转多基因1年生库安托杨对溃疡病的抗性分析

[目的]本研究对转多基因库安托杨株系的溃疡病抗病性检测及抗病相关基因的表达分析,为通过基因工程手段培育抗病林木新树种提供有价值的参考。[方法]以转JERF36等多个外源基因的1年生库安托杨株系D5-9、D5-20、D5-21及非转基因受体D5-0为材料,对主干人工接种溃疡病菌的各株系病情指数进行了测定,同时对接种5 d后树皮组织中5个抗病基因的表达情况进行了实时定量PCR(qPCR)分析。[结果]在溃疡病菌胁迫下,3个转基因株系对溃疡病菌的抗性显著优于非转基因受体株系,转基因株系间抗病性也具有一定差异,D5-21的病情指数显著低于其它2个转基因株系;肉桂醇脱氢酶基因在3个转基因株系树皮中表达量均高于对照,类甜蛋白基因仅在D5-19树皮中的表达量高于对照,其他3个基因在转基因株系中的表达量或与对照相当或低于对照。[结论]转多基因库安托杨株系的溃疡病抗性与非转基因对照相比均有显著提高,且转基因株系间差异显著;同时转基因株系中不同抗病基因的表达模式也有很大差异,说明溃疡病菌胁迫下转多基因库安托杨抗病调控的复杂性,其分子调控机制还有待深入研究。     

转抗菌肽基因毛白杨的培育及其对溃疡病的抗性

以毛白杨为转基因受体材料,利用根癌农杆菌介导法转化来源于益母草的阳离子抗菌肽基因LJAMP2。经卡那霉素筛选,共获得50株抗性植株。GUS组织染色和PCR检测显示有30株抗性植株呈阳性,初步证明外源目的基因已整合到毛白杨基因组中。RT-PCR证实抗菌肽基因LJAMP2在转基因植株中能大量表达。离体抗病性试验表明:转基因毛白杨细胞粗提液的抑菌能力明显强于非转基因植株。进一步将溃疡病菌接种在转基因和野生型毛白杨茎段上培养30天,转基因植株的病级指数均低于非转化植株。上述抗性试验结果表明:在毛白杨中超量表达益母草抗菌肽基因LJAMP2能显著提高其溃疡病抗病性。     

转TSRF1基因尾叶桉的培育及其广谱抗病性

以尾叶桉U6无性系无菌种子苗下胚轴为外植体,通过对不同生长调节剂的优化组合,建立尾叶桉高效再生体系.应用正交试验设计,优化尾叶桉遗传转化体系.经PCR和Southern杂交检测表明,TSRFI基因已整合到尾叶桉基因组中;转基因植株灌根接种青枯菌后,转基因植株表现出发病延迟,抗性提高,抗性相关酶活性显著增强.转化植株离体叶片对疫霉菌抗性明显增强.荧光定量PCR的结果表明转基因植株接种致病菌后TSRF1基因表达量得到提高.研究结果表明,转TSRF1基因尾叶桉表现出一定的广谱抗病能力.     

柿树炭疽病菌接种实验

采用分离自广西桂林市‘恭城水柿’发病枝条、叶片炭疽菌GXGCLMLL-1分别接种9个柿品种的叶片和枝条,比较不同接种方法的发病情况及不同柿品种的抗性。结果表明,有伤接种较为理想,接种方便,发病速度快,病情稳定,易于观察;‘牛心柿’、‘盖柿’对炭疽病抗病性较高,‘尖柿’、‘贡柿’、‘次郎’对炭疽病抗病性较低。     

泡桐炭疽病和楠木白绢病的防治

泡桐炭疽病泡桐炭疽病主要在实生苗上发生,也可侵染根蘖苗和大树上发生丛枝病的部分.病菌侵染嫩茎、叶柄、叶脉和叶片.茎和叶柄上的病斑呈椭圆形,直径1-2毫米到3-5毫米,病斑中央稍坳陷.雨天,病斑内产生粉红色分生孢子盘.叶片上的病斑近圆形,直径1毫米左右,黄褐色,常成穿孔状.发病严重时,病斑连成片,常引起嫩梢干缩枯死,俗称扭耳病.     

过量表达LAR3和Bbchit1以提高毛白杨对叶枯病的抗性

利用转基因技术将多种抗病基因共同转入毛白杨中以提高其抗性,从而获得毛白杨抗病新品种是目前解决杨树真菌病害的主要研究方向之一。本研究通过根癌农杆菌介导的二次遗传转化,将来源于球孢白僵菌几丁质酶基因Bbchit1转入过量表达无色花色素还原酶基因LAR3转基因毛白杨中,实时定量PCR显示Bbchit1与LAR3均能有效表达,离体抗病试验显示Bbchit1+LAR3共表达转基因毛白杨细胞粗提液对杨树叶枯病菌具有明显抑制作用,进一步将叶枯病菌接种在转基因和非转基因毛白杨叶片上培养30天,转基因植株的感病面积均低于非转基因植株且Bbchit1+LAR3共表达转基因株系抗病效果更明显。上述抗病试验结果表明:LAR3和Bbchit1在杨树中共表达可提高其对叶枯病的抗性。     

干旱胁迫对转SacB基因、转BADH基因的美丽胡枝子的影响

以同时培育的转SacB基因、转BADH基因及未转基因的美丽胡枝子盆栽苗为材料,研究干旱条件下3种试材的脯氨酸、甜菜碱、可溶性糖、过氧化物歧化酶、丙二醛、叶绿素含量的变化.结果表明:在正常土壤水分状态下,3种试材的这几项指标含量没有明显差异,但随着干旱胁迫强度的增加,2类转基因的美丽胡枝子所积累的脯氨酸、可溶性总糖量明显高于非转基因植株,在甜菜碱积累方面.转入BADH基因的美丽胡枝子要强于非转基因植株及转入SacB基因的植株.尽管在干旱状态下,3种试材的SOD活性增强,但2种转基因植株的SOD活性并没有明显大于非转基因植株.不论是转入SacB基因,还是转入BADH基因,转基因植株明显可抑制丙二醛在植物体内的快速积累和叶绿素的降解.田间观测的结果也进一步表明:转基因植株的耐旱性明显强于非转基因植株.     

成龄转基因银中杨试验林外源基因水平转移及土壤微生物连年监测

【目的】对成龄转JERF36基因银中杨——抗逆1号杨试验林进行连年生物安全性监测，评价抗逆1号杨可能引起的生态风险，为评估转基因杨树及转基因植物土壤环境安全性提供依据。【方法】以11年生抗逆1号杨、非转基因银中杨对照、林下杂草、林下土壤、试验林旁行道树下杨树实生苗叶片、土壤中筛选出的卡那霉素抗性菌株为材料，采用PCR扩增方法检测外源基因JERF36稳定性及水平转移潜在性，利用稀释平板法对9～11年生抗逆1号杨与非转基因杨对照林地的可培养微生物(细菌、真菌和放线菌)的种群数量进行分析。【结果】外源基因JERF36在抗逆1号杨中稳定存在，林下杂草、林下土壤、试验林旁行道树下杨树实生苗叶片、土壤中筛选出的卡那霉素抗性菌株的DNA样品中均未出现目的基因片段。9～11年生抗逆1号杨与非转基因对照间林下土壤中微生物总数量均无显著差异；不同年份间的抗逆1号杨和非转基因对照的林下土壤微生物数量均略有差异且趋势一致，其中6—8月土壤中微生物总量为9年生>10年生>11年生；9～11年生抗逆1号杨和非转基因对照林地土壤3大类微生物(细菌、真菌和放线菌)数量均无显著差异，各年变化趋势一致，在树木...     

杨树溃疡病接种方法的比较

以五种不同的接种方法，在感病的杨树植株上，接种杨树溃疡病菌（Dothiorella gregaria Sacc.)的试验结果表明，烫伤法和伤皮法均能诱致发病，两者在接种发病率，病斑扩展的速度，范围等方面无显著性差异，后者由于比较接近自然状况，创伤范围，接种菌量相对一致，便于比较。     

水杨酸诱导油茶抗炭疽病的研究

应用水杨酸溶液喷洒油茶叶片可有效诱导植株产生对炭疽病的局部性和系统性抗性.浓度150 mg·L-1诱导抗性效果最好,其对嫩叶的局部诱导病斑减小率为54.15%,对老叶的病斑减小率为61.55%.该浓度的水杨酸溶液喷洒对油茶炭疽病的系统性诱导抗性可使病斑减小率达46.7%;田间试验防治效果达到45.32%.水杨酸诱导油茶产生的局部性抗性可持续20 d而抗性水平无明显下降.扫描电镜显示在诱导处理的油茶叶片上,分生孢子能够萌发,但是芽管的生长受抑制,甚至异常变形卷曲.     

STUDY ON THE HOST BIOLOGICAL INDUCED RESISTANCE OF BACTERIAL CANKER OF POPLAR

ThefirstrePortabouttheprotectiveeffeetofless-aggrssivestrainswerethatSalamaninoculatedtheless-aggrssivcstrainsofI`tfnchingIobaccoontobaccoandIeaditob-tainpartresistance.ANerthen,thcexistofinducedresistancehavebeenrcPortonseveralkindsofplantssuchasPnIms,(*cstnut.Ma-hl.v.,1tcumis.lIlmus.lI'4'5'7.8.9.l().14.l5.16.I7.l8.1'J2lj.21=4.2s.2f'.=7.2x.3()IThei11ducedresistanceofplantsisassocl-atcdt`,iththeincrcaseofthelc17clofthepllellem*tl1eprocesscsofphellemaredcpcndonthcactivelevcloftheperoxidas…     

Research on poplar canker (Xanthomonas populi subsp. populi) in the Netherlands. Part III

A new inoculation method for testing the resistance of poplars to bacterial canker caused by Xanthomonas populi subsp. populi (Ridé) Ridé & Ridé is discussed. The procedure is based on a more natural infection method and involves spraying plants in spring with bacterial suspensions.     

泡桐丛枝病类菌原体(MLO)病原传染长春花研究初报

菟丝子(Cuscuta spp.)是一种寄生植物,利用菟丝子为介体可以将植物病毒或类菌原体(mycoplasma like organism)从一株植物传染到另一株植物上。杨一朗陈景耀分别用大豆菟丝子和南方菟丝子为媒介成功地将甘薯丛枝病从甘薯传到长春花上,产生花器叶化、侧枝丛生病状。     

A severe canker disease of Corymbia ficifolia caused by Quambalaria coyrecup in native and urban forests of Western Australia

A serious canker disease is affecting the health of Corymbia ficifolia in urban areas of Perth, Western Australia. Quambalaria coyrecup was frequently associated with diseased trees and its presence confirmed by morphological characters and DNA sequencing. A number of Quambalaria species have previously been identified as causal agents of canker and shoot blight diseases on a range of Eucalyptus and Corymbia species. It was therefore the aim, using glasshouse studies, to determine whether Q. coyrecup was the primary causal agent of the cankers on C. ficifolia and whether other Quambalaria species could also be associated with the disease. All seedlings inoculated with Q. coyrecup produced canker symptoms within 1 month after inoculation. Canker lesions typical of those observed in the field also occurred in the four subsequent months, after which time the trial was terminated. Inoculation with Q. cyanescens and Q. pitereka (isolated from C. ficifolia shoots) did not result in lesion development. This study establishes that Q. coyrecup is a serious pathogen of C. ficifolia and that wounding is required for canker development to occur. The management of Quambalaria canker must therefore include the minimization of artificial wounding of trees in the nursery and field.     

Characterization of Lasiodiplodia theobromae (Pat.) Griffon & Maubl causing stem canker on Teak (Tectona grandis L.) in Ecuador

Teak is a very important tropical timber in Ecuador. In 2017, teak samples displaying stem canker symptoms were collected in two provinces of Ecuador. From 11 symptomatic trees, 11 isolates resembling a species of Lasiodiplodia were obtained. All isolates obtained induced stem canker on teak plants after artificial inoculation, confirming them as the cause of the observed canker symptoms. Bayesian inference with concatenated sequences of complete ITS and partial TEF-α and β-TUB gene sequences from two representative isolates clustered teak isolates with other sequences of Lasiodiplodia theobromae available in Genbank. This is the first report of L. theobromae causing stem canker on teak plants in Ecuador.     

不同松树感染松材线虫后NO和核酸酶变化与其抗病性的关系

以黑松、马尾松和火炬松为研究对象,对其接种松材线虫后4,12,24,48,72,96和120 h的NO和核酸酶的活性进行测定.3种松树接种松材线虫后,针叶内NO平均含量的积累为火炬松＜马尾松＜黑松,这种差异在12 ～48 h尤为明显;48h后较其割伤处理(CK1)的增加幅度为火炬松＞马尾松＞黑松.同时3种松树接种后的核酸酶变化趋势存在差异,单链核酸酶活性在4～48 h为火炬松＞马尾松＞黑松,而火炬松双链核酸酶活性在4～96h时却一直低于马尾松和黑松,这表明不同松树体内单链或双链核酸酶的活性变化与其抗病性有关.且3种松树在接种松材线虫后,表现出症状的时间顺序为黑松最早,马尾松次之,火炬松最晚.这表明NO和核酸酶的变化与松树感染松材线虫后的病程发展密切相关.     

Infection of Norway spruce container seedlings by Gremmeniella abietina

First‐ and second‐year containerized Norway spruce seedlings were inoculated with conidia of type A (large tree type) and type B (small tree type) of Gremmeniella abietina var. abietina at different times during the summer. The appearance of symptoms after artificial inoculation and natural infection on spruce seedlings were recorded the following spring and compared with the disease symptoms on Scots pine seedlings. The proportion of diseased seedlings after inoculation reached as high as 80%. The susceptible period during the summer began later on the first‐year seedlings than on the second‐year seedlings, and was similar for the pine seedlings. Susceptibility of first‐year seedlings was highest in August and on second‐year seedlings in July. The accumulated temperature sum, relative humidity and height growth for first‐ and second‐year seedlings was assessed. Natural infection in 2002 caused more disease on pine than on spruce seedlings. Experimental thinning of seedlings had no effect on disease incidence. In a preliminary comparison between the ability of A and B types to cause disease in Norway spruce seedlings, type B caused more damage than type A after inoculation. However, type A caused a high disease frequency in other experiments in this study. Symptoms on Norway spruce seedlings often first occurred in the mid‐section of the shoot, and were similar to those observed on pine seedlings: needles turned brown, starting at the needle base, in the spring following inoculation. On first‐year spruce, diseased needles were shed rapidly, in contrast to a slower rate of shedding on first‐year pine seedlings. Pycnidia developed about 2 years after inoculation (on pine 1 year after inoculation). On Norway spruce seedlings the lower part of the shoot, including the lateral shoots, often remained alive. The experiments show that G. abietina can cause disease on containerized Norway spruce seedlings under nursery conditions in Finland. The coincidence of spore dispersal, seedling susceptibility and predisposing factors are important in disease development.     

Susceptibility of adult trees of the endangered species Pinus armandii var. amamiana to pine wilt disease in the field

Adult trees of Pinus armandii var. amamiana (PAAm) and P. thunbergii grown in the field were inoculated with 100000 or 1000 of the nematode Bursaphelenchus xylophilus to evaluate their susceptibility to pine wilt disease. PAAm trees inoculated with 100000 nematodes started to show disease symptoms 2 weeks after inoculation, and all died within 29 weeks. Although the PAAm trees inoculated with 1000 nematodes tended to show delayed disease symptoms compared with those inoculated with 100000 nematodes, all of them died within 33 weeks after inoculation. All P. thunbergii trees inoculated with 1000 nematodes had died 6 weeks after inoculation. In the nematode-inoculated PAAm trees, death of branches distal to the nematode inoculation site was the first visible symptom, followed by the systemic discoloration of needles, whereas the whole tree wilted simultaneously in P. thunbergii trees. In nematode-inoculated PAAm trees, the period from inoculation to death was longer than that in P. thunbergii. These results suggest that adult PAAm trees are susceptible to pine wilt disease, but are less vulnerable than P. thunbergii.