,1,2Effects of Oxytetracycline Treatment on the Control of Sweet Orange Huanglongbing and the Expression of PP2 Gene
YAO TingShan1,2, ZHOU Yan1, DIANN ACHOR3, WANG Nian3, ZHOU ChangYong,1,2通讯作者:
收稿日期:2018-11-23接受日期:2019-01-23网络出版日期:2019-04-01
| 基金资助: |
Received:2018-11-23Accepted:2019-01-23Online:2019-04-01
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姚廷山,Tel：023-68349005;E-mail：
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姚廷山, 周彦, DIANNACHOR, 王年, 周常勇. 土霉素处理对柑橘黄龙病的防治效果及PP2基因表达的影响[J]. 中国农业科学, 2019, 52(7): 1227-1236 doi:10.3864/j.issn.0578-1752.2019.07.010
YAO TingShan, ZHOU Yan, DIANN ACHOR, WANG Nian, ZHOU ChangYong.
0 引言
【研究意义】柑橘黄龙病（Huanglongbing,HLB）是世界柑橘产业的毁灭性传染病害,病原为一种寄生于韧皮部筛管组织的革兰氏阴性细菌,可导致叶片黄化、果实畸形早落、种子败育及根系腐烂,直至树体死亡[1,2,3]。HLB病原菌分为亚洲种Ca. L. asiaaticus（Las）[4]、非洲种 Ca. L. africanus（Laf）[5]和美洲种Ca. L. americanus（Lam）[6,7],其中Las及其传播媒介昆虫亚洲柑橘木虱（ACP,Diaphorina citri）在全球分布最普遍,危害也最严重。迄今为止,HLB无抗病品种且病原仍无法分离培养,导致其有效防控研究进展缓慢,给柑橘产业带来了前所未有的挑战[8]。因此有效抑制植株中黄龙病菌的含量是当前研究的重点内容[9],开展对目前唯一明确能直接杀死HLB病菌的抗生素的效果评价及作用机制具有重要意义。【前人研究进展】研究表明,大量喷施农药杀灭亚洲柑橘木虱及移除HLB病树的联合防控方法花费巨大且效果不明显,尤其地势平坦或飓风、台风等影响地区,无法控制亚洲柑橘木虱的扩散[9]。柑橘抗病育种和亚洲柑橘木虱的遗传转化途径研究为HLB的防控提供了希望,但离田间有效应用还有较大距离[10,11]。抗生素是目前唯一明确能直接杀死HLB病菌的化学药物,在温网室试验中,土霉素（OTC）可高效抑制HLB病菌[12,13],但喷施的OTC很难进入叶片组织,只能通过抑制叶表面的其他细菌来保护植株生长[14],光照和降雨会加速OTC的流失,影响周边环境[15],因此需要建立替代方法来提高OTC的防治效果,同时减少对环境的影响。树干注射是直接将化学物质或营养物质输送到植物体内的目标精确、环境友好的方法,在植物营养供给和病虫害防控中被广泛应用[16,17]。亚洲和南非曾用树干注射杀菌剂进行HLB防控,但因其劳动力及设备费的高昂而推广受限。此外,树干注射OTC后一旦停止用药,次年HLB还会复发[18,19]。近期迫于HLB的严重威胁,美国佛罗里达州已放开对OTC的使用,并开展药剂筛选、施用方法等研究[9,13]。HLB病原菌在柑橘韧皮部繁殖及扩展,因此具有维持植物形态、运转内部物质及愈合伤口等功能的韧皮部蛋白成为研究焦点[20,21]。尤其是韧皮部蛋白中含量最多的一种通过二硫键共价连接的二聚体多聚GlcNAc结合凝集素,即韧皮部蛋白2（phloem protein 2,PP2）[22]。免疫细胞化学定位研究显示,PP2不仅分布于伴胞,在植物筛管分子中也有分布[23,24],由于堵塞韧皮部中的筛管是黄龙病菌致病的主要方式,因此PP2蛋白可能成为HLB防控的关键。【本研究切入点】然而,OTC对HLB发病程度不同柑橘的治疗效果尚未研究,施用OTC与柑橘韧皮部关键基因PP2的关系也未见报道。【拟解决的关键问题】应用qRT-PCR、高效液相色谱（HPLC）等监测植株中Las、OTC和PP2基因的含量变化,评价OTC对不同患病程度柑橘的治疗效果,探索OTC防控HLB的可能作用机制。1 材料与方法
试验于2017年在美国佛罗里达大学柑橘研究与教育中心完成。1.1 供试材料
供试柑橘为4年生Valencia夏橙（Citrus sinensis）,由美国佛罗里达大学柑橘研究与教育中心提供。2017年3月放置于温室中保存。定期浇水并每3个月使用“Citrus gain”肥料（Bougainvillea Growers International,St. James City,FL,USA）提供矿物质营养。药剂：Agro-OTC（FireLine 17WP,AgroSource,Inc.）。
1.2 供试材料分组
根据检测的Las基因组拷贝数进行分组,分为初感染、轻度发病及重度发病3组,每组3株Valencia重复。试验选择Valencia夏橙成熟叶片（1年叶）,使用直径0.9 cm打孔器在植株东西南北中5个方位各选取1片叶打孔取样,Wizard Genomic DNA纯化试剂盒（Promega）提取基因组DNA。纯度及浓度检测后稀释至10 ng?μL-1作为qRT-PCR扩增模板。20 μL qRT-PCR反应体系：2 μL模板,2×mix 10 μL（Qiagen,Valencia,CA）,10 μmol?L-1上/下游引物CQULA04F（5′-TGGAGGTGTAAAAGTTGCCAAA）/CQULA04R（5′-CCAACGAAAAGATCAGATATTCCTCTA）各1 μL[25],10 μmol?L-1 Probe（序列）0.5 μL（Qiagen,Valencia,CA）,ddH2O 5.5 μL[26]。反应程序：95℃ 15 min预变性;94℃ 15 s,60℃ 1 min,40个循环进行扩增。所有qRT-PCR反应在ABI PRISM 7500实时PCR仪中进行（Applied Biosystems,Foster City,CA）。通过质粒制作标准曲线来计算样品中的Las基因组拷贝数[26]。应用软件QuantStudioTM Design&Analysis Softwarev1.4.1进行数据分析。1.3 树干注射OTC处理及Las含量分析
选用Chemjet tree Injector注射器于2017年7月10日在发病程度不同的3组柑橘树上注射Agro-OTC 0.25 g/树（相当于盐酸土霉素活性成分0.1 g/树）,每处理重复3次,每组处理均设清水对照。健康植株与患HLB植株的物候期不同[27,28],选择Valencia夏橙成熟叶片（1年叶）,注射OTC后7、30、60及90 d使用直径0.9 cm打孔器在植株东西南北中5个方位各选取1片叶打孔取样,参考1.2应用qRT-PCR检测柑橘体内Las的含量,并做统计分析。1.4 淀粉含量测定
注射后7、30、60及90 d,从同株树的3个不同方向各选1片成熟叶（1年叶）,打孔得到3个直径0.6 cm的叶盘,重复3次,叶盘混合并用钢珠均质化后,沸水加热10 min,2 500 r/min离心2 min,上清液用于淀粉含量检测。淀粉检测参照I2/KI方法进行,以水稻淀粉（Sigma Aldrich,St. Louis,MO,USA）为标样,加入I2/KI 50 μL使淀粉显色,594 nm处监测淀粉含量[26,29]。1.5 淀粉染色观察（light microscopy,LM）
OTC注射前及注射后90 d,分别采集HLB初感染及HLB严重患病两组植株的成熟叶（1年叶）、嫩叶及茎组织,用洁净刀片将茎和叶片主脉切2 mm×3 mm小块,置于组织固定液（3%戊二醛溶于0.1 mol?L-1 pH 7.2的磷酸钾缓冲液）固定,4℃过夜。经脱水、渗透、包埋及超薄切片后,用光学显微镜观察组织内部结构及淀粉粒分布[30]。1.6 韧皮部PP2基因表达
应用轻度发病Valencia夏橙植株为供试植物,参照RAWAT等[31]对DNA的微阵列（Microarray）差异表达基因的探针信息设计引物,分析Valencia夏橙植株在注射OTC后,韧皮部关键基因PP2的变化情况。探针ID为Cit_35955_1_S1_at_C,引物序列为23F（5′-TCGTTGCCATCAGAAGTATCAC）,23R（5′-CCAACGCAAATAAACTGTCCC）。在Valencia植株树干注射OTC 0.1 g/树后的0、30、60及90 d分别取样,迅速带回实验室中于-80℃冰箱中保存,设置Las含量相当的Valencia清水对照,均3株树重复,每株取东西南北中5张叶片混样。采用试剂盒RNeasy Mini Kit（Qiagen,Valencia,CA）抽提叶片总RNA,TURBO DNA-free kit（Ambion,Austin,TX）去除其中的DNA,经ND-8000 Nanodrop分光光度计（NanoDrop Technologies,Wilmington,DE）测定RNA的浓度和纯度,A260/A280>2.0,浓度>100 μg?μL-1为符合试验要求RNA。取RNA样品产物2 μL反转录为cDNA,用于qRT-PCR对cDNA样品进行基因表达水平检测。候选内参基因选择柑橘甜橙甘油醛-3-磷酸脱氢酶基因（glyceraldehyde-3-phosphate dehydrogenase,GAPDH）（5′-GGAAGGTCAAGATCGGAATCAA/5′- CGTCCCTCTGCAAGATGACTCT）,按ΔCT法分析目标基因的相对表达量及其标准差[32]。运用SPSS 13.0进行单因素方差分析和Duncan检验,采用Prism 7.0进行数据分析及图表绘制。1.7 营养阶段发育参数
不同发病程度的Valencia夏橙组树干注射OTC 0.1 g/树后80 d,即2017年9月30日调查植株新梢和叶片数,记录后用Prism7.0进行数据分析及图表绘制。每个不同发病程度组分别设置清水注射为对照,并且设置健康Valencia植株树干注射OTC 0.1 g/树与注射清水为对照,3次重复。2 结果
2.1 供试材料Valencia植株分组
供试Valencia植株的Las含量差异较大,发病程度也不相同。本研究依据Valencia叶片中的Las含量,将植株分为初感染、轻度发病及重度发病3组,每组3个重复。3组发病程度不同植株各设注射清水对照,每组3个重复。具体为初感染HLB的Valencia植株的叶片Las含量为<9.00×105 cells/g叶片,轻度发病植株的叶片Las含量为9.00×105—9.00×107 cells/g叶片,重度发病植株的叶片Las含量为>9.00×107 cells/g叶片（表1）。Table 1
表1
表1基于植株叶片中Las含量的Valencia分组
Table 1
| 编号 Number | 植株 Plant | Las含量 Las content (cells/g leaf) | 编号 Number | 植株 Plant | Las含量 Las content (cells/g leaf) | 编号 Number | 植株 Plant | Las含量 Las content (cells/g leaf) | ||
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 初感染-T Newly infected-T | 1.58×105 | 7 | 轻度发病-T Mild infected-T | 2.60×106 | 13 | 重度发病-T Severely infected-T | 4.60×108 | ||
| 2 | 1.32×105 | 8 | 1.54×106 | 14 | 3.59×108 | |||||
| 3 | 1.23×105 | 9 | 9.04×105 | 15 | 4.10×108 | |||||
| 平均 Average | 1.38×105 | 平均 Average | 1.68×106 | 平均 Average | 4.10×108 | |||||
| 4 | 初感染-CK Newly infected-CK | 1.01×105 | 10 | 轻度发病-CK Mild infected-CK | 2.10×107 | 16 | 重度发病-CK Severely infected-CK | 4.31×108 | ||
| 5 | 1.21×105 | 11 | 4.59×106 | 17 | 2.60×108 | |||||
| 6 | 9.34×104 | 12 | 9.05×106 | 18 | 7.19×108 | |||||
| 平均 Average | 1.07×105 | 平均 Average | 1.16×106 | 平均 Average | 4.70×108 |
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2.2 树干注射OTC后叶片Las含量变化
注射OTC后病株中Las的含量显著下降,其中对初感染HLB的植株治疗作用最明显,药后7、30、60及90 d检测结果均为阴性（图1-A）。其他两组Valencia夏橙植株在注射后7 d,Las含量均明显减少,药后90 d,轻度发病组的叶片Las含量从（1.68×106±858884）cells/g叶片减至（7.21×104±30981）cells/g叶片,下降幅度极为明显,部分植株在注射药剂后60 d及90 d的检测结果为阴性,但轻度发病对照组Las含量从（1.15×107±8489766）cells/g叶片增至（5.62×107± 2.10×107）cells/g叶片（图1-B）;重度发病组从（4.10× 108±3.04×108）cells/g叶片减至（2.80×107±2.70×107）cells/g叶片,而重度发病对照组从（4.70×108± 2.32×108 ）cells/g叶片增至（9.71×108±4.61×108 ）cells/g叶片（图1-C）。结果表明,注射OTC 0.1 g/株后,可有效降低Las在柑橘体内的含量,初感染HLB植株在3个月内基本可以治愈,轻度发病植株部分可表现为阴性,重度发病植株体内Las含量有效减少。图1
新窗口打开|下载原图ZIP|生成PPT图1患HLB夏橙注射OTC后叶片中Las的含量变化
不同小写字母表示差异显著（P<0.05）。下同
Fig. 1The change of Las content in leaves of Valencia with HLB after OTC trunk injection
Different lowercases indicate significant difference (P<0.05). The same as below
2.3 淀粉含量变化
注射OTC后,HLB发病程度不同的植株淀粉含量均有所下降,初感染HLB植株叶片的淀粉含量在注射后7 d、轻度发病植株在注射后60 d下降最明显。重度发病植株在注射后7 d与30 d下降且叶片内淀粉含量相当,在注射后60 d及90 d降至更低水平,且60 d与90 d时叶片内淀粉含量相当。初感染HLB植株在注射OTC 0.1 g/株后90 d内,叶片内淀粉含量均保持在<2 μg?mm-2;轻度发病植株叶片注射后90 d比60 d略有升高;重度发病植株叶片注射后7 d,叶片内淀粉含量变化明显,但7—90 d淀粉含量变化较小（图2）。图2
新窗口打开|下载原图ZIP|生成PPT图2患HLB夏橙注射OTC后叶片中的淀粉含量变化
Fig. 2The change of starch content in leaves of Valencia with HLB after OTC trunk injection
显微观察表明,夏橙HLB重度发病组的老叶、嫩叶及茎中的淀粉含量及密度皆明显高于初感染HLB的柑橘组,在3种组织中茎观察的淀粉粒沉积量最大,也最密集。注射OTC后,HLB患病严重程度不同的各处理淀粉粒清除作用也不相同。注射OTC对夏橙各组织的影响不同,初感染HLB的嫩叶在注射前后均观察不到淀粉粒的存在;注射前的老叶上有少量淀粉粒沉积,注射后90 d,淀粉粒沉积有所减少但在老叶组织内仍有明显分布;茎中的淀粉粒在注射OTC前后均有较多分布;HLB重度发病植株的老叶、嫩叶及茎中在注射之前均有大量淀粉粒分布,注射OTC后90 d,老叶及茎中的淀粉粒含量有所减少,但是仍有较多淀粉粒分布（图3）。
图3
新窗口打开|下载原图ZIP|生成PPT图3OTC处理前后Valencia组织横截面光学显微照片
A—F：初感染Valencia植株树干注射OTC前后对比图。其中A为处理前成熟叶片;B为处理后90 d成熟叶片;C为处理前嫩叶;D为处理后90 d嫩叶;E为处理前的茎;F为处理后90 d的茎。G—L：重度发病Valencia植株树干注射OTC前后对比图。其中G为处理前成熟叶片;H为处理后90 d成熟叶片;I为处理前嫩叶;J为处理后90 d嫩叶;K为处理前的茎;L为处理后90 d的茎。M—O：健康Valencia植株对照。其中M为健康老熟叶片;N为健康幼嫩叶片;O为健康的茎。非木质化/纤维素细胞壁染色为红色或紫色,胞浆蓝色,淀粉颗粒红黄色。蓝色箭头指示的蓝色点代表韧皮部堵塞,黄色箭头表示的红黄色颗粒代表淀粉。Co：皮层;P：韧皮部;Fi：纤维;Pi：髓;X：木质部
Fig. 3Light micrographs of Valencia tissue cross sections before and after OTC treatment
A-F: Contrast charts of newly infected Valencia before and after OTC injection. Among them, A is the mature leaf before treatment; B is the mature leaf after treatment 90 days; C is the tender leaf before treatment; D is the tender leaf after treatment 90 days; E is the stem before treatment; F is the stem after treatment 90 days. G-L: Contrast charts of severely infected Valencia before and after OTC injection. Among them, G is the mature leaf before treatment; H is the mature leaf after treatment 90 days; I is the tender leaf before treatment; J is the tender leaf after treatment 90 days; K is the stem before treatment; L is the stem after treatment 90 days. M-O: Healthy Valencia plant control. Among them, M is healthy mature leaf; N is healthy young leaf; O is healthy stem. Non-lignified/cellulose cell walls are stained red or purple, cytoplasm blue, and starch granules reddish yellow. The blue spots indicated by blue arrows represent phloem plugging. The reddish yellow granules indicated by yellow arrows represent starch. Co: Cortex; Fi: Fiber; P: Phloem; Pi: Pith; X: Xylem
2.4 嫩梢（叶）抽发
2017年7月10日Valencia夏橙春梢叶片近成熟时注射0.1 g/株OTC,注射后80 d调查秋梢嫩梢和叶抽发情况。结果表明,Valencia夏橙注射OTC 0.1 g/树后,初感染HLB和轻度发病两组嫩梢抽发量显著高于未注射OTC的植株,HLB重度发病注射组的嫩梢抽发多于未注射组,但差异不显著。初感染HLB和轻度发病两组注射OTC后的嫩梢抽发量无显著差异,但显著高于HLB重度发病注射组。嫩叶抽发与嫩梢的分析结果较为相似,但发病重的Valencia夏橙的嫩叶抽发数量会减少。统计分析结果表明,OTC注射HLB轻度发病组的嫩叶抽发数显著少于HLB初感染的OTC注射组,而显著多于HLB重度发病的OTC注射组。值得注意的是,HLB感染程度不同的3组未注射OTC的Valencia夏橙的嫩梢抽发和嫩叶抽发均无显著差异（图4）。图4
新窗口打开|下载原图ZIP|生成PPT图4Valencia夏橙注射OTC后嫩梢（叶）抽发变化
1：初感染植株（处理）Newly infected plant (Treatment);2：初感染植株（对照）Newly infected plant (CK);3：轻度发病植株（处理）Mild infected plant (Treatment);4：轻度发病植株（对照）Mild infected plant (CK);5：重度发病植株（处理）Severely infected plant (Treatment);6：重度发病植株（对照）Severely infected plant (CK);7：健康植株（处理）Healthy plant (Treatment);8：健康植株（对照）Healthy plant (CK)
Fig. 4The change of new shoot (leaf) of Valencia after OTC trunk injection
2.5 韧皮部PP2基因表达
注射OTC 0.1 g/树后30 d,供试Valencia夏橙的PP2基因表达量显著降低。注射后30、60及90 d处理株和健康对照株的PP2基因表达均无显著差异。Valencia夏橙植株注射OTC后,韧皮部关键诱导表达基因PP2的表达量大幅下降,推测注射OTC后显著降低了植株组织内Las的含量及淀粉沉积量,减少了柑橘韧皮部所受的胁迫,引起PP2的表达量减少（图5）。图5
新窗口打开|下载原图ZIP|生成PPT图5患HLB夏橙注射OTC后PP2基因表达变化
Fig. 5The change of PP2 gene expression of Valencia with HLB after OTC trunk injection
3 讨论
柑橘黄龙病（HLB）因其病原菌不能培养,导致防控难度极大。目前,科研工作者已提出激素处理、肥力措施及抗病性育种等方法防控HLB,也尝试通过遗传措施改造其传播媒介亚洲柑橘木虱,这些方法虽然具有解决HLB问题的潜力,但离田间应用还有较大差距[10,26,33]。前人研究表明,单用或混用OTC皆可较好防控HLB[9,34-36],但一旦停止用药,HLB会再次发生。本研究将患有HLB Valencia植株通过qRT-PCR检测体内Las丰度后分为初感染、轻度发病及重度发病3组。3组不同患病程度的Valencia植株分别注射OTC 0.1 g/株后,应用qRT-PCR、淀粉染色观察、淀粉含量测定等技术检测注射后90 d内OTC对患病程度不同的3组夏橙的作用效果,并对韧皮部关键基因PP2表达进行了测定。结果表明,患有不同程度HLB的Valencia夏橙植株在注射OTC 0.1g/株后,体内Las含量均下降明显,淀粉粒含量也有较大下降。初感染HLB的4年生Valencia植株,通过注射0.1 g/株OTC,可以在90 d内基本清除植株体内Las危害;HLB轻度发病植株在注射OTC后,开始时Las的清除作用不明显,60—90 d有显著下降,90 d个别植株基本恢复健康;重度患病的柑橘植株体内Las含量及淀粉粒含量仍保持较高水平,推测这是前期报道的树干注射OTC后HLB会复发的重要原因之一。增加OTC注射次数或注射量,或许可更有效防控HLB,但需监控OTC在植物组织尤其是果实内的残留量,应综合考虑OTC的剂量和靶标树的平衡关系,寻找最佳OTC注射量。Las危害柑橘后,可造成淀粉累积及根系损坏,吸收能力下降,营养缺乏,从而抽梢困难。柑橘抽不出新梢即为生长受到阻挠,进而影响挂果。本研究表明,注射OTC 0.1 g/树于4年生Valencia上,可增加秋梢的发生量,初感染HLB植株在注射OTC 0.1 g/树后的嫩梢抽发量与健康植株相当。后续研究可聚焦于OTC树干注射后对HLB发病植株根系的影响。值得注意的是,通过人工或化学方法杀梢,可减少HLB传播媒介柑橘木虱的产卵场所,是目前国内防控HLB的有效方法之一,可大大减少木虱虫源,从而降低黄龙病的发生。如何来平衡促进新梢抽发及杀梢减少柑橘木虱虫源之间的关系,也是未来研究值得关注的问题。
韧皮部蛋白因Las在韧皮部危害和繁殖而成为研究的重点,存在于筛管分子中并具有ATPase活性,被认为与筛管中的物质运输有关[37]。研究表明,感染HLB的植株在筛板的筛孔中伴随有胼胝质和P蛋白沉积,导致韧皮部堵塞,光合产物运输受阻,叶肉中淀粉过度积累,叶绿素被破坏,最终失绿[38,39]。PP2基因沉默或胼胝质沉积相关基因沉默可能是减缓柑橘黄龙病症状的有效途径。本研究表明,注射OTC后,患HLB植株PP2基因表达量大大减少,而健康植株的表达量基本不变,推测注射OTC后减少了柑橘韧皮部所受的胁迫,该结果可为今后防控机理研究及防治方法改进提供一定的参考。
4 结论
树干注射0.1 g/株土霉素（OTC）可有效治疗4年生Valencia夏橙黄龙病（HLB）初感染植株及部分轻度发病植株,使其检测结果转为阴性;不能使重度发病柑橘植株恢复健康,但可有效减少其体内的Las含量及沉积的淀粉粒含量。树干注射OTC后,可促进患HLB植株嫩梢抽发,减少韧皮部关键基因PP2的表达量,并至少90 d可维持这一水平,表明OTC可有效减少韧皮部病菌等的胁迫压力。（责任编辑 岳梅）
参考文献 原文顺序
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被引期刊影响因子
DOI:10.4454/jpp.v88i1.828URL [本文引用: 1]
A detailed account is given of the history, aetiology, biology, epidemiology, detection, geographical distribution, and control of huanglongbing (HLB), a destructive disease of citrus that represents a major threat to the world citrus industry, and is slowly invading new citrusgrowing areas. HLB, whose name in Chinese means "yellow dragon disease", was first reported from southern China in 1919 and is now known to occur in next to 40 different Asian, African, Oceanian, South and North American countries. The agent is a phloem-restricted, non cultured, Gram-negative bacterium causing crippling diseases denoted "greening" in South Africa, "mottle leaf" in the Philippines, "dieback" in India, "vein phloem degeneration" in Indonesia. The HLB bacterium belongs to the genus Candidatus Liberibacter, three species of which are currently known, Candidatus Liberibacter asiaticus, occurring in Asian countries and, to a lesser extent, in Brazil and the USA (Florida), Candidatus Liberibacter africanus with its subspecies "capensis", recorded from African countries, and Candidatus Liberibacter americanus present in Brazil. The suggestion is that each liberibacter species has evolved in the continent after which it is named. HLB symptoms are virtually the same wherever the disease occurs. Infected trees show a blotchy mottle condition of the leaves that results in the development of yellow shoots, the early and very characteristic symptom of the disease. Trees are stunted, declining and bear a few, small-sized, and deformed (lop-sided) fruits, that are poorly coloured (greening) and with coloration starting at the peduncular end (colour inversion). HLB can be transmitted by grafting from citrus to citrus and by dodder to periwinkle. The psyllids Trioza erytreae and Diaphorina citri are natural vectors. Two different types of HLB are known: the heat-sensitive African form transmitted by T. erytreae, which develops at temperatures of 22-25掳C, and the heat-tolerant Asian form, transmitted by D. citri, which stands temperatures well above 30掳C. Although the HLB pathogen can be identified by electron microscopy, other laboratory methods are used for routine detection. ELISA with monoclonal antibodies is not recommended. Better systems are dot blot hybridization with a DNA probe, and various PCR formats (one-step, nested, multiplex) using species-specific primers based on 16S rRNA or rplKAJL-rpoBC operon sequences. Because no curative methods of HLB are available, control is preventive and largely based on inoculum elimination by removal of infected trees and chemical treatments against vectors. Strict quarantine measures must be implemented to impair further international spread of HLB agents and their vectors.
DOI:10.1094/PHYTO-12-12-0331-RVWURLPMID:23441969 [本文引用: 1]
Abstract Citrus huanglongbing (HLB) is one of the oldest citrus diseases and has been known for over a century. HLB is caused by 'Candidatus Liberibacter' spp. that are phloem-limited, fastidious 脦卤-proteobacteria and infect hosts in different Kingdoms (i.e., Animalia and Plantae). When compared with well-characterized, cultivatable plant-pathogenic Gram-negative bacteria, the interactions of uncultured insect-vectored plant-pathogenic bacteria, including 'Ca. Liberibacter' spp., with their hosts remain poorly understood. 'Ca. Liberibacter' spp. have been known to cause HLB, which has been rapidly spreading worldwide, resulting in dramatic economic losses. HLB presents an unprecedented challenge to citrus production. In this review, we focus on the most recent research on citrus, 'Candidatus Liberibacter asiaticus', and psyllid interactions, specifically considering the following topics: evolutionary relationships among 'Ca. Liberibacter' spp., genetic diversity, host range, genome analysis, transmission, virulence mechanisms, and the ecological importance of HLB. Currently, no efficient management strategy is available to control HLB, although some promising progress has been made. Further studies are needed to understand citrus, 'Ca. L. asiaticus', and psyllid interactions to design innovative management strategies. Although HLB has been problematic for over a century, we can only win the battle against HLB with a coordinated and deliberate effort by the citrus industry, citrus growers, researchers, legislatures, and governments.
DOI:10.1128/JB.00413-13URLPMID:3754606 [本文引用: 1]
Miniature inverted-repeat transposable elements (MITEs) are nonautonomous transposons (devoid of the transposase gene tps) that affect gene functions through insertion/deletion events. No transposon has yet been reported to occur in "Candidatus Liberibacter asiaticus," an alphaproteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease). In this study, two MITEs, MCLas-A and MCLas-B, in "Ca. Liberibacter asiaticus" were detected, and the genome was characterized using 326 isolates collected in China and Florida. MCLas-A had three variants, ranging from 237 to 325 bp, and was inserted into a TTTAGG site of a prophage region. MCLas-A had a pair of 54-bp terminal inverted repeats (TIRs), which contained three tandem repeats of TGGTAACCAC. Both "filled" (with MITE) and "empty" (without MITE) states were detected, suggesting the MITE mobility. The empty sites of all bacterial isolates had TIR tandem repeat remnants (TRR). Frequencies of TRR types varied according to geographical origins. MCLas-B had four variants, ranging from 238 to 250 bp, and was inserted into a TA site of another "Ca. Liberibacter" prophage. The MITE, MCLas-B, had a pair of 23-bp TIRs containing no tandem repeats. No evidence of MCLas-B mobility was found. An identical open reading frame was found upstream of MCLas-A (229 bp) and MCLas-B (232 bp) and was predicted to be a putative tps, suggesting an in cis tps-MITE configuration. MCLas-A and MCLas-B were predominantly copresent in Florida isolates, whereas MCLas-A alone or MCLas-B alone was found in Chinese isolates.
DOI:10.1094/PD-90-1360CURL [本文引用: 1]
Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed.
DOI:10.1111/aab.12149URL [本文引用: 1]
Abstract Candidatus Liberibacter species are Gram-negative bacteria that live as phloem-limited obligate parasites in plants, and are associated with several plant diseases. These bacteria are transmitted by insects called psyllids, or jumping plant lice, which feed on plant phloem sap. Citrus huanglongbing (yellow shoot) or citrus greening disease is associated with three different species of Ca . Liberibacter02–02 Ca . L. asiaticus, Ca . L. africanus and Ca . L. americanus02–02all originally found on different continents. Ca . L. asiaticus is the most severe pathogen, spread by Asian citrus psyllid Diaphorina citri and causing devastating epidemics in several countries. Ca . L. africanus occurs in Africa where it is spread by the African citrus psyllid Trioza erytreae . Ca . Liberibacter solanacearum is associated with diseases in several solanaceous plants, and transmitted by potato psyllid Bactericera cockerelli . Zebra chip disease is causing large damage in potato crops in North America. In Europe Ca . Liberibacter solanacearum is associated with diseases of the Apiaceae family of plants, carrot and celery, and transmitted by psyllids Trioza apicalis and Bactericera trigonica . When Ca . Liberibacter is suspected as the disease agent, the diagnosis is confirmed by DNA-based detection methods. Ca . Liberibacter-associated plant diseases can be controlled by using healthy plant propagation material, eradicating symptomatic plants, and by controlling the psyllid populations spreading the disease.
DOI:10.1094/PDIS-92-11-1547URL [本文引用: 1]
Abstract Huanglongbing (HLB), also known as citrus greening disease. is a devastating, disease of citrus caused by phloem-limited bacteria that have not been grown ill Culture. Three species. 'Candidatus Liberibacter asiaticus. 'Ca. L africanus', and 'Ca. L. americanus', are known, 'Ca L. asiaticus' and its insect vector. the psyllid Diaphorina citri. have been recently introduced into Florida. We attempted to isolate 'Ca. L. asiaticus' using media formulations developed in response to the growth of another bacterium that appears to be related to the liberibacters based on 16S rRNA gene identities. Cultures were obtained that were polymerase chain reaction (PCR) positive for 'Ca. L. asiaticus'. However, transmission electron microscope examination of the Culture. PCR using generic primers, and sequencing of the PCR products revealed the presence of other bacteria in the cultures. These were actinobacteria related to Propionibacterium acnes based on 16S rRNA identities. The co-cultures remained after attempts to purify the Cultures by singe-colony isolation, suggesting that the bacteria might be Mutually beneficial to each other in culture. The co-cultures have survived more than 10 weekly passages to fresh medium. PCR using P acnes-specific primers indicated that actinobacteria are common inhabitants Of Citrus and psyllids. whether or not 'Ca. L. asiaticus' is present.
DOI:10.1016/j.cropro.2012.01.004URL [本文引用: 1]
The use of an enhanced nutritional programs (ENPs) to minimize the deleterious effects of the vector transmitted bacterial disease, citrus huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (Las), has been a topic of considerable discussion and debate since the discovery of HLB in Florida. Most reports of the putative effects of ENPs are either anecdotal or based on non-replicated trials lacking non-treated controls or proper experimental design and analysis with sufficient statistical rigor. Even so, Florida citrus producers use this unproven and non-validated approach for HLB management in lieu of conventional integrated control of inoculum which includes rouging symptomatic trees to reduce inoculum and vector control using insecticide. The formulation of the ENPs varies considerably, but usually consists of foliar applications of standard essential micronutrients, salts of phosphite, and in some programs, salicylate salts. Two field trials were conducted on Valencia sweet orange [Citrus sinensis (L.) Osbeck] to test efficacy of widely used ENPs. The first trial consisting of a randomized complete block design with 3 blocks and 4 replicate trees/block was conducted from 2008 to 2010. All trees were PCR+ for Las at the onset of the trial, but exhibited only mild HLB symptoms. This stage of infection was chosen based on claims that the ENPs maintain the health and productivity of HLB-infected trees, thereby extending the orchard's commercial viability. Combinations of components were compared with a control consisting of a standard fertilization and control program for psyllids. Additional treatments consisted of phosphite with Mn-carbonate, Mn-metalosate, Cu-metalosate, or Zn-metalosate, and injection treatments using soluble copper or silver mixed with a polymer. After two seasons of three applications each, there were no significant differences in bacterial titer dynamics, fruit yield (number of fruit/tree, kg fruit/tree, proportion of fruit dropped), or juice quality (Brix, acid, Brix:acid ratio) between treated trees and non-treated control trees. In a second trial of six commercial citrus blocks containing 40,885 trees wherein enhanced vector control and rouging of diseased trees was practiced, the ENP in three blocks was compared to conventional fertilization in three blocks. In this commercial trial, yields, disease progress, and epidemic dynamics did not differ between the ENP and conventional fertilization treatments. Results of the large commercial trial corroborated the experimental results of the first trial with more diverse micronutrient treatments. Considering both trials together, the ENP did not sustain tree health, yield, or fruit quality of Las-infected HLB-symptomatic trees. Moreover, since the nutritional supplements had no effect on Las titer, a major concern is that existing ENP strategies have promoted area-wide buildup of inoculum and increased disease spread within and between citrus orchards.
DOI:10.1007/s10340-016-0804-1URL [本文引用: 1]
Delottococcus aberiae De Lotto (Hemiptera: Pseudococcidae), a species of mealybug that causes the deformation of fruit, is a new invasive citrus pest in Spain. Here, we examined the potential of five native or naturalized Encyrtid (Hymenoptera) parasitoids—Acerophagus angustifrons (Gahan), Anagyrus sp. near pseudococci (Girault), Cryptanusia comperei (Timberlake), Leptomastix algirica... [Show full abstract]
DOI:10.1094/PHYTO-02-16-0114-RURLPMID:27482624 [本文引用: 4]
Abstract Citrus Huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing 'Candidatus Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days post injection. High levels of OTC (>850 g/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 g/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e. 1.76 and 2.19 log reduction) between 2 and 28 DPI. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.
DOI:10.1653/024.093.0408URL [本文引用: 2]
The Asian citrus psyllid, Diaphorina citri, is a vector of the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus, a pathogen associated with the economically important citrus disease known as huanglongbing. Knowledge of the molecular genetics of D. citri and other insects provides insights into the basic biology of insects. For example, insects can be subjected to stressful conditions and then screened to determine if the conditions promote specific genetic responses. Such information, by identifying critical genetic responses linked to survival, can then be used in the development of genetic tools and in novel management strategies aimed at reducing psyllids populations. In this study, transcriptional responses of D. citri adults against 3 stress factors were investigated: physical wounding, heat stress, and exposure to low doses of the insecticide imidacloprid. No measureable transcriptional activity was observed for genes (cyp, gst, CuZn-SOD, hsc70, or hsp90), which, in other insects, have been shown to respond to either physical wounding, heat stress, or exposure to insecticides. However, increased transcriptional activity of a heat-shock gene, hsp70, was found in adult psyllids exposed to 42°C, although 6 h of exposure to this temperature was lethal to psyllids. These results suggest that hsp70 may play a role in response to heat stress of D. citri. Summer temperatures can exceed 37°C in Florida, Texas and California areas where the psyllids now occurs. Natural temperature fluctuations and gradual increases provide enough time for psyllids to acclimate to hot summer temperatures. We propose that the development of a method to disrupt gene expression, such as hsp70, may be applicable for future strategies to suppress psyllids populations. El sílido asiático de los cítricos, Diaphorina citri, es un vector de la bacteria, Candidatus Liberibacter asiaticus que habita el floema y que es un patógeno asociado con una enfermedad de importancia económica conocida como huanglongbing. El conocimiento de la genética molecular de D. citri y otros insectos provee una perspectiva a la biología básica de insectos. Por ejemplo, los insectos pueden ser sometidos a condiciones de estrés y luego evaluados para determinar si estas condiciones promueven una respuesta genética específica. Dicha información, que identifica respuestas genéticas críticas que estan relacionadas a la sobrevivencia, puede ser usada en el desarrollo de herramientas genéticas y estrategias novedosas de manejo dirigidas a reducir poblaciones de sílidos. Investigamos las respuestas transcripcionales de los adultos de D. citri contra 3 factores de estrés: heridas físicas, estrés de calor, y exposición a las dosis bajas del insecticida imidacloprid. No actividad transcripcional medible fue observada en los genes (cyp, gst, CuZn-SOD, hsc70, o hsp90) que han muestrado respuestas a heridas físicas, estrés de calor, o exposición a las dosis bajas de insecticidas en otros insectos. Sin embargo, un aumento en la actividad transcripcional del gene al choque de calor, hsp70, fue encontrado en adultos de sílidos expuestos a 42°C, aunque la exposición de 6 horas a esta temperatura fue letal para los sílidos. Estos resultados indican que que el gene hsp70 puede jugar un papel en la respuesta de D. citri al estrés de calor. Las temperaturas de verano pueden ser mas de 37°C en areas de la Florida, Texas, y California donde el sílido ahora ocurre. Fluctaciones naturales y aumentos graduales de temperatura provee suficiente tiempo para que los sílidos se aclimaten a las temperaturas calorosas de verano. Proponemos que el desarrollo de un método para perturbar la expressión genética, como en el gene hsp70, puede ser aplicable a estrategias para suprimir poblaciones de sílidos en un futuro.
[本文引用: 1]
DOI:10.1094/PHYTO-09-11-0265URLPMID:22568814 [本文引用: 1]
Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.
[本文引用: 2]
DOI:10.1094/PDIS-04-10-0282URL [本文引用: 1]
Oxytetracycline (OTC), a member of the tetracycline antibiotics, is used as a foliar spray to control Xanthomonas arboricola pv. pruni on stone fruits and Erwinia amylovora on pome fruits. We studied the dynamics of OTC residues on attached peach (Prunus persica) leaves treated with 300 ppm active ingredient of an agricultural OTC in relation to temperature, natural sunlight, and simulated rain...
[D].
[本文引用: 1]
DOI:10.1007/BF02277580URL [本文引用: 1]
X. pruni was effectively controlled in the cv. Blenril by a single trunk infusion of oxytetracycline (OTC). In Sept. 1975 6-yr-old trees, severely defoliated from X. pruni infection, were injected with an aqueous solution containing 0.6, 0.8 or 1 g a.i. OTC/tree through 3 holes (7 mm diam.) drilled 5 cm deep at about a 45 deg angle in a spiral around the trunk base. All concs. gave disease cont...
DOI:10.3389/fpls.2015.00016URLPMID:25717330 [本文引用: 1]
Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1-2 apple tree injections of either streptomycin, potassium phosphites (PH) or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water- or non-injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2 and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control.
DOI:10.3969/j.issn.0529-1542.2014.02.033URL [本文引用: 1]
为筛选有效防治罹病柑橘植株体内黄龙病菌的药剂,选取6种药剂进行韧皮部输液,利用实时定量PCR对病株体内带菌量进行动态监测以确定相对防效.结果表明,连续施药8个月后,硫酸铜、布罗波尔处理组和对照组带菌量下降了20％～30％,2,2-二溴-3-次氮基丙酰胺、异噻唑啉酮以及氨苄青霉素带菌量减少了80％以上;ZnSO4处理带菌量下降了65.60％.各观察期内相对防效都超过50％的药剂有2,2-二溴-3-次氮基丙酰胺(≥50％)和氨苄青霉素(≥70％).相对防效分析表明,施药后8个月1 g/L 2,2-二溴-3-次氮基丙酰胺、异噻唑啉酮、氨苄青霉素相对防效分别达到了89.75％、83.72％和87.27％,初步判断这3种药剂对黄龙病病树有防控效果.
DOI:10.3969/j.issn.0529-1542.2014.02.033URL [本文引用: 1]
为筛选有效防治罹病柑橘植株体内黄龙病菌的药剂,选取6种药剂进行韧皮部输液,利用实时定量PCR对病株体内带菌量进行动态监测以确定相对防效.结果表明,连续施药8个月后,硫酸铜、布罗波尔处理组和对照组带菌量下降了20％～30％,2,2-二溴-3-次氮基丙酰胺、异噻唑啉酮以及氨苄青霉素带菌量减少了80％以上;ZnSO4处理带菌量下降了65.60％.各观察期内相对防效都超过50％的药剂有2,2-二溴-3-次氮基丙酰胺(≥50％)和氨苄青霉素(≥70％).相对防效分析表明,施药后8个月1 g/L 2,2-二溴-3-次氮基丙酰胺、异噻唑啉酮、氨苄青霉素相对防效分别达到了89.75％、83.72％和87.27％,初步判断这3种药剂对黄龙病病树有防控效果.
URL [本文引用: 1]
Citrus yellow shoot (CYS, Huanglongbing) is a highly destructive disease of citrus in South China. Yellow shoots and leaf mottling are the characteristic symptoms of the disease. The bud transmissibility and the viruslike nature of the disease were established in 1956. In 1975-79, electron microscope studies revealed that a pleomorphic organism with a unit membrane of thickness about 20 nm was ...
[本文引用: 1]
DOI:10.1105/tpc.105.031419URLPMID:15863519 [本文引用: 1]
The phloem macromolecular transport system plays a pivotal role in plant growth and development. However, little information is available regarding whether the long-distance trafficking of macromolecules is a controlled process or passive movement. Here, we demonstrate the destination-selective long-distance trafficking of phloem proteins. Direct introduction, into rice (Oryza sativa), of phloem proteins from pumpkin (Cucurbita maxima) was used to screen for the capacity of specific proteins to move long distance in rice sieve tubes. In our system, shoot-ward translocation appeared to be passively carried by bulk flow. By contrast, root-ward movement of the phloem RNA binding proteins 16-kD C. maxima phloem protein 1 (CmPP16-1) and CmPP16-2 was selectively controlled. When CmPP16 proteins were purified, the root-ward movement of CmPP16-1 became inefficient, suggesting the presence of pumpkin phloem factors that are responsible for determining protein destination. Gel-filtration chromatography and immunoprecipitation showed that CmPP16-1 formed a complex with other phloem sap proteins. These interacting proteins positively regulated the root-ward movement of CmPP16-1. The same proteins interacted with CmPP16-2 as well and did not positively regulate its root-ward movement. Our data demonstrate that, in addition to passive bulk flow transport, a destination-selective process is involved in long-distance movement control, and the selective movement is regulated by protein-protein interaction in the phloem sap.
DOI:10.1186/1471-2148-10-79URLPMID:20236552 [本文引用: 1]
Background Lectins are a class of carbohydrate-binding proteins. They play roles in various biological processes. However, little is known about their evolutionary history and their functions in plant stress regulation. The availability of full genome sequences from various plant species makes it possible to perform a whole-genome exploration for further understanding their biological functions. Results Higher plant genomes encode large numbers of lectin proteins. Based on their domain structures and phylogenetic analyses, a new classification system has been proposed. In this system, 12 different families have been classified and four of them consist of recently identified plant lectin members. Further analyses show that some of lectin families exhibit species-specific expansion and rapid birth-and-death evolution. Tandem and segmental duplications have been regarded as the major mechanisms to drive lectin expansion although retrogenes also significantly contributed to the birth of new lectin genes in soybean and rice. Evidence shows that lectin genes have been involved in biotic/abiotic stress regulations and tandem/segmental duplications may be regarded as drivers for plants to adapt various environmental stresses through duplication followed by expression divergence. Each member of this gene superfamily may play specialized roles in a specific stress condition and function as a regulator of various environmental factors such as cold, drought and high salinity as well as biotic stresses. Conclusions Our studies provide a new outline of the plant lectin gene superfamily and advance the understanding of plant lectin genes in lineage-specific expansion and their functions in biotic/abiotic stress-related developmental processes.
DOI:10.1104/pp.100.3.1433URL [本文引用: 1]
DOI:10.1007/BF00028856URLPMID:8000002 [本文引用: 1]
Abstract The phloem of pumpkin and squash contains a dimeric chitin-binding lectin called PP2 (phloem protein 2). We have isolated three genomic clones from pumpkin (Cucurbita maxima Duch.) that encoded PP2. One clone, lambda gPC13-1, contained two PP2 genes that were 99.8% identical over a region of 3055 nucleotides. This conserved region included 1922 bp of 5' non-coding sequence, 844 bp of protein coding sequence (including two introns), and 289 bp of 3' non-coding sequence. To examine the conservation of the phloem lectin within the genus Cucurbita, we analyzed nine different species for PP2, its mRNA, and the genes that encode PP2. DNA blot analysis indicated that each species contained genes that encoded PP2, however, there was considerable restriction fragment length polymorphism (RFLP) among the species. PP2 gene copy number reconstructions indicated that PP2 is encoded by a small gene family (two to eight genes). Although a high level of PP2 DNA polymorphism existed among species, a single mRNA (ca. 1 kb) was detected in each species. PP2, affinity-purified from the vascular exudate of each species, reacted with PP2-specific antibodies; five species contained a single PP2 polypeptide while four species contained two PP2 polypeptides.
DOI:10.1111/j.1365-3059.2006.01438.xURL [本文引用: 1]
Conventional PCR and two real-time PCR (RTi-PCR) methods were developed and compared using the primer pairs CQULA03F/CQULA03R and CQULA04F/CQULA04R, and TaqMan probe CQULAP1 designed from a species-specific sequence of the rplJ/rplL ribosomal protein gene, for diagnosis of citrus huanglongbing (HLB) disease in southern China. The specificity and sensitivity of the three protocols for detecting ' Candidatus Liberibacter asiaticus' in total DNA extracts of midribs collected from infected citrus leaves with symptoms in Guangxi municipality, Jiangxi Province and Zhejiang Province, were tested. Sensitivities using extracted total DNA (measured as copy number, CN per 08 L of recombinant plasmid solution) were 439·0 (1·30 × 10 5 CN 08 L 611 ), 4·39 (1·30 × 10 3 CN 08 L 611 ) and 0·44 fg 08 L 611 (1·30 × 10 2 CN 08 L 611 ) for conventional PCR, TaqMan and SYBR Green I (SGI) RTi-PCR, respectively. SGI RTi-PCR was the most sensitive, but its specificity needed to be confirmed by running a melt-curve assay. The TaqMan RTi-PCR assay was rapid and had the greatest specificity. Concerning the correlation of PCR detection results with the various HLB symptoms, uneven mottling of leaves had the highest positive rate (96·50%), indicating that leaf mottling was the most reliable symptom for field surveys. Dynamic analysis results from the TaqMan assays showed that the titre (CN) g 611 citrus tissue of ' Ca. L. asiaticus' was highest between October and December (threshold cycle ( C t ) average = 29·3, CN = 3·35 × 10 7 ) and lowest between March and May ( C t average = 32·0, CN = 5·10 × 10 6 ) in 2004 and 2005. The optimized molecular-based assays should prove useful for presymptom diagnosis of HLB disease, monitoring and identification of ' Ca. L. asiaticus', and field epidemic regulation.
DOI:10.1007/s10658-009-9439-xURL [本文引用: 4]
Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 70302bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri . To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.
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DOI:10.1016/j.scienta.2018.04.017URL [本文引用: 1]
Two-year-old greenhouse-grown Huanglongbing (HLB)-affected and healthy ‘Hamlin’ sweet orange ( Citrus sinensis L. Osbeck) trees were spray treated with 1062μM strigolactone (SL) during the floral induction period (November 2016 and January 2017), and the flowering and fruit-setting period (April and June of 2017). Trees were grouped in four treatments: SL-treated HLB-affected trees; untreated HLB-affected trees; SL-treated healthy trees, and healthy control trees. Strigolactone application on HLB-affected trees during floral induction accelerated and augmented reproductive growth by inducing a 2.3-fold increase in flower bearing branches and an average of 2.6-fold increase in flowers per tree within two months from application. On the contrary, the same trees produced 31% less vegetative branches, 55% less flower bearing branches and 85% fewer flowers than HLB trees during the spring flush (April 2017). For the entire season, the number of fruit was higher in HLB62+62SL trees compared to untreated HLB-affected trees. Strigolactone application on healthy trees increased sucrose and starch content in mature leaves by 26% and 166%, respectively, with a 47% increase in the number of flowers compared to untreated trees. Strigolactone application during the flowering and fruit-setting period induced additional summer flush. The results of iodine staining and analytical observations demonstrated a decline in starch content in leaves and an increase in roots of SL-treated HLB-affected trees. We conclude that strigolactone ameliorates the effect of HLB by balancing vegetative and reproductive developmental response in HLB-affected citrus trees through the partitioning of stored carbohydrate.
DOI:10.1016/0003-2697(68)90317-5URL [本文引用: 1]
DOI:10.3923/ppj.2010.56.64URL [本文引用: 1]
This study was undertaken to develop a better understanding of the relationship between symptom development and the nature of the citrus disease, huanglongbing. The most characteristic symptom of huanglongbing (HLB) is the non-symmetrical mottled chlorosis of leaf blades. Starch accumulation and phloem collapse have been associated with symptom development in this disease presumed to be caused by ICandidatus /ILiberibacter asiaticus. Several hypotheses regarding phloem disruption to starch accumulation to chlorosis evolved concerning symptom development. These were tested using light and Transmission Electron Microscopy (TEM). Samples collected and fixed for TEM with various stages of HLB symptoms, revealed the following: starch accumulation occurred after phloem plugging and cell collapse and therefore, localized carbohydrate deficiency may be a factor. Starch packing of chloroplasts did not rupture the outer membranes, but the inner grana structure was disrupted thus, leading to chlorosis. This occurred only in parts of the leaf where phloem plugging occurred. Sieve elements were obstructed by both amorphous and filamentous materials and both occurred in readily observed amounts, while bacteria were insufficient to directly cause plugging. The amorphous material was positively identified as callose by immunoassay with gold labeling. Phloem protein 2 was identified in the filamentous plugging material using immunoassay with gold labeling. This information supports the development of HLB symptoms in the following sequence: phloem plugging and necrosis with cell wall swelling of sieve elements and companion cells followed by some phloem cell collapse, presumed sugar backup in localized leaf blade areas leading to starch accumulation until chloroplast structure is disrupted with resulting chlorosis.
DOI:10.1186/s12870-015-0568-4URLPMID:4517500 [本文引用: 1]
Background Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus???HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus???HLB interactions using meta-analysis and gene co-expression network modelling. Results Twenty-two publically available transcriptome studies on citrus???HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. Conclusion Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus???HLB transcriptome data that will help to elucidate the holistic picture of the citrus???HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation.
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URL [本文引用: 1]
Citrus tristeza virus (CTV), a plus-sense ssRNA virus, is member of the genus Closterovirus, family Closteroviridae. RNA viruses are inducers as-well-as targets of gene silencing defense mechanism of host plants and this has been exploited as a tool in functional genomics. CTV was developed into virus-induced gene silencing (VIGS) or RNA-interference (RNAi) vector, which interferes with expression of endogenous genes in citrus or GFP-transgene in Nicotiana benthamiana (16c) in a sequence specific manner. Photobleaching phenotype indicative of silencing of endogenous gene, phytoene desaturase in citrus, and red color under UV indicative of silencing of transgene GFP in N. benthamiana (16c) was observed using CTV-RNAi vector. CTV-RNAi vector has great potentials in combating huanglongbing (HLB) disease through (1) enhancing basal defense of citrus by silencing of auxin signaling F-Box receptor genes while simultaneously overexpressing microRNAs; (2) down-regulation of overexpressed genes, callose synthase and phloem protein-2, responsible for phloem-plugging in citrus by HLB; (3) expressing dsRNA specific to essential genes of insect vector psyllid (Diaphorina citri) to disable transmission of 'Candidatus' Liberibacter asiaticus pathogen. Simultaneous silencing of multiple endogenous genes of a metabolic pathway is possible through tandem engineering of potential siRNA eliciting regions in CTV-RNAi vector.
DOI:10.1094/Phyto-66-1038URL [本文引用: 1]
DOI:10.1146/annurev.phyto.40.120301.093927URL
DOI:10.1016/S0065-2113(05)87001-4URL [本文引用: 1]
DOI:10.1038/nrm1470 [本文引用: 1]
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DOI:10.1186/1471-2164-14-247URLPMID:23586643 [本文引用: 1]
Citrushuanglongbing(HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria namedCandidatusLiberibacter africanus (CaLaf),Ca.L. asiaticus (CaLas), andCa.L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector,Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam. The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes fromArabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas. Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.
