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.

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.

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.

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.

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.

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.

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.

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.

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。

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。

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。

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.

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。

土霉素对豫杂一号泡桐丛枝病幼苗形态和DNA甲基化水平的影响

The effects of oxytetracycline on the morphology of the seedling with witches’ broom and global DNA methylation level of Paulownia tomentosa × Paulownia fortunei were investigated.The results indicated that the infected seedlings by phytoplasma recovered to normal morphology of the healthy seedlings after being treated with more than 20 mg L-1 oxytetracycline,and the pathogen could not be detected with PCR in the seedlings.The global DNA methylation level of diseased seedlings was lower than that of healthy one.The oxytetracycline treatment was able to increase the DNA methylation level,and the methylation level of the diseased seedlings increased with the dose of applied oxytetracycline.However the increased methylation of infected seedlings with oxytetracycline treatment could still not reach to level of healthy seedling（CK）,even the seedlings treated with lethal oxytetracycline concentration.

植物生长调节物质对泡桐丛枝病株幼苗形态和叶片蛋白质含量变化的影响

利用患丛枝病的豫杂一号泡桐组织培养苗,研究了植物生长调节物质对其幼苗形态和蛋白质变化的影响.结果表明,植物生长素类物质在一定程度上可以减轻豫杂一号泡桐丛枝病发病的症状,但不同种类的生长素对其减轻的程度存在一定的差异.ABA减轻效果甚微.此外,经植物生长调节物质处理的患丛枝病豫杂一号泡桐幼苗叶片蛋白质凝胶电泳中出现了在对照幼苗叶片中观察不到pI 6.3,31 kD的蛋白质(多肽).这意味着植物生长调节物质处理对患丛枝病泡桐苗木的基因表达产生了影响.

泡桐种源抗丛枝病性状的遗传变异

泡桐丛枝病是影响泡桐生长的一种主要病害,种源试验表明在毛泡桐种源中,除甘肃平凉未见发病以外,其它 种源均有病害发生。江苏南京、湖北十堰、黄冈、陕西商县和辽宁大连5个种源发病较重,病情指数超过30％;白花 泡桐丛枝病发病率较低,自然分布区南部的种源很少见到丛枝病发生,分布区北部与毛泡桐分布区有重叠的种源, 丛枝病发病相对较重。毛泡桐起源靠西的种源发病较轻,随着经度的增加,丛枝病发病有增大趋势;白花泡桐发病 与种源经度无关,而与纬度相关明显,呈现出纬度越高,发病率越低,病情指数越小的趋势。泡桐品种间丛枝病发病 率和病情指数差异明显,通过品种选择可以获得抗病优良品种。

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。

泡桐丛枝病病树周围几种植物上植原体的分子检测

【目的】初步明确自然条件下可能感染泡桐丛枝病植原体的寄主植物。【方法】用植原体16S rRNA基因的通用引物,对从泡桐丛枝病病树周围采集的表现黄化、小叶、皱叶、丛枝等症状或无症状的16种植物样品的DNA进行巢式PCR扩增,对所扩增的片段进行序列测定和分析。并利用泡桐从枝病植原体延伸因子的抗血清对部分样品进行间接免疫荧光观察。【结果】巢式PCR结果显示从牛筋草(Eleusine indica)、辣椒(Capsicum annuum)、狗尾草(Setaria viridis)、山药(Dioscorea opposita)、灯笼泡(Physalis angulata)、花生(Arachis hypogaea)及南瓜(Cucurbita moschata)共7种植物样品和阳性对照PaWB菏泽分离物(PaWB-HZ)中均得到了约1.2kb的特异性片段。序列分析表明这7种植原体分离物和PaWB-HZ属于翠菊黄化组的16SrI-D亚组。对所采集的植原体侵染的寄主植株辣椒、山药、花生、南瓜和泡桐进行间接免疫荧光观察结果显示,仅在PaWB-HZ侵染的泡桐中发现翠绿色特异荧光,在健康泡桐及其它4种分离物侵染的植物中均未检测到明显的植原体特异荧光。【结论】首次从泡桐丛枝病病树周围存在的7种其它植物中检测到植原体,并且测定其植原体16S rDNA核苷酸序列与泡桐丛枝植原体相关基因片段高度同源,初步推测这7种植物可能是泡桐丛枝病植原体自然寄主或是通过昆虫偶尔被感染。