Effects of phylloxera infestation on the root ultrastructure of grape cultivars with different resistance

[目的]探讨不同抗性葡萄品种对葡萄根瘤蚜Daktulosphaira vitifolia Fitch侵染后的组织结构响应.[方法]以砧木140Ru(Vitis rupestris×V.berlandieri)和栽培品种赤霞珠Vitis vinifera cv.Cabernet Sauvignon离体三级根及140Ru盆栽苗为试材接种根瘤蚜卵,接种两周后取刺吸位点组织作超薄切片并观察超微结构变化,取盆栽140Ru不同时期根结测定多酚含量.[结果]超微结构显示,砧木140Ru根系周皮层最外层细胞壁的厚度(1 031.25 nm)及周皮层细胞层数(6～7层)显著高于赤霞珠品种的周皮层细胞厚度(543.75 nm)及层数(3～4层),140Ru根系韧皮部酚类物质含量比赤霞珠高出35％;侵染后,砧木140Ru周皮层细胞第3层以内的细胞壁加厚并积聚大量的多酚类物质,140Ru葡萄新根被根瘤蚜侵染后多酚含量呈升高趋势,在接种20d时是对照的2.4倍;赤霞珠粗根被根瘤蚜侵染后薄壁细胞中细胞质变浓,出现大量淀粉粒,线粒体及内质网数量增多.[结论]砧木140Ru的周皮层组织结构较赤霞珠不利于根瘤蚜口针穿刺,被根瘤蚜侵染后发生了不利于根瘤蚜侵染取食的变化.     

根瘤蚜侵染对不同抗性葡萄品种根系超微结构的影响

【目的】探讨不同抗性葡萄品种对葡萄根瘤蚜Daktulosphaira vitifolia Fitch侵染后的组织结构响应。【方法】以砧木140Ru (Vitis rupestris×V. berlandieri)和栽培品种赤霞珠Vitis vinifera cv. Cabernet Sauvignon离体三级根及140Ru盆栽苗为试材接种根瘤蚜卵, 接种两周后取刺吸位点组织作超薄切片并观察超微结构变化, 取盆栽140Ru不同时期根结测定多酚含量。【结果】超微结构显示, 砧木140Ru根系周皮层最外层细胞壁的厚度（1 031.25 nm）及周皮层细胞层数（6～7层）显著高于赤霞珠品种的周皮层细胞厚度（543.75 nm）及层数（3～4层）, 140Ru根系韧皮部酚类物质含量比赤霞珠高出35％; 侵染后, 砧木140Ru周皮层细胞第3层以内的细胞壁加厚并积聚大量的多酚类物质, 140Ru葡萄新根被根瘤蚜侵染后多酚含量呈升高趋势, 在接种20 d时是对照的2.4倍; 赤霞珠粗根被根瘤蚜侵染后薄壁细胞中细胞质变浓, 出现大量淀粉粒, 线粒体及内质网数量增多。【结论】砧木140Ru的周皮层组织结构较赤霞珠不利于根瘤蚜口针穿刺, 被根瘤蚜侵染后发生了不利于根瘤蚜侵染取食的变化。     

根瘤蚜对不同抗性葡萄的选择性及葡萄根系挥发性物质的鉴定

为了探讨不同抗性水平砧木及品种对根瘤蚜吸引及驱避效应以及鉴定挥发性物质组分,采用离体根段培养法测定葡萄根瘤蚜Phylloxera viticola Fitch对砧木5BB,140Ru及栽培品种巨峰的选择性,发现根瘤蚜对栽培品种巨峰的根段具有很强的选择性,至接种后3 d,67.13％的根瘤蚜优先选择巨峰;而对5BB和140Ru的选择性却很差,分别有15.29％和17.58％的根瘤蚜选择5BB及140Ru。根瘤蚜在不同品种根段上的生长发育和繁殖也存在明显差异。对砧木5BB和巨峰根系的挥发性组分进行测定,发现2个品种挥发物独有组分数量均较高,巨峰独有组分为23个,占总成分数的48.94％,5BB的独有组分为32个,占总成分数的57.14％。5BB和巨峰的共有组分为24个,主要成分均为亚油酸甲酯和反油酸甲酯,但含量有所不同,其中,5BB以倍半萜类的丁香醇及脂肪酸甲酯类的亚油酸甲酯,反油酸甲酯,棕榈油酸甲酯含量较高,高于巨峰中相应物质含量的4.23%~6.46％,而巨峰以邻苯二甲酸二丁酯及胆甾烷含量较高,分别高于5BB相应组分含量的6.81％和1.07％。结果说明葡萄根瘤蚜对不同抗性水平砧木及品种确实存在不同的选择性,并且不同抗性水平的砧木及品种的挥发性物质存在显著差异。     

部分葡萄品种和砧木抗葡萄根瘤蚜性能鉴定

为了检测我国葡萄品种和砧木对中国生态型葡萄根瘤蚜Daktulosphaira viticola (Fitch) 的抗性,在实验室条件下采用离体根接种法鉴定了10个葡萄砧木、18个葡萄栽培品种,测定了根瘤蚜存活率、发育历期、产卵量、种群龄期结构指数和成虫大小等指标。结果表明： 砧木品种SO4,5BB,1103P,420A,Lot,101-14MG,3309C,140Ru和110R抗根瘤蚜,接种后蚜虫陆续死亡,尤其是SO4,5BB,1103P和420A对根瘤蚜高抗,使根瘤蚜滞育仅发育至1龄。我国当作砧木使用的贝达不抗根瘤蚜,接种后蚜虫存活率始终保持在38.82％～47.5%,日产卵量达9.01;欧美杂交种中的康克、康拜尔和卡它巴对根瘤蚜稍有抗性,根瘤蚜日产卵量在6.00～6.85粒之间,其中康克、康拜尔葡萄上蚜虫的存活率随时间的延长而明显降低,由最初的60％下降至25％～30％; 欧美杂交种中的黑虎香和白香蕉虽然在一定程度上能够限制根瘤蚜的存活和发育进程,但对根瘤蚜的繁殖量没有影响。所有供试欧亚种和欧美杂交种的栽培品种都对根瘤蚜高度敏感,接种后第29 d 蚜虫存活率仍保持在40％～60％,且前后变化较小,日产卵量变化于7～12.35粒之间,总产卵量均在190粒以上。由此可见,我国供试栽培品种均不抗根瘤蚜,在根瘤蚜疫区需要用抗性砧木嫁接苗建园。     

烟草水浸提液对葡萄根瘤蚜侵染危害的控制效应

【目的】检疫害虫葡萄根瘤蚜Daktulosphaira vitifoliae已经在我国多个地区葡萄园发现为害, 直接影响葡萄生长; 前期实验发现烟草水浸提液可有效控制离体根上根瘤蚜的存活率, 本研究进一步开展盆栽验证, 探讨烟草水浸提液对葡萄根瘤蚜侵染的控制效应及葡萄植株生长抑制的缓解作用。【方法】以两年生温室盆栽‘巨峰’自根苗为实验材料, 接种根瘤蚜虫卵35 d后进行烟草水浸提液处理, 处理后测定供试植株的根结数、生长量、根系活力、叶片光合速率及叶绿素荧光等指标。【结果】20 mg/mL和50 mg/mL的烟草水浸提液对根瘤蚜均有一定程度的抑制作用, 施药21 d后, 葡萄根部的新增根结数分别比侵染对照下降了42.9%和52.8%。烟草水浸提液处理还在一定程度上缓解了根瘤蚜侵染对植株的光合能力和荧光的抑制作用。20 mg/mL和50 mg/mL烟草水浸提液处理的株高分别比侵染对照提高了57.1%和7.7%; 根瘤蚜侵染降低了土壤中真菌、细菌和放线菌的数量, 烟草水浸提液处理进一步降低了真菌和放线菌的数量, 降低了由微生物带来的次生侵染几率。【结论】烟草水浸提液对葡萄根瘤蚜的侵染具有控制效果, 并可对葡萄根瘤蚜侵染已造成的寄主危害有一定的缓解作用。     

外源水杨酸和茉莉酸诱导巨峰葡萄抗根瘤蚜

【目的】水杨酸和茉莉酸在植物诱导防御虫害反应中发挥着重要作用。本研究旨在探讨水杨酸和茉莉酸诱导葡萄对根瘤蚜的抗性。【方法】以盆栽巨峰为试材,在接种葡萄根瘤蚜Daktulosphaira vitifoliae的同时喷施水杨酸和茉莉酸,测定和评估了对根瘤蚜生长发育及产卵量的影响,以及对葡萄根系抗氧化相关酶［过氧化物酶(POD)和过氧化氢酶(CAT）］活性、丙二醛（MDA）含量、新梢生长量及光合速率的影响。【结果】水杨酸和茉莉酸诱导处理显著降低了根瘤蚜卵量及下代1,2龄若蚜总数,接种35 d后根瘤蚜的产卵量分别减少了41.35％和50.00％,1龄和2龄若蚜总数分别减少了42.31％和50.00％;根瘤蚜侵染后根系中POD和CAT活性均呈先升高后降低的趋势,且水杨酸和茉莉酸诱导处理在各测定时期均高于仅接种根瘤蚜处理;水杨酸和茉莉酸处理的根系中MDA含量在各测定时期均低于仅接种根瘤蚜处理;水杨酸和茉莉酸处理降低了根瘤蚜侵染对植株地上部生长及光合的抑制。接种处理后第30天,仅接种根瘤蚜处理的植株地上部生长量减少了48.11%,光合速率降低了58.77％,而水杨酸和茉莉酸处理后的新梢生长量分别减少了31.57％和25.71％,光合速率分别降低了32.89％和24.67％。【结论】叶片喷洒茉莉酸和水杨酸能够降低根瘤蚜种群密度,并提高葡萄根系活性氧清除能力和防御酶活性,缓解树势衰退。     

钙肥对葡萄根瘤蚜生长发育和繁殖的影响

【目的】为明确葡萄所需的大量元素钙对葡萄根瘤蚜 Daktulosphaira vitifoliae(Fitch）生长发育和繁殖的影响。【方法】在温室中,设置不同 Ca⁽²⁺⁾浓度（0、2 和 4mmol·L(2+)浓度（0、2 和 4mmol·L^(-1)）的钙全营养液于葡萄全生育期浇灌葡萄,评价离体根段对葡萄根瘤蚜的存活率、若蚜、成蚜寿命和繁殖力的影响。【结果】随着钙离子浓度的增加,葡萄根瘤蚜在葡萄根上的 1 龄若蚜历期缩短 0.80 倍以上,成蚜历期增加超过 1.88倍,寿命显著延长超过 1.18 倍,产卵量提高 3.34 倍以上。与 0 mmol·L(-1)）的钙全营养液于葡萄全生育期浇灌葡萄,评价离体根段对葡萄根瘤蚜的存活率、若蚜、成蚜寿命和繁殖力的影响。【结果】随着钙离子浓度的增加,葡萄根瘤蚜在葡萄根上的 1 龄若蚜历期缩短 0.80 倍以上,成蚜历期增加超过 1.88倍,寿命显著延长超过 1.18 倍,产卵量提高 3.34 倍以上。与 0 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾和 2 mmol·L(2+)和 2 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾处理相比,葡萄根瘤蚜在 4 mmol·L(2+)处理相比,葡萄根瘤蚜在 4 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾处理下的葡萄根上,内禀增长率 r_m、周限增长率 λ 和净增殖率 R_0均最高,分别为（0.191 ± 0.003）、（1.210 ± 0.004）和（169.070 ± 11.897)(P < 0.05）,且世代发育历期显著缩短（P <0.05）。特定年龄龄期存活率 S_(xj)、种群特定年龄繁殖力 m_x和种群特定年龄繁殖值 l_xm_x在 4mmol·L(2+)处理下的葡萄根上,内禀增长率 r_m、周限增长率 λ 和净增殖率 R_0均最高,分别为（0.191 ± 0.003）、（1.210 ± 0.004）和（169.070 ± 11.897)(P < 0.05）,且世代发育历期显著缩短（P <0.05）。特定年龄龄期存活率 S_(xj)、种群特定年龄繁殖力 m_x和种群特定年龄繁殖值 l_xm_x在 4mmol·L^(-1)Ca(-1)Ca⁽²⁺⁾处理种群中保持较高水平,随着钙浓度降低,葡萄根瘤蚜的各生命参数明显降低。【结论】钙元素对葡萄根瘤蚜的生长发育和繁殖能力具有促进作用,葡萄根瘤蚜发生葡萄园更应合理使用钙肥。     

两个葡萄杂交后代根系抗葡萄根瘤蚜及抗寒性鉴定

【目的】为了筛选抗葡萄根瘤蚜Daktulosphaira vitifoliae Fitch且抗寒的葡萄砧木以适应我国葡萄生产需求。【方法】以山葡萄Vitis amurensis Rupr.左山1号×SO4杂种F1代的45个株系(A系列)和左山1号×101-1杂种F1代27个株系(B系列)为试材,采用离体根接种鉴定法进行抗葡萄根瘤蚜鉴定及抗性分级;采用差热分析系统(differential thermal analysis,DTA)进行各株系根系的低温放热(low temperature exotherms,LTE)分析,建立各株系根系韧皮部及木质部的温度-伤害度(LT-I)回归方程,评估各株系根系的抗寒性。【结果】葡萄根瘤蚜在杂交株系根系上的产卵量均显著低于敏感品种巨峰,筛选出被葡萄根瘤蚜侵染后不能形成根瘤,抗葡萄根瘤蚜级别为0级的A系列杂交株系18个和B系列株系11个。被葡萄根瘤蚜侵染后形成根瘤比例低于10%的抗葡萄根瘤蚜级别为1级的A系列杂交株系9个和B系列株系4个;筛选出A系列综合低温放热温度隶属度函数、韧皮部和木质部低温放热温度隶属度函数3个指标均低于贝达的株系27个,B系列各指标均低于贝达的株系3个。【结论】本研究筛选出抗寒性强且对葡萄根瘤蚜抗性强的A系列株系15个和B系列株系2个。其中,A14,A16,A18,A22,A23,A28,A34,A35,A38,A44,A50,B24和B26对葡萄根瘤蚜抗性级别为0;A11,A15,A17和A27对葡萄根瘤蚜抗性级别为1。     

抗性砧木对赤霞珠葡萄叶片白藜芦醇合成过程及含量的影响

为了明确不同抗性砧木对赤霞珠葡萄叶片白藜芦醇含量及其合成过程中前体物质和相关代谢酶活性的影响,分析不同抗性砧木与白藜芦醇合成的关系,以获得提高接穗品种赤霞珠葡萄叶片白藜芦醇含量的抗性砧木。该研究选择弗卡(Fercal)、5C、140R、3309M、3309C、SO4、抗砧3号(Kangzhen3)、5BB为砧木与赤霞珠(CS)葡萄进行嫁接,以赤霞珠自根苗为对照(CK),采用高效液相色谱技术,测定成熟葡萄叶片白藜芦醇以及合成白藜芦醇前体物质苯丙氨酸、肉桂酸、香豆酸含量,并对合成白藜芦醇相关代谢酶苯丙氨酸解氨酶(PAL)、肉桂酸-4-羟化酶(C4H)、4-香豆-辅酶A连接酶(4CL)、过氧化物酶(POD)以及多酚氧化酶(PPO)的活性进行测定。结果表明:(1)不同砧木均能提高接穗赤霞珠葡萄叶片白藜芦醇含量11%~46%。(2)在各砧穗组合中,CS/140R嫁接苗叶片的白藜芦醇含量最高达到18.24μg/g,其合成白藜芦醇前体物质苯丙氨酸和香豆酸含量也最高,分别达到38.61和1.06μg/g。(3)在各砧穗组合中,白藜芦醇相关代谢酶PAL活性以CS/3309C嫁接苗叶片最高;嫁接苗叶片代谢酶C4H和4CL活性均高于赤霞珠自根苗;POD和PPO活性均以CS/Fercal的接穗赤霞珠叶片最强。研究发现,不同抗性砧木能显著提高赤霞珠葡萄叶片白藜芦醇含量,相关代谢酶C4H活性对葡萄白藜芦醇的合成至关重要,PPO活性与葡萄叶片白藜芦醇合成也密切相关,CS/140R是8个砧穗组合中提高赤霞珠葡萄叶片白藜芦醇含量最具优势的砧穗组合。     

我国根瘤蚜mtDNA COⅠ遗传多样性与系统发育

通过线粒体DNA COⅠ多态性研究了我国根瘤蚜Daktulosphaira vitifoliae Fitch的遗传分化与系统发育,并将我国根瘤蚜单倍型与GenBank中已发表的85个单倍型进行了聚类分析。结果表明：序列中A,C,T,G 4种核苷酸的比例分别为34.8%,15.8%,39.2%和10.2%, 29 个变异位点中单一多态位点 12 个,简约信息位点17 个。确定了 13 种单倍型,检测5种单倍型,其中上海群体单倍型相对丰富,且上海葡萄根瘤蚜群体与其他3个群体之间没有共享单倍型。同时上海群体与其他3个群体的遗传距离最大（0.039~0.040）,Nm最小（0.02）,在分子系统发育树和单倍型网络图上为独立分支,说明我国根瘤蚜至少有两个独立的起源。     

Preference and performance responses of California grape phylloxera to different Vitis rootstocks

Laboratory experiments on host preference and performance were simultaneously conducted with Cabernet Sauvignon, AXR#1, and 5C Vitis rootstocks for the California biotypes A and B grape phylloxera. Preference bioassays recorded over a 3-day period indicated that phylloxera exhibit differential host choice. AXR#1 is antixenotic to biotype A, and 5C is antixenotic to both phylloxera biotypes. Preferences observed with biotype B for Cabernet Sauvignon and AXR#1 were not statistically different, whereas biotype A had shown a significant preference for Cabernet Sauvignon. On 5C the preferences scored were significantly lower than on either of the other two rootstocks, and there was no significant difference beetween biotypes. Performance bioassays recorded over a 29-day period indicated that survival, development, and reproduction of both biotypes were affected by Vitis rootstock. Both biotypes were unable to survive or develop on 5C suggesting the presence of antibiotic resistance in 5C against these phylloxera biotypes. The antibiotic effects observed with AXR#1 against biotype A were expressed as significant reductions in survival, development, and reproduction. Performance of biotype B on AXR#1 was similar to its performance on Cabernet Sauvignon. For both AXR#1 and 5C rootstocks and both phylloxera biotypes A and B the antibiotic mechanism was considerably stronger than the antixenotic mechanism. This research indicated that host preference and performance are positively correlated in grape phylloxera with the rootstocks and phylloxera biotypes tested.     

Evaluating resistance of some rootstocks to grape phylloxera with in vitro and excised root testing systems

Summary The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Rul40, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Rul 40 rootstock was more susceptible than R99 and 3309C rootstocks.     

Metarhizium anisopliae, a potential agent for the control of grape phylloxera

This investigation deals with the control effects of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae on Daktulosphaira vitifoliae. In pot experiments, the soil surrounding phylloxera-infected grapes was inoculated with barley colonised with M. anisopliae. After thirty-two days, ineight of ten Metarhizium-applied pots nofresh phylloxera infections could be observed.In two of ten plants, a few fresh nodositiessingly occupied with phylloxera or phylloxeraeggs could be found. In all untreated plants,fresh nodosities with either single (two of sixplants) or multiple (four of six plants)occupation with phylloxera could be observed.M. anisopliae could be re-isolated in aconcentration of <1 × 10³ CFU g^$minus;1 soil dry weight from those pots with phylloxera-infected plants that had been treated with the fungus. The potential role of M. anisopliae in grape phylloxera management is discussed.     

Variation in performance on two grape cultivars within and among populations of grape Phylloxera from wild and cultivated habitats

When an indigenous insect becomes a pest, comparisons of performance of pest and non-pest populations on crop plants and of genetic variation in that performance may provide insight into the evolution of pest populations. To measure such genetic variation, 8–15 clones of the grape phylloxera (Daktulosphaira vitifoliae Fitch) were collected from wild grapevines in each of 3 geographically isolated sites (populations) and from commercial vineyards in northern California. A complete life table was made for clonal replicates from populations collected from wild grapevines on each of two commercial grape cultivars, the susceptibleVitis vinifera (L.) cultivar Cabernet Sauvignon, and the phylloxera-resistant rootstock ‘AxR # 1’. Variation in mean performance on these two hosts was partitioned among clones within collection sites and among sites. Performance measures included an individual analog to the intrinsic rate of increase (r), age at first oviposition, fecundity in the first ten days of reproduction, total fecundity, and longevity. The overall performance of phylloxera from the wild grapevines on the resistant cultivar AxR # 1 was greater than or equal to that on the susceptible cultivar Cabernet Sauvignon. There was significant variation among clones within populations from wild grapes in the rate of increase on ‘AxR # 1’ and marginally significant clonal variation in some of the component paramters. There was no significant variation among clones within populations on ‘Cabernet Sauvignon’ and no significant differences between populations on either crop in any trait. In a second experiment we compared the relative performance of 15–17 clones from wild grapevines and from commercial vineyards when reared on ‘Cabernet Sauvignon’ and ‘AxR # 1’. Phylloxera from commercial vineyards had much higher overall performance on ‘Cabernet Sauvignon’ than did phylloxera from the wild grapevines. Phylloxera from the commercial vineyard also had higher performance on ‘Cabernet Sauvignon’ than on ‘AxR′ 1’ but the performance of the phylloxera from wild and commercial grapes did not differ on ‘AxR # 1’. Our results show that there is genetic variation in traits related to performance on a resistant rootstock within these indigenous non-pest populations of phylloxera, but not among them. The pattern of performance of pest and non-pest populations on two commercial cultivars suggests that current levels of phylloxera performance on crop cultivars are the result of adaptation to those cultivars which has occurred while phylloxera has been associated with viticulture. Implications of these results for understanding the recent adaptation of phylloxera to ‘AxR # 1’ in California are also discussed.