柑橘溃疡病菌免疫荧光和生物学检测技术研究

本研究通过对7个不同柑橘品种的离体叶片接种及致病性观察筛选出了柑橘溃疡病菌敏感寄主材料，并建立了柑橘溃疡病菌的生物学检测方法；还初次尝试建立了一种简便易行的柑橘溃疡病菌免疫荧光快速鉴定方法。柑橘离体叶片接种实验结果表明：福本、红肉脐橙、纽荷尔和枳壳对柑橘溃疡病菌表现出一定的感病性，而粗柠檬和邓肯葡萄柚未表现出明显感病迹象，反而出现了坏死性应激反应。免疫荧光试验结果显示：浓度为10^3cfu／mL的菌液在30℃下用BSA封闭2h，30℃下抗体结合1h，荧光抗体浓度为4μg,／mL，室温放置40min后镜检，就可以在荧光显微镜下清晰地看到绿色的柑橘溃疡病菌体，而用该方法检测不到水稻白叶枯病菌。用免疫荧光抗体检测柑橘溃疡病菌操作简便，仅需一台荧光显微镜，整个检测过程仅需要4h。     

4种杀菌剂对柑橘溃疡病菌的室内毒力测定

利用紫外分光光度计测定液体培养细菌菌液吸光度值(OD值)的方法,测定了27.12%碱式硫酸铜SC、37.5%氢氧化铜SC、30%络氨铜AS、0.3%四霉素AS共4种杀菌剂对柑橘溃疡病菌的抑制作用,结果表明:27.12%碱式硫酸铜SC、37.5%氢氧化铜SC、30%络氨铜As和0.3%四霉素AS对柑橘溃疡病菌的EC50值分别为572.33μg/mL、819.16μg/mL、1442.55μg/mL和0.47μg/mL,0.3%四霉素AS对柑橘溃疡病菌的毒力最强,30%络氨铜AS最弱。     

柑橘溃疡病菌外膜蛋白XAC1347在耐受铜杀菌剂中的功能

 细菌性溃疡是柑橘上的一种重要细菌病害,施用含铜化学药剂是防治该病的主要措施。本研究报道柑橘溃疡病菌外膜蛋白XAC1347在铜化学药剂耐药性中的功能。在平板抑菌试验中,46% Cu(OH)₂散粒剂和33%王铜·Cu(OH)₂悬浮剂的的最大稀释倍数分别为2 000倍和2 500倍,相对应的有效成分氢氧化铜和王铜的浓度分别为0. 023%和0.013 2%时,柑橘溃疡病菌的生长受到抑制。在2 000倍稀释浓度条件下,两种杀菌剂对外膜蛋白XAC1347突变体的抑菌圈分别增加了10倍和5倍。接种柑橘植物后,突变体ΔXAC1347中耐铜基因copA和copB基因的表达水平分别下降了37%和15%;在铜离子胁迫条件下,copA和copB基因的表达水平分别下降了33%和51%。在突变体中组成型表达XAC1347基因,均能部分恢复突变体的表型。这些结果表明,外膜蛋白XAC1347对柑橘溃疡病菌耐受含铜杀菌剂有重要作用。     

柑橘溃疡病菌检测方法及其在检疫中的优选

采用常规检测法、免疫斑点法、酶联免疫检测法和PCR检测法对柑橘溃疡病菌纯培养和柑橘叶片提取液进行平行测定。结果表明,对柑橘叶片提取液的检测,4种检测法检出的样品带菌率分别为11.12%、27.22%、18.69%、20.31%;对柑橘溃疡病菌纯培养的检测,4种方法检测结果一致。本实验同时比较了几种方法的灵敏度、特异性和稳定性,以及不同方法在各级检验检疫中的适应性。     

柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立

建立柑橘溃疡病菌的普通LAMP和快速LAMP检测方法,使其能应用于基层检验检疫部门对病害的快速检测.利用柑橘溃疡病菌基因组特有的保守区域设计LAMP引物,通过优化反应条件,建立柑橘溃疡病菌的普通LAMP检测体系;在普通LAMP引物的基础上设计一对环引物,建立柑橘溃疡病菌的快速LAMP检测体系,并以多种参比菌DNA以及健康柑橘叶片基因组DNA为模板对普通LAMP和快速LAMP检测体系的特异性进行了验证,利用柑橘溃疡病菌菌液和DNA溶液梯度稀释液对普通LAMP和快速LAMP检测体系的灵敏度进行了验证.普通LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25×104 cfu和2.03×10-1 ng,快速LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25 cfu和2.03×10-5ng.在特异性测试中,普通LAMP检测体系与快速LAMP检测体系均仅对柑橘溃疡病菌进行扩增,对非靶标菌和柑橘叶片基因组DNA不产生扩增,普通LAMP与快速LAMP检测体系特异性测试结果一致.快速LAMP检测体系在0.5h内就可以达到普通LAMP检测体系的扩增量,是普通LAMP检测体系反应时间的一半,大大提高了检测的效率;快速LAMP检测体系菌悬液和DNA检测灵敏度均比普通LAMP检测体系提高了10 000倍.成功地建立了柑橘溃疡病菌的普通LAMP及快速LAMP检测方法,为柑橘溃疡病菌的检测提供了一种新的简便、快速的检测手段.     

十一种杀菌剂防治马铃薯晚疫病的适宜施用时期

为了明确当前市场上具有代表性的11种防治马铃薯晚疫病杀菌剂的适宜施用时期,采用人工接种马铃薯叶片的方法,测定了其中5种药剂在接种晚疫病菌前10 d内不同时间施用的预防效果和其中7种药剂在接种后24 h内不同时间施用的治疗效果。结果显示:在保证良好防治效果的前提下,药剂在病原菌接种前保护性施用的适宜时期比在接种后治疗性施用的适宜时期要长。在测试浓度下,接种前5种供试药剂保持100%防治效果的适宜施用时期为接种前3~10 d;而接种后7种供试药剂保持相同防效的适宜施用时期仅为接种后6~12 h,在生产实际中难以操作。研究结果说明,在田间马铃薯晚疫病的化学防治中,为了取得良好防治效果,每次用药均应在病菌侵入之前采用保护性施用,尽量避免在病菌侵入之后进行治疗性施用。     

春雷·噻霉酮WG对柑橘溃疡病的防治效果

为研究柑橘溃疡病的防治效果,采用8％春雷·噻霉酮WG、46％氢氧化酮WG、47％春雷·王酮WP分别施药3次,每次间隔7 d.通过对比发现8％春雷·噻霉酮WG 750倍液、1000倍液和1500倍液对柑橘叶片溃疡病有较好的防治效果,防效分别为86.3％、84.2％和84.5％,可作为柑橘溃疡病防治的选择药剂.     

4种剂型噻虫胺对柑橘木虱的田间防治效果

2018年—2019年,采用田间小区试验评价了噻虫胺4种剂型(30%悬浮剂、18%包埋颗粒剂、13.5%壳聚糖微球和0.5%颗粒剂)及2种施用方式(喷雾和沟施)对柑橘木虱的防治效果。结果表明:叶面喷施30%噻虫胺悬浮剂60 mg/kg处理对幼年树及成年树上柑橘木虱的控制效果好且持效期约30 d,而药后46~96 d的防治效果大幅下降,难以控制其为害。在幼年树根部周围拌毒土沟施时,噻虫胺18%包埋颗粒剂和13.5%壳聚糖微球0.1~0.2 g/株处理对柑橘木虱的控制效果较好,且持效期长达96 d;0.5%噻虫胺颗粒剂0.1 g/株处理对柑橘木虱的效果好且持效期达46 d,而药后66~96 d的防治效果下降明显,难以控制其为害;在0.1 g/株剂量下,0.5%噻虫胺颗粒剂药后7~30 d的效果最好、18%包埋颗粒剂次之、13.5%壳聚糖微球稍差,而18%包埋颗粒剂药后46~96 d的效果最佳、13.5%壳聚糖微球次之、0.5%颗粒剂稍差。在成年树根部周围沟施时,噻虫胺18%包埋颗粒剂、13.5%壳聚糖微球0.6~0.8 g/株处理对柑橘木虱的控制效果好且持效期长达89 d,而0.5%噻虫胺颗粒剂0.2 g/株处理的效果较好且持效期长达89 d;在0.2 g/株剂量下,不同剂型噻虫胺对柑橘木虱的防效差异与幼年树基本一致。此外,当剂型、剂量及施用方式均相同时,噻虫胺对柑橘木虱的控制作用略低于噻虫嗪。综上所述,在柑橘生产实践中,若田间柑橘木虱发生量大,应喷施1次30%噻虫胺悬浮剂60 mg/kg;为有效控制幼年树柑橘木虱为害,应于初发期拌毒土沟施1次,使用药剂为18%噻虫胺包埋颗粒剂,用量为0.2 g/株;针对成年树柑橘木虱,则需将其用量增至0.6 g/株。     

30%螺虫乙酯·噻虫嗪悬浮剂对柑橘木虱的田间药效试验

30%螺虫乙酯·噻虫嗪SC防治柑橘木虱的田间药效试验结果表明,在柑橘木虱发生初期施用30%螺虫乙酯·噻虫嗪SC,施药后10d,试验药剂浓度为50mg/kg、60mg/kg和75mg/kg对柑橘木虱的防效分别达到74.41%、79.36%和91.77%,均有较好的防治效果,且试验药剂对柑橘安全,推荐使用浓度为75mg/kg。     

3种杀菌剂对柑橘溃疡病的防治效果

溃疡病是柑橘的主要病害之一,也是广西南宁区域沃柑的主要病害,严重影响沃柑品质.本试验选用47％春雷·王铜WP、20％噻唑锌SC、20％噻菌铜SC等3种杀菌剂,对2 a生沃柑溃疡病进行了防效对比试验,结果表明,47％春雷·王铜WP 500倍液对柑橘溃疡病的防治效果较好,防治效果达到84.25％,并且对柑橘安全、无负面影响...     

基于两性生命表评价取食柑橘木虱对六斑月瓢虫和龟纹瓢虫生长发育及种群的影响

为有效防控重要害虫柑橘木虱Diaphorina citri,在实验室条件下测定取食柑橘木虱后六斑月瓢虫Cheilomenes sexmaculata和龟纹瓢虫Propylea japonica各虫态的发育历期,利用构建的年龄-阶段两性生命表对取食柑橘木虱后六斑月瓢虫、龟纹瓢虫生长发育及其种群参数进行研究。结果显示,取食柑橘木虱后六斑月瓢虫羽化前的发育历期为14.80 d,显著长于取食柑橘木虱后龟纹瓢虫的14.41 d;取食柑橘木虱后六斑月瓢虫和龟纹瓢虫产卵期分别为15.88 d和10.23 d,前者显著长于后者;取食柑橘木虱后六斑月瓢虫净增殖率、内禀增长率和周限增长率分别为25.48、0.10 d-1和1.10 d-1,取食柑橘木虱后龟纹瓢虫净增殖率、内禀增长率和周限增长率分别为14.13、0.06 d-1和1.06 d-1,均低于取食柑橘木虱后六斑月瓢虫;取食柑橘木虱后六斑月瓢虫和龟纹瓢虫的平均世代周期分别为33.87 d和44.95 d,前者显著短于后者。表明取食柑橘木虱后六斑月瓢虫和龟纹瓢虫均能完成整个世代,且六斑月瓢虫种群增长优于龟纹瓢虫,可大规模繁殖六斑月瓢虫,可用于柑橘木虱...     

Effect of the duration of inoculum exposure on development of citrus canker symptoms on seedlings of Swingle citrumelo

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is one of the most serious diseases citrus in Florida, and elsewhere in the world. The disease causes yield loss and some fresh fruit trade restrictions may apply. Cultural management techniques such as windbreaks may work by not only reducing wind speed, but also reducing the period of exposure of susceptible foliage or fruit to those wind speeds that support infection from incoming inoculum. To investigate the effect of exposure period to inoculum of Xcc, seedlings of canker-susceptible Swingle citrumelo were exposed to sprayed inoculum for increasing periods at different wind speeds. The incidence and severity of citrus canker was assessed. In three experiments the incidence and severity of citrus canker most often increased with longer periods of exposure to inoculum, especially so at wind speeds of ≥16 m/s compared to wind speeds of ≤5 m/s (wind speed also increased disease incidence and severity). Regression analysis demonstrated relationships between period of exposure to inoculum and the percent infected leaves per plant, the number of lesions per plant, the number of lesions per infected leaf, and for the percent of infected leaves with lesions on the petioles at wind speeds of ≥16 m/s (R²?=?0.16–0.72). Due to the effect of inoculum exposure period and wind speed, attempts should be made to minimize exposure of canker-susceptible citrus when wind speed is highest and inoculum is available. Windbreaks should help minimize periods of exposure to splashed inoculum in high winds.     

龟纹瓢虫对柑橘木虱的捕食功能反应及猎物偏好性

为评估龟纹瓢虫Propylea japonica对柑橘木虱Diaphorina citri的生物防治潜能,在实验室条件下测定龟纹瓢虫1~4龄幼虫和成虫对柑橘木虱5龄若虫的捕食功能反应、搜寻效应、自身密度干扰反应以及捕食偏好性。结果表明,龟纹瓢虫1~4龄幼虫和成虫对柑橘木虱5龄若虫的捕食功能反应均符合Holling II型功能反应类型,其各虫态龟纹瓢虫的瞬时攻击率分别为0.313、0.610、0.234、0.585和0.675,处理时间分别为0.088、0.077、0.013、0.008和0.016 d,龟纹瓢虫4龄幼虫对柑橘木虱5龄若虫的捕食能力最大,为73.125,龟纹瓢虫成虫、3龄幼虫、2龄幼虫、1龄幼虫次之,捕食能力分别为42.188、18.000、7.922和3.557。各虫态龟纹瓢虫对柑橘木虱5龄若虫的搜寻效应随柑橘木虱5龄若虫密度升高而下降;在柑橘木虱5龄若虫密度一定的条件下,各虫态龟纹瓢虫对柑橘木虱5龄若虫的捕食率随自身密度升高而下降,其自身密度干扰方程为E=0.421P^-0.52;当柑橘木虱5龄若虫和豆蚜Aphis craccivora 4龄若虫2种猎物共存时,龟纹瓢虫成虫对其食物选择性指数均小于1,表明龟纹瓢虫成虫对这2种猎物无捕食偏好性。表明龟纹瓢虫对柑橘木虱5龄若虫有较强的防控潜能。     

Over-expression of the Arabidopsis <Emphasis Type="BoldItalic">NPR1</Emphasis> gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.     

Alternaria Brown Spot of Minneola in Greece; Evaluation of Citrus Species Susceptibility

During the last three years, a new disease was observed in northwestern Greece on Minneola trees, hybrid of mandarin and grapefruit. On May small brown necrotic leaf spots surrounded by yellow halo areas of various sizes appeared and covered a major portion of the leaves with extension of necrosis into the veins. On young fruits small, slightly depressed black spots were the first symptoms, which later became 2–7 mm in diameter. Brown spots were observed on the leaves and fruits in several orchards in the same area, causing leaves and fruits to drop. In some orchards over 50% of the fruits were affected. From the fruit and leaf spots the typical small-spore species Alternaria alternata was isolated. Pathogenicity tests were performed by artificially inoculating fruits of Minneola, common mandarin and Clementine. The symptoms of the disease were reproduced only on fruits of Minneola hybrids by the specific strain of the fungus Alternaria alternata pv. citri. Different citrus susceptibility tests indicated that mandarins Minneola, Nova and Page were very susceptible to tested isolates while Clementine SRA and Poros Clementine were not. All lemons and lime Seedless were not susceptible. Grapefruit New Hall was not susceptible, while the Star Ruby was. Orange Lane Late, Navel Late, Oval Poros, Olinda, Navel Athos were not susceptible and only Moro showed reaction being slightly susceptible only to one isolate.     

丽草蛉对柑橘木虱成虫的捕食功能反应及捕食偏好

为明确丽草蛉Chrysopa formosa对柑橘木虱Diaphorina citri成虫的生物防控潜能,在实验室条件下测定丽草蛉1、2、3龄幼虫和成虫对柑橘木虱成虫的捕食功能、搜寻效应及其种内干扰效应,并测定其对不同猎物的捕食偏好。结果显示,丽草蛉1～3龄幼虫和成虫对柑橘木虱成虫均有捕食行为,捕食功能反应均符合Holling II模型。丽草蛉1、2、3龄幼虫和成虫对柑橘木虱成虫的瞬时攻击率分别为0.247、0.393、0.769和0.509,处理时间分别为0.228、0.048、0.015和0.013 d,理论日最大捕食量分别为4.386、20.833、66.667和76.923头,控害效能分别为1.083、8.188、51.267和39.154。各虫态丽草蛉对柑橘木虱成虫的搜寻效应均随着猎物密度的增加而降低,其中3龄丽草蛉幼虫的搜寻效应下降趋势最大。当柑橘木虱成虫密度固定时,单头丽草蛉的日捕食量随柑橘木虱密度的增加而逐渐降低,其捕食作用受到自身密度的干扰,干扰效应方程为E=0.260P-0.351。当存在柑橘木虱与豌豆蚜Acyrthosiphon pisum两种捕食对象时,丽草蛉3...     

Correlation of ethylene synthesis in Citrus fruits and their susceptibility to Alternaria alternata pv. citri

The susceptibility of Fortune (Citrus clementina × Citrus reticulata), Citrus paradisi and Citrus limon fruits to Alternaria alternata pv. citri was investigated using different artificial inoculation methods. The results obtained reveal that the C. paradisi and C. limon fruits are less susceptible to A. alternata pv. citri than Fortune fruits, although all showed symptoms of Alternaria brown spot when the cuticle was broken and the flavedo or flavedo + albedo was removed. Furthermore, it was seen that susceptibility to the fungus decreased as the age of the fruit increased. There was a positive correlation between the susceptibility of the different Citrus fruits to A. alternata pv. citri and their “in vivo” ethylene levels, the most susceptible fruit (Fortune) producing more ethylene during growth than the less susceptible C. limon and C. paradisi. This suggests that ethylene may well be considered as a possible marker of Citrus fruit susceptibility to A. alternata pv. citri. Disease development increased when the Fortune fruits were treated with 1 mM ACC (a precursor of ethylene biosynthesis) or 1 mM Ethephon (an ethylene-releasing compound) prior to inoculation with A. alternata pv. citri. The role of ethylene as a factor involved in disease development is discussed.     

云杉矮槲寄生的侵染对青杄光合与蒸腾作用的影响

 云杉矮槲寄生(Arceuthobium sichuanense)属半寄生性多年生种子植物,是青海省云杉天然林毁灭性生物灾害之一。本研究首次报道了云杉矮槲寄生在自然条件下可以侵染青杄,并对健康与染病青杄(Picea wilsonii)针叶形态及其光合日变化进行了测定。结果表明:1)染病青杄的针叶长度和宽度显著小于健康青杄(P＜0.001),而染病青杄的针叶比叶面积(SLA)显著大于健康青杄(P＜0.001);2)云杉矮槲寄生的侵染显著降低了青杄叶片的净光合速率、蒸腾速率以及气孔导度(P＜0.05),但不影响其胞间CO₂浓度(P =0.32);3)冗余分析(RDA)表明,云杉矮槲寄生的侵染导致青杄对不同环境因子的依赖程度发生变化,健康青杄表现为环境CO₂浓度(CO₂)＞空气温度(T_air)＞叶片温度(T_l)＞蒸汽压亏缺(Vpd)＞空气相对湿度(RH)＞光合有效辐射(PAR),而染病青杄则出现T_l＞RH＞T_air＞PAR＞Vpd＞CO₂,且T_l成为影响染病青杄叶片光合与蒸腾作用(P_n、T_r)的主要环境因子,且染病青杄对RH和PAR变化的响应更加敏感。此外,染病青杄叶片的水分利用率(WUE)显著低于健康寄主(P＜0.05)。因此,云杉矮槲寄生的侵染可降低青杄对环境的适应能力,从而加速寄主的衰老死亡。     

Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness

The combined effect of temperature (15°C, 20°C, 25°C, 30°C, 35°C, 40°C and 42°C) and leaf wetness duration (0, 4, 8 12, 16, 20 and 24 h) on infection and development of Asiatic citrus canker (Xanthomonas citri subsp. citri) on Tahiti lime plant was examined in growth chambers. No disease developed at 42°C and zero hours of leaf wetness. Periods of leaf wetness as short as 4 h were sufficient for citrus canker infection. However, a longer leaf duration wetness (24 h) did not result in much increase in the incidence of citrus canker, but led to twice the number of lesions and four times the disease severity. Temperature was the greatest factor influencing disease development. At optimum temperatures (25–35°C), there was 100% disease incidence. Maximum disease development was observed at 30–35°C, with up to a 12-fold increase in lesion density, a 10-fold increase in lesion size and a 60-fold increase in disease severity.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.