应用白僵菌进行生物防治具有重要意义。     

三裂叶豚草锈菌侵染寄主叶片的显微和超微观察

【背景】三裂叶豚草是我国重要的外来入侵植物之一,其传播速度快,已给我国造成巨大的经济损失。近年来发现的三裂叶豚草锈菌是一种对其具有生物防治潜力的病原菌。【方法】本文利用显微技术研究了三裂叶豚草锈菌的侵染过程及其对寄主结构的影响。【结果】三裂叶豚草锈菌菌丝可从多处侵入同一个叶肉细胞,胞间菌丝与叶肉细胞相接触可使部分细胞壁增厚。锈菌侵染使三裂叶豚草叶脉末梢导管分枝增多,造成三裂叶豚草水分代谢失调;叶片细胞内膜系统破碎化,细胞器结构受到不同程度的破坏,导致细胞内膜系统紊乱,细胞器结构稳定性降低。【结论与意义】豚草锈菌侵染破坏了三裂叶豚草叶片的细胞结构。本研究为深入研究豚草锈菌的致病机理奠定了基础。     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不穿透寄主质膜与原生质,而是被其所包围。但随着菌丝进一步扩展,叶片组织发生了一系列的病理变化,其中包括叶肉细胞肿胀、细胞质消解、叶绿体等细胞器解体以及寄主细胞坏死塌陷,并最终在叶表面产生典型的褐斑病症状。     

不同抗性泡核桃对褐斑病病原菌侵染的生理生化响应北大核心CSCD

以抗病和感病泡核桃无性系为实验材料,人工接种褐斑病病原菌后测定不同时期叶片中保护酶活性、总酚、类黄酮、叶绿素含量等相关生理生化指标,探讨不同抗性泡核桃响应褐斑病病原菌侵染的生理生化差异。结果表明:(1)接种病原菌后,感病无性系64叶片带菌率随着侵染时间的增加而升高,且显著高于抗病无性系199(P<0.05)。(2)抗病无性系199和感病无性系64叶片的SOD、POD、CAT、APX和PPO活性随着侵染时间均呈现先升高后降低的变化趋势,其中SOD、POD和APX活性均在16 d时达到最大值;与较感病无性系相比,接种后抗病无性系的POD和APX活性较强;在接种前期(1~16 d),感病无性系PPO活性高于抗病无性系,后期(16~34 d)CAT活性也较抗病无性系高。(3)抗病无性系叶片叶绿素含量始终高于感病无性系;抗病无性系MDA含量在接种后无明显变化,而感病无性系先增加后降低,其细胞膜脂过氧化较重。(4)两个无性系叶片可溶性蛋白和可溶性糖含量变化较平缓,且差异不显著,在接种后期(34 d)有升高的趋势;接种5 d以后,感病无性系叶片类黄酮和总酚含量始终显著高于抗病无性系。研究发现,泡核桃抗病无性系叶片带菌率较低,较难受到侵染,并且通过提高POD和APX活性以及积累较多叶绿素、可溶性蛋白和可溶性糖来应对病原菌侵染引起的氧化胁迫,抑制病原菌的繁殖,从而提高其抗病能力。     

苹果盘二孢的分离培养研究

苹果盘二孢Marssonina coronaria引起的褐斑病是造成我国苹果树早期落叶的主要原因,由于该病菌分离培养困难,阻碍了对其生物学特性的研究,进而影响了对其防治机理和流行规律的研究。本研究应用4种培养基质,探索了3种方法对苹果盘二孢的分离效果。结果表明,3种方法均可分离到病原菌,但组织块分离法和分生孢子团分离法成功率仅有10%左右,而单孢分离法污染少,成功率高达到90%以上,明显优于其他两种方法。不同培养基上菌落形态、大小和产孢情况差异也很大,培养1个月(25℃)后PDA上菌落黑褐色隆起,表面蚯蚓粪状,无气生菌丝,无子实体和基内菌丝;10%V8培养基上菌落中央隆起,黑褐色,表面生少量气生菌丝,边缘放射状,基内菌丝深褐色,有子实体;苹果叶片葡萄糖琼脂培养基(LDA)上菌落平坦,黄褐色,表面生茂密的金黄色气生菌丝,基内菌丝深褐色,有子实体;苹果叶片煎汁葡萄糖琼脂培养基(LEDA)上菌落有明显的不规则隆起,黄褐色至黑褐色,表面生少许气生菌丝,菌落生长缓慢,无基内菌丝,分生孢子盘菌落表面生,菌落直径仅2mm左右,而在其他培养基上的菌落直径可达6-8mm,说明培养基质、分离方法均对苹果盘二孢的分离培养和生长发育有明显的影响。     

气象因素对陕西省苹果褐斑病流行的影响及预测模型

基于对1999-2008年陕西省气温、相对湿度以及陕西省苹果主产区苹果褐斑病发生、流行的监测,对苹果褐斑病发生时间、发病规律及流行程度进行了分析,并构建了旬平均气温(T)和旬平均相对湿度(Hm)影响下苹果褐斑病一维和三维动态预测模型.结果表明：研究区苹果褐斑病受环境因素影响严重,7、8月在田间扩展迅速并引起大量落叶,危害可持续至9月,初霜后不再有新病斑扩展;构建模型所用的T和Hm与田间实际条件基本一致,苹果褐斑病三维动态预测模型为：f(T,Hm)=-0.0172T³+0.9497T²-16.2209T+88.9923-0.00001Hm³+0.00354Hm²-0.15554Hm+2.36578［f(T,Hm)为病情指数］.模型结果表明,田间引起苹果褐斑病发生的T为15 ℃,大流行条件为7、8月T为 23 ℃、Hm在90%以上.     

Serological studies on the accumulation and localisation of three tomato leaf curl geminiviruses in resistant and susceptible Lycopersicon species and tomato cultivars

Accessions of wild Lycopersicon spp. and selected Fl hybrid tomato cultivars were compared for their resistance to three whitefly-transmissible geminiviruses: Indian tomato leaf curl virus (ITmLCV) and tomato yellow leaf curl viruses from Sardinia (TYLCV-Sar) and Senegal (TYLCV-Sen). The resistance of different plant lines was expressed in different ways but in most instances a given line reacted similarly to graft inoculation with the three viruses. L. pimpinellifolium LA1478 produced as much virus antigen, assessed by triple antibody sandwich-ELISA, as the susceptible cv. Moneymaker but developed only very mild symptoms and is therefore tolerant of infection. In L. hirsutum LA1777 and L. peruvianum CMV-INRA, very mild or no symptoms developed but antigen concentrations were substantially less than in Moneymaker. L. chilense LA1969 remained symptomless and its antigen concentration was < 1% of that in Moneymaker. Symptoms were mild or barely evident in the Fl hybrid cultivars. Cultivars Tyking and Fiona had antigen concentrations about 5–10% of those of Moneymaker, whereas TY20, Top 21 and Tyger had intermediate antigen concentrations. In a few instances, the extent to which virus accumulation was restricted depended on the challenge virus. Accumulation of TYLCV-Sen in TY20, Top 21 and Tyger was less affected than that of the other two viruses, and accumulation of TYLCV-Sar in accessions LA1777 and CMV-INRA was less affected than that of TYLCV-Sen or ITmLCV. Tissue-printing tests showed that ITmLCV and TYLCV-Sen antigens were confined to phloem tissue. In Tyking, the number of virus antigen-containing phloem traces and the antigen content of individual traces were less than in Moneymaker but the partitioning of antigen between internal and external phloem was unaffected.     

美洲黑杨抗黑斑病基因的RAPD标记筛选和连锁分析

以美洲黑杨（Populus deltoides Bartr.cv.“Lux”(I-69/55)）为母本,欧美杨（P.euramericana cv.I-45）为父本得到的F1为材料,其中I-69对黑斑病表现为高度抗病,I-45为高度感病。利用分离群体混合分析（bulked segregrants analysis,BSA）技术建立两个DNA池（感病池和抗病池）,筛选出了与抗黑斑病性状基因相连锁的RAPD标记:OPAI17―1550、OPAI13―900。利用选择性基因型分析法进行标记―抗黑斑病基因连锁分析,两标记与抗黑斑病基因遗传距离分别为29.9cM和37.4cM。 Identification of Markers Linked to Resistance Locus of Marssonina Leaf Spot in Poplars by Bulked Segregant Analysis(BSA) ZHANG Bo1,HUANG Min-ren1,ZHUGE Qiang1,HAN Zheng-min1,YIN Tong-ming1,PAN Hui-xin1,ZHU Li-huang2,WU Rong-ling3,WANG ming-xiu１ 1.The Key Laboratory of Forest Tree Genetic Engineering,Nanjing Forestry University,Nanjing,210037,China; 2.The Institute of Genetics and Developmental Biology,Chinese Academy of Science,Beijing 100101,China; 3.Department of Statistics,University of Florida,Gainesville,Florida 32611,USA Abstract:DNA markers linked to resistance locus of Marssonina leaf spot in poplars were found by bulked segregant analysis(BSA).The bulks consisted of individual with a extreme phenotype taken from a population of 91 F1 clones,which is a progeny of Populus deltoides Bartr.cv.“Lux”(I-69/55)(Resistance) and P.euramericana cv.I-45(Susceptible).Out of 114 RAPD primers,four markers showed polymorphisms between the resistance-bulk and the susceptible-bulk.By using selective genotype linkage analysis,OPAI17-1550 and OPAI13-900 were found linked to the resistance locus.The genetic distances between the two markers and the resistance locus were 29.9cM and 37.4cM,respectively. Key words：BSA; RAPD; selective genotype analysis; Marssonina leaf spot     

Influence of bacterial leaf blight on the photosynthetic characteristics of resistant and susceptible rice

The effect of crop disease on photosynthetic characteristics is important for disease control. Two varieties, Shenzhou 98 and Neiwuyou 8015 with resistance and susceptibility to bacterial leaf blight (BLB), respectively, were selected, and the responses of the net photosynthetic rate (P_N) to active photon flux density (PPFD) and intercellular carbon dioxide concentration (C_i), as well as chlorophyll fluorescence, pigments and stomatal resistance (SR), were measured. The results showed that BLB infection greatly decreased the maximum photosynthetic rate (P_max), light saturation point (LSP), carboxylation efficiency (CE), maximal fluorescence (F_m) and actual photochemical efficiency of PSII ( Φ _PSII) but increased the light compensation point (LCP) and dark respiratory rate (R_D), which suggested that the performance of rice photosynthesis was decreased by BLB infection. The BLB infection had a lower effect on resistant rice Shenzhou 98 than on susceptible rice Neiwuyou 8015. The reduction of pigment and increased SR caused by BLB infection may have resulted in the decline in the photosynthetic rate. Significant effects of the BLB infection were observed on chlorophyll fluorescence F_m and Φ_PSII in resistant and susceptible rice. These parameters may be useful for noninvasive monitoring of plant disease considering the negative effect caused by other stresses.     

Molecular interactions between rosy apple aphids, Dysaphis plantaginea, and resistant and susceptible cultivars of its primary host Malus domestica

The use of crop varieties resistant or tolerant to insect pests or other stress factors is one approach in non‐chemical crop‐protection. Knowledge of the biochemical and molecular background of insect–plant interactions is a prerequisite for optimizing breeding for resistance. However, the resistance genes involved in plant–aphid interactions have so far only been identified and characterized in very few plant species. Our work aims to elucidate the molecular and biochemical mechanisms involved in resistance of apple trees, Malus domestica L. (Rosaceae), against its primary aphid pest, the rosy apple aphid, Dysaphis plantaginea (Passerini) (Homoptera: Aphididae), which is considered a serious economic pest of apple. Gene expression in both resistant and susceptible apple cultivars after infestation with rosy apple aphids was investigated by employing the cDNA‐AFLP method (cDNA–Amplified Fragment Length Polymorphism). From approximately 12 500 cDNA fragments detected on polyacrylamide gels, 21 bands were apparently up‐ or down‐regulated only in the resistant cultivar ‘Florina’ after aphid infestation compared to the susceptible cultivar ‘Topaz’ and/or mechanically wounded or non‐infested leaves. These fragments were cloned, sequenced, and the pattern of gene expression for six fragments was subsequently verified by virtual Northern blots. Sequence comparisons of these fragments to GenBank accessions revealed homologies to already known genes, most of them isolated from Arabidopsis thaliana L. Among them, a putative RNase‐L‐inhibitor‐like protein, a pectinacetylesterase, an inositol‐phosphatase‐like protein, a precursor of the large chain of the ribulose‐1,5‐biphosphate‐carboxylase, and defence‐related genes such as a vacuolar H(+)‐ATPase subunit‐like protein and an ADP‐ribosylating enzyme were identified. The results are discussed in relation to a putative role of these genes in conferring aphid resistance in apple trees.