Use of random amplified polymorphic DNA （RAPD） analysis to detect jujube witches＇ broom phytoplasmas

Jujube witches＇ broom is a devastating disease of Ziziphusjujube that occurs in various jujube regions of China. Nucleic acid extracted from midribs of samples collected from three jujube varieties （＂Suanzao＂, ＂Lajiaozao＂ and ＂Langzao＂） from symptomatic and asymptomatic shoots were tested by random amplified polymorphic DNA analyses. Using 13 different 10 and 11-bp random primers the amplification of jujube DNA was achieved from all the samples; AMI4 primer provided amplification of specific DNA fragments of about 400 bp, only from samples collected from symptomatic plants. No genetic variations in these varieties were identified using the other 11 arbitrary primers; only with primer AL07 it was possible to differentiate ＂Langzao＂ from the other two varieties tested. All the experiments were repeated 2 times and the results were consistent. Compared with PCR analyses with phytoplasma-specific primers, RAPD techniques resulted to be an alternative rapid and sensitive method for detecting jujube phytoplasmas presence in different jujube varieties.

Utilization of Changyun Jujube, a Cultivar with Resistance to Witches Broom Disease

In 2021,the Shanting District Fruit Industry Service Center conducted an application test of a treatment for jujube witches broom disease using Changyun jujube.The results demonstrated that when diseased Changhong jujube plants were grafted with Changyun jujube in the spring using bark or cleft grafting,the majority of the new shoots of Changyun jujube exhibited no symptoms of witches broom disease,while a few exhibited symptoms of the disease.With the growth of new shoots,the symptoms of witches broom disease gradually abated,returning to normal growth and development.Similarly,the symptoms of witches broom disease on the rootstock below the grafting mouth also gradually abated,returning to normal.The Changyun jujube rootstock was utilized as the intermediate rootstock to grafting the jujube cultivars Qiyuexian and Fucuimi.The two cultivars were subsequently affixed with branch bark from the witches broom disease.The two cultivars did not exhibit any symptoms of witches broom disease,thus providing an opportunity to investigate potential treatments for this disease in jujube.Finally,the cultivation techniques of the Changyun jujube were presented.

Technical Studies of Controlling Jujube Witches Broom Disease with Resistant Rootstock

[Objectives]The paper was to screen and identify the control effect of resistant rootstock on jujube witches broom disease.[Methods]The seeds or branches of disease-free plants were collected from wild jujube bushes infected by witches broom disease,and 5434 seedlings were obtained by sowing or cutting.The virus was inoculated by grafting diseased bark.After 6 years of preliminary screening,40 plants with resistance to jujube witches broom disease were obtained.Taking these resistant plants as the rootstocks,the susceptible variety‘long red jujube’was grafted at a height of 40 cm.When the plants grew to appropriate thickness,diseased bark was grafted to‘long red jujube’and rootstock to transmit the virus.After 18 years of observation and judgment,4 rootstock strains with high resistance or immunity to jujube witches broom disease were finally determined,namely 90801,90803,90806 and 908011.The susceptible variety‘long red jujube’was grafted to 4 rootstocks and inoculated the virus through diseased bark grafting method.[Results]The resistant rootstock had obvious disease resistance effect.When the branches of 90801 and 90803 rootstocks were grafted to the trunk of diseased trees,the symptoms of jujube witches broom disease gradually alleviated till disappeared.[Conclusions]Using germplasm with high resistance or immunity to jujube witches broom disease as rootstock can effectively prevent the spread of jujube witches broom disease,and bridging the trunk of diseased trees with branches from resistant rootstock has certain therapeutic effect.

Identification and Control of Paulownia fortunei Witches Broom Disease

Pathogen and characteristics,occurrence law,damage symptom of Paulownia fortunei witches broom disease were simply described,and the control method of the disease was summarized.

Spatial pattern of paulownia witches' broom and its effects on the growth of Paulownia elongata in Shaanxi Province,China

The spatial pattern of paulownia witches＇ broom was investigated using an Iwao＇s （m^＊-m） model in Shaanxi Province, China. Iwao＇s （m^＊-m） model revealed that spatial pattern of paulownia witches＇ broom exhibited a uniform distribution in sample plots. In this model, characteristics of contagiousness coefficient （a） justified the probability of infection by virus-transmission vectors. For tree age less than six, susceptible individuals were aggregated as clusters in the plots and then mutually repelled over age. A preliminary survey of stand volume show that from age five to six, stand volumes of disease grade II showed excessive vegetative growth and returned to normal level between ages ten and 11. Loss rate of volume increment with disease grade II decreased, and then returned to raising trend both at grades III and IV. This phenomenon is possibly caused by an over-compensation effect and by environmental factors. The redundancy of growth hormones in branches, shoots, leaves, buds and roots increased the stem volume of entire trees.

Relationship between Witches' Broom Protein and Dynamic of Some Amino Acids in Paulownia Tree Leaves

This paper dealt with the SDS polyacrymide gel electrophoresis of proteins ahd analysis of the amino acids in the diseasedand healthy leaves with the same strain respectively, which were at the same side and height, of Paulownia catalpifolia, P. elongata, P. albiphloea and P. kauakamii respectively. The results indicated that the leaves of 4 species of Paulownia trees with witches' broom had one protein band, of which molecular weight was 12 kD, which did not appear in the healthy leaves free of phytoplasmas. Moreover, the protein quantity in the affected leaves was more than that in the healthy leaves free of phytoplasmas, At the same time, there was significant difference on the amino acids between the healthy and diseased leaves of P. catalpifolia and P. kawakamii. The amount of cystine in the affected leaves was higher than that in the healthy leaves, but the change of amount of phenylalanine in the affected and healthy leaves was contrary.These changes of proteins and amino acids in the leaves might be related to the witches' broom of the Parlounia trees.

叶下珠黄化植原体和丛枝植原体的分子鉴定

2022年8月在海南省文昌市一个槟榔黄化病园内,分别发现叶片黄化和丛枝症状的叶下珠,前期检测均为植原体感染。为了明确叶下珠黄化植原体和丛枝植原体的分类鉴定,本研究通过克隆16S rDNA基因和核糖体蛋白(rp)基因,并进行基因序列一致性、系统发育树及虚拟RFLP等分析。结果显示:克隆获得叶下珠黄化植原体16S rDNA片段1246 bp,rp基因1212 bp;叶下珠丛枝植原体16S rDNA片段1827 bp,rp基因1240 bp。基因序列一致性显示,叶下珠黄化植原体与丛枝植原体的16S rDNA基因序列均与16SrⅠ组的植原体一致性高达98%以上,与槟榔黄化植原体海南株系的16S rDNA序列一致性达100%;而二者的rp基因序列均与rpⅠ组的植原体一致性高达99%以上。系统发育树分析显示,叶下珠黄化植原体和丛枝植原体16S rDNA基因均与翠菊黄化组(16SrⅠ)植原体聚于一个大分支,且与16SrⅠ-B亚组的槟榔黄化植原体聚于同一个小分支,亲缘关系接近;而rp基因均与翠菊黄化组(rpⅠ)植原体集聚于一个大分支,且与rpⅠ-B亚组的翠菊黄化植原体聚于同一个小分支,亲缘关系接近。虚拟RFLP分析显示,叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列虚拟RFLP图谱与16SrⅠ-B的洋葱黄化植原体的参考图谱相同,且相似系数为1.00。综上表明,叶下珠黄化植原体和丛枝植原体均属于16SrⅠ-B亚组成员。研究结果可对采用铲除槟榔黄化病中间寄主的防控手段提供理论依据。

陕北枣区枣疯病、枣裂果病及桃小食心虫发生危害调查

陕北枣区是我国重要的红枣种植区,枣疯病、枣裂果病和桃小食心虫的危害已成为制约当地红枣产业健康发展的主要因素。本研究对陕北枣区“两病一虫”的发生程度及分布特点进行全面调查,旨在为当地制定红枣重大病虫害的预警和综合防控策略提供数据支持。根据陕北枣区红枣的分布特点,调查了延川、清涧、吴堡和佳县4县15个乡镇60个行政村枣园的枣疯病、枣裂果病及桃小食心虫的发生危害情况。结果表明,枣疯病在陕北枣区普遍发生,呈现偏南部发病率高、偏北部发病率低的特点,15个乡镇枣疯病平均发病率在0.17%~33.63%,感病最严重的枣园发病率高达61.00%。枣脆熟期的裂果率在10.33%~77.40%,也呈现枣区偏南部裂果重、偏北部裂果轻的特点。枣裂果率与枣园所处纬度呈负相关,与8月-9月降雨量呈正相关。此外,枣裂果率的高低与枣树品种有关,‘骏枣’和‘团枣’的平均裂果率显著高于‘木枣’;‘骏枣’裂果以纵裂和不规则裂为主,‘团枣’以不规则裂为主、环裂次之,‘木枣’则以不规则裂和纵裂为主。陕北枣区桃小食心虫蛀果率较低,介于0.50%~10.40%,‘团枣’和‘骏枣’的蛀果率显著高于‘木枣’。在陕北枣区枣树病虫害防控中,应以预防和治理枣裂果病和枣疯病为重点,通过防治媒介昆虫叶蝉和加强田间管理延缓枣疯病的发生蔓延,在桃小食心虫发生严重的枣区应采取综合防治措施,降低一代幼虫的蛀果率。

枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达

枣疯病(jujube witches-broom,JWB)俗称枣树的“癌症”,是由枣疯病植原体导致的侵染性病害,给我国的枣栽培与生产造成了严重危害,对枣疯病植原体增殖基因的研究有助于枣疯病的有效防治。为获得枣疯病植原体胸苷激酶基因(tdk),利用PCR方法扩增该基因并测序,结果表明,该基因的开放阅读框长度为576 bp,编码191个氨基酸,其蛋白质分子量为21.76 ku。序列分析和预测表明,该蛋白质为植原体细胞质中性质相对稳定的亲水蛋白质。遗传距离和进化树分析显示,植原体tdk基因比其16S rDNA基因的保守程度低,tdk基因和16S rDNA基因序列的进化树高度相似,表明tdk基因适于植原体分子分类。为获得枣疯病植原体TDK蛋白,成功构建了原核表达载体pET-28a-JWB-Heze-tdk;在诱导条件为0.1 mmol·L^(-1)IPTG、20℃、140 r·min^(-1)时,该原核表达载体在Escherichia coli BL21(DE3)菌株中可表达出大量可溶性的N端融合了6×His标签的JWB-Heze TDK蛋白;利用Ni-IDA琼脂糖树脂纯化出了可溶性的JWB-Heze TDK融合蛋白。研究结果为JWB-Heze TDK体外酶活性测定和抗体制备奠定了基础。

柽柳丛枝植原体鉴定及16S rRNA基因多样性分析

【目的】研究柽柳丛枝病在新疆南疆地区的分布危害、分类、形态特征及病原物16S rRNA基因的遗传多样性,为该病害的检测和鉴定提供理论依据。【方法】调查新疆南疆地区柽柳丛枝病的分布及危害,采用形态学与分子生物学相结合的方法,对柽柳丛枝病病原物进行透射电镜观察、16S rRNA基因和rp基因扩增,研究其形态特征及分类地位,并对新疆南疆不同地区柽柳丛枝病病原物16S rRNA基因遗传多样性。【结果】柽柳丛枝病在新疆南疆地区的平均发病率为9.06%,平均病情指数为5.66;在柽柳丛枝韧皮部组织中观察到有植原体颗粒的存在;16S rRNA基因和rp基因巢式PCR分别获得1219 bp和1174 bp大小的条带,柽柳丛枝病属于植原体病害,在分类地位上属于植原体16SrXXX-A亚组;新疆南疆柽柳丛枝植原体主要可分为4个株系,16S rRNA基因核苷酸相似度在96.5%~99.8%。【结论】柽柳丛枝病在新疆南疆地区主要分布在阿克苏市、温宿县、巴楚县、图木舒克市、阿拉尔市、伽师县,阿拉尔市12团等地,且发生情况和危害程度均较轻,不同地区之间的发病率及病情指数存在显著差异性;柽柳丛枝病存在植原体颗粒并主要在韧皮部组织中,有球形、椭圆形和不规则形态,大小300~600 nm不等。

Transgenic Paulownia Expressing shiva-1 Gene Has Increased Resistance to Paulownia Witches＇ Broom Disease

Stem segments from diseased Paulownia tomentosaXP, fortunei and leaves from healthy control were transformed with the expression vector p438PRSI via Agrobacterium tumefaciens. The p438PRSI vector contained shiva-1 gene, which encodes an antibacterial peptide under the control of a CaMV35S promoter. The regenerated plants from transformed explants were planted in a greenhouse and nursery. PCR and Southern blotting analysis showed that the shiva-1 gene was successfully integrated into the Paulownia genome. Transcription of the integrated shiva-1 gene was confirmed by RT-PCR. Bioassay in the green house and phytoplasma DNA-dot blotting demonstrated that resistance to Paulownia witch＇s broom disease （PWB） increased significantly in shiva-l-transgenic Paulownia. Further investigations indicated that higher Shiva- 1 expression correlated with fewer phytoplasma and less symptoms in diseased transgenic Paulownia. Together, our findings strongly suggest that breeding shiva-1-Paulownia is an effective strategy to control PWB disease.

Identification of the SERK Gene Family in Paulownia fortunei and Its Involvement in the Response to Biotic and Abiotic Stresses

Somatic embryogenesis receptor-like kinases(SERKs)are receptor-like proteins that contain leucine-rich repeats and are involved in various signaling pathways.This study identified SERK family members in the Paulownia fortunei genome and analyzed their characteristics and expression profiles using bioinformatics methods.We identified 12 SERK genes with relatively conserved gene structures and motifs that were distributed unevenly on eight Paulownia chromosomes.The gene promoters contained various cis-acting elements that regulated the expression of the PfSERK genes in response to hormones and abiotic stresses.Synteny analysis indicated that 10 segmental duplication events had occurred during evolution of the PfSERK family.The expression profile of PfSERKs in various tissues of Paulownia fortunei was detected by quantitative real-time PCR(qRT-PCR).Many PfSERK genes can respond to drought and salt stress.Combined with RNA-seq and protein interaction network,it is speculated that PfSERK3/11 may participate in the occurrence of Paulownia witches’broom(PaWB)by regulating the plant height of Paulownia.

枣疯病和酸枣丛枝病植原体16S rDNA和tuf基因的序列同源性分析

【目的】枣疯病是枣树上由植原体引起的一种毁灭性病害,遍布于中国华北、西北、华东、华南等25个省(市)的枣区,造成了巨大的经济损失。【方法】经PCR扩增,分别对中国陕西的彬县、阎良、武功、佳县、杨凌,河北沧州和山东德州7个枣区的枣疯病样品和杨凌4个酸枣丛枝病样品植原体16SrDNA基因保守序列和延伸因子tuf基因进行克隆和测序。【结果】获得枣疯病和酸枣丛枝病植原体的16SrDNA基因片段均为1239bp,tuf基因均为851bp。通过序列同源性比较,结果表明中国陕西、河北、山东的枣疯病的病原一致,归属于植原体16SrⅤ-B组。由于枣疯病和酸枣丛枝病的植原体16SrDNA有5个碱基的差异,tuf基因的同源性为99.6%,推测为同一个种的不同寄主生物学型。【结论】首次报道了中国枣疯病和酸枣丛枝病植原体16SrDNA和延伸因子tuf基因的序列,确定了枣疯病和酸枣丛枝病植原体的分类地位,为研究枣疯病植原体的致病分子机理、遗传本质提供理论依据。

枣疯病病树中内源激素的变化研究

【目的】揭示枣疯植原体对枣树内源激素的影响,弄清枣疯病病树中内源激素的变化趋势。【方法】应用HPLC法分别对健株、病株和盐酸土霉素治疗后转健植株中的细胞分裂素(玉米素,Zeatin)、生长素(吲哚乙酸,IAA)、赤霉素(GA3)和脱落酸(ABA)的含量进亚周年检测。【结果】在根部,健株、治疗株和病株中IAA、GA3和ABA的含量没有明显区别,但在7、8月份病株根部中Zeatin的含量要明显高于健株;在叶部,健株、治疗株和病株中的IAA、GA3和ABA的含量也没有明显区别,但在生长后期病株叶片中Zeatin含量显著高于健株。不同患病程度叶片中激素的比较结果表明,患病程度越重,Zeatin/IAA(C/A)比值越高。【结论】植原体侵染枣树植株后致使其内源激素失衡,主要是细胞分裂素含量的增加,最终导致了枣疯病症状表现。

极抗枣疯病枣新品种‘星光’

‘星光’是从骏枣变异单株中系统选育出的高抗枣疯病新品种。在太行山枣疯病重发区,将其高接到病树上能正常生长结果。‘星光’果实大(22·9g),制干率高(56·4%),既可直接作为抗病品种进行栽培,也可用于染病植株和感病品种的高接改造。

枣疯植原体的分布特点及周年消长规律

Jujube Witches’ Broom (JWB), caused by phytoplasma, is the most serious and destructive disease of Chinese Jujube. The distribution and year_round concentration variation of JWB phytoplasma were studied under fluorescence microscope using DAPI. The results showed that phytoplasma might exist in the sieve tubes of all organs, phytoplasma contents varied with organs, sides of organs and growing seasons, phytoplasma usually existed in the roots of the same direction with diseased branches. The uneven_distribution could be observed much often in lightly diseased trees than in seriously diseased one. In roots, the content of phytoplasma was highest in May, relatively low in June, July and August, and lowest in December to March. In branches, the content of phytoplasma increased gradually with the rising of the temperature after bud sprouting in April and May, then increased dramatically and reached peak in July and August, thereafter decreased in autumn. From December to February, there was still a large amount of phytoplasma in diseased branches. The content of phytoplasma in branches kept higher than in roots throughout a year.

泡桐丛枝病发生相关蛋白质的电泳分析

对毛泡桐和白花泡桐同龄、同方位的病株健叶、病株病叶和健株健叶蛋白质进行了单向和双向SDS聚丙烯酰胺凝胶电泳分析研究。单向电泳结果表明 ,毛泡桐和白花泡桐病株健叶、病株病叶和健株健叶的蛋白质在种类和数量上存在一定的差异 ,其明显的蛋白质凝胶扫描谱带分别有 2 2、2 0和 1 7以及 2 7、2 1和 2 2条 ;双向电泳结果表明 ,毛泡桐与白花泡桐在健株健叶、病株健叶和病株病叶蛋白质变化方面具有一定相似性 ,即在两种泡桐健株健叶和病株健叶中存在的一种pI6.8、MW2 4KD蛋白多肽在病株病叶中观察不到。

枣疯病发病因子和防治技术研究

枣疯病病情与距侧柏林远近、间作物种类、树龄大小及品种等有关。距侧柏林近发病重,发病率达52.2%～90.6%;与小麦、玉米间作发病较重,为14.4%;与花生、红薯、芝麻间作发病轻,为3.6%;树龄小的(20a生以内)发病重,为42.9%,树龄大的(100a生)发病轻,为5.7%;品种之间抗病性和酶活性有显著差异,灰枣、扁核酸为感病品种,发病率为71.0%和70.0%,灵宝枣和九月青抗病,发病率为0.6%和4%,鸡心枣、广洋枣介于中间。田间防治试验证明,用“去丛灵”钻孔施药带和曲颈瓶施药法疗效显著,治愈率12.5%～25%,总有效率为50%～56.3%。采取综合措施和伐除重病株,可预防该病害的扩展蔓延。

抗生素对泡桐丛枝病植原体和发病相关蛋白质的影响

The effects of different concentrations of 5-fluorouracil, rifampicin and tetracycline on the phytoplasmas in the Paulownia tomentosa×Paulownia fortunei seedling and the pathogenic protein related to the witches’ broom symptom were studied with PCR based on 16S rRNA gene of the phytoplasmas and 2-dimensional electrophoresis of proteins. The results indicated that the plantlets in vitro culture treated with 0.5～300 mg·L -1 5-fluorouracil had little effect on the symptom remission, but those plantlets treated with 100 and 150 mg·L -1 rifampicin for 30 d showed obvious effect on the symptom remission respectively, and the phytoplasmas in the plantlets treated with 150 mg·L -1 rifampicin could not be detected with PCR of 16S rDNA of the phytoplasmas. Moreover, the protein (pI6.8,m 24 ku) which appeared in both the healthy seedling and diseases ones treated with 150 mg·L -1 rifampicin disappeared in the infected seedling and the those without the symptom after treating with rifampicin and tetracycline, and in which existed with phytoplasma through PCR detection.