菠萝洁粉蚧的分子鉴定、遗传结构及其控制基础研究
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摘要
粉蚧类昆虫隶属于半翅目Hemiptera粉蚧科Pseudococcidae,可以为害多种作物、森林树种和观赏植物,是一类重要的经济害虫。菠萝洁粉蚧Dysmicoccus brevipes (Cockerell)、新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley均隶属于半翅目粉蚧科洁粉蚧属Dysmicoccus,是菠萝的主要害虫。菠萝洁粉蚧、新菠萝灰粉蚧不仅可以刺吸菠萝汁液,使菠萝植株长势衰弱,果实发育不良,分泌的蜜露容易引起煤烟病,同时还是菠萝重要病害凋萎病病毒(PMWaV)的主要传播媒介昆虫
目前,由于受到粉蚧分类、鉴定及饲养技术的限制,关于我国南方粉蚧系统发育及生物学方面的研究较少,而关于菠萝洁粉蚧生物学、生态学及种群遗传与进化的研究更是鲜有报道。研究南方常见粉蚧的核糖体及线粒体基因序列是南方粉蚧分子鉴定的基础。研究菠萝洁粉蚧D. brevipes、新菠萝灰粉蚧D. neobrevipes遗传分化、生物学、生态学及控制技术,不仅可以为我国南方粉蚧的研究提供参考,更是菠萝重要病害——菠萝凋萎病有效控制的前提。
本学位论文围绕菠萝粉蚧的遗传结构及综合防治技术这一主题,明确了菠萝洁粉蚧D. brevipes、新菠萝灰粉蚧D. neobrevipes在我国的分布和危害；对包括菠萝洁粉蚧、新菠萝灰粉蚧在内的我国12种粉蚧的分子鉴定进行了初步研究；并在我国菠萝洁粉蚧不同地理种群的遗传结构及其与夏威夷、巴西、菲律宾、泰国菠萝洁粉蚧亲缘关系分析及生物学、生态学研究的基础上,对我国菠萝洁粉蚧的起源和综合防治技术进行了探讨。主要内容如下：
1.我国热区常见粉蚧调查的及分子鉴定
采用形态学和核糖体18S、28S及线粒体COI基因片段序列比对相结合的方法对我国热区采集的粉蚧进行鉴定,初步明确了菠萝洁粉蚧、新菠萝灰粉蚧在我国的分布和危害。菠萝洁粉蚧在我国海南、广东、广西、云南和福建5省区均有分布,主要为害菠萝根部、叶片和果实。新菠萝灰粉蚧目前主要分布在海南和广东,主要为害剑麻叶片的基部,在海南乐东发现可为害菠萝的叶片和果实,但一般不为害根部；此外,还在粉蕉上发现新菠萝灰粉蚧的为害。
对菠萝洁粉蚧、新菠萝灰粉蚧等9种粉蚧(14个种群)核糖体基因18S、28S进行测序,结合GenBank中同源序列,对不同粉蚧的18S、28S序列进行了多重排比和聚类分析。序列分析结果表明,18S、28S适合于粉蚧种级的分子鉴定,榕树粉蚧Pseudococcus baliteus Lit两个种群核糖体基因18S、28S序列遗传距离约0.014,该蚧虫可能是至少含有两个隐存谱系或姊妹种的复合种。臀纹粉蚧属Planococcus的3种粉蚧柑橘臀纹粉蚧Planococcus citri (Risso)、大洋臀纹粉蚧Planococcus minor (Maskell)、无花果臀纹粉蚧Planococcus ficus (Signoret)的核糖体基因18S、28S序列具有高度相似性,但线粒体COI基因序列有33个变异位点,可作为3个近缘种分子鉴定的依据。
2.我国菠萝洁粉蚧遗传分化和原产地分析
对我国菠萝产区10个县市14份菠萝洁粉蚧标本及夏威夷1份菠萝洁粉蚧标本的核糖体ITS2、线粒体COI、Cytb基因部分片段序列进行了测序,结合巴西、泰国、菲律宾的菠萝洁粉蚧标Genebank中同源序列对我国菠萝洁粉蚧遗传结构及原产地进行了分析。结果表明,菠萝洁粉蚧D. brevipes以孤雌生殖为主,其遗传变异较小。我国菠萝洁粉蚧D. brevipes不同地理种群可以分为3个不同的枝系,即：以曲界(Qujie)为代表的大陆枝系、以海南琼海(Qionghai)为代表的琼海枝系和以海南万宁(Wanning)为代表的万宁枝系。夏威夷种群与巴西、泰国种群可能拥有最近的亲缘关系,其次是夏威夷种群与菲律宾种群,然后是夏威夷种群与我国大陆种群和海南琼海种群,而我国海南万宁种群与其它各种群均具有较远的亲缘关系,我国海南万宁种群可能是菠萝洁粉蚧的一个隐存谱系。
我国菠萝洁粉蚧各单倍型的分布揭示了我国菠萝洁粉蚧的扩散路径,我国大陆菠萝洁粉蚧、海南琼海菠萝洁粉蚧很可能来自菲律宾,这与19世纪末期20世纪初我国从南洋大量引进菠萝种苗的史实相符；我国菠萝洁粉蚧海南万宁谱系可能来源于夏威夷、巴西、泰国、菲律宾之外的某个区域,亦或者我国菠萝洁粉蚧海南万宁谱系早在上世纪初从南洋大量引进菠萝种植品种前就已经在我国海南万宁地区定殖。我国菠萝洁粉蚧单倍型的分布揭示了我国菠萝洁粉蚧的扩散路径,其扩散路径与菠萝种苗调运路径一致。
3.菠萝洁粉蚧饲养技术及生物学生态学研究
采用南瓜和蛭石相结合的方法,成功建立了菠萝洁粉蚧实验种群。饲养1个月后菠萝洁粉蚧增长系数达20倍以上。菠萝洁粉蚧在我国以孤雌生殖为主,鲜见雄虫；菠萝洁粉蚧为不完全变态昆虫,若虫3龄,年发生5-7代。不同温度(恒温、梯度温度)对菠萝洁粉蚧的生长发育和种群数量均有一定的影响。供试温度下,南瓜饲养菠萝洁粉蚧雌虫世代历期介于42.40-205.00天之间,高于29℃及低于21℃时菠萝洁粉蚧生长发育受到一定的抑制；菠萝洁粉蚧种群趋势指数分析表明,菠萝洁粉蚧最适恒温为24℃(I=39.86),最适梯度温度为21～26℃(I=57.36),梯度温度比恒温更有利于菠萝洁粉蚧的生长发育和种群的增长。
菠萝洁粉蚧喜欢在集聚在隐蔽环境处生活,常集聚在根部或者叶腋及果实凹陷处为害,菠萝洁粉蚧田间分布呈聚集分布,自然种群年发生动态呈单峰型,雷州半岛地区10～12月份是菠萝洁粉蚧自然种群数量增长最快的时期,菠萝洁粉蚧自然种群数量高峰期位于12月下旬至于1月上旬。气温和降雨量是影响菠萝洁粉蚧自然种群数量的关键因素。
4.菠萝洁粉蚧的综合防治技术
就8种化学农药对菠萝洁粉蚧雌性成虫及2龄若虫的毒力进行了测定,各药剂对菠萝洁粉蚧的致死浓度表现出一定的差异。从LC50来看,各药剂对菠萝洁粉蚧成虫毒力大小依次为：啶虫脒>毒死蜱>杀扑磷>氧化乐果>噻嗪酮>乐果>吡虫啉>哒螨灵,对菠萝洁粉蚧2龄若虫毒力大小依次为：啶虫脒>毒死蜱>杀扑磷>噻嗪酮>氧化乐果>吡虫啉>乐果>哒螨灵。而对菠萝洁粉蚧成虫LC90浓度所对应的稀释倍数从高到底依次是：48%毒死蜱EC(1286.73×)、3%啶虫脒EC(843.17×)、40%杀扑磷EC(750.20×)、70%吡虫啉WG(647.19×)、40%氧化乐果EC(606.52×)、40%乐果EC(350.66×)、25%噻嗪酮WP(275.15×)、15%哒螨灵EC(31.79×),对菠萝洁粉蚧2龄若虫LC90浓度所对应的稀释倍数从高到底依次是：48%毒死蜱EC(2739.88×)、40%杀扑磷EC(1991.04×)、3%啶虫脒EC(1252.61×)、70%吡虫啉WG(1021.51×)、25%噻嗪酮WP(639.58×)、40%氧化乐果EC(475.93×)、40%乐果EC(309.86×)、15%哒螨灵EC(59.85×)。吡虫·噻嗪酮等对菠萝洁粉蚧具有良好的防治效果,种苗浸泡处理效果尤为明显。
几种常用的杀虫杀螨剂对菠萝洁粉蚧的防治效果及其在菠萝上的安全间隔期的研究表明：种苗浸泡处理比田间喷雾处理对菠萝洁粉蚧的防治效果好,吡虫·噻嗪酮、吡虫啉、杀扑磷(速扑杀)等对菠萝洁粉蚧具有良好的防治效果,吡虫·噻嗪酮、吡虫啉、杀扑磷等药剂浸泡处理的菠萝植株种植4个月后未发现菠萝洁粉蚧为害,对菠萝洁粉蚧的防治效果达100%；氧化乐果、噻嗪酮、炔螨特、吡虫啉等药剂在菠萝上的降解速度慢,安全间隔期长,杀扑磷、毒死蜱、哒螨灵等药剂在菠萝上降解速度快,安全间隔期短。菠萝种前进行种苗浸泡处理,收获时果实均未检出农药残留。
在田间用蚂蚁毒饵诱杀蚂蚁可以降低菠萝洁粉蚧的种群数量,且蚂蚁毒饵诱杀蚂蚁的地块菠萝凋萎病发病率明显低于对照区。49℃处理2h后各龄菠萝洁粉蚧大量死亡,处理4～5h后各龄菠萝洁粉蚧全部死亡。经过45℃处理24h及49℃处理12h后的菠萝吸芽种植后能够正常生长,因此通过菠萝吸芽(腋芽)高温(49℃)处理可以达到菠萝洁粉蚧植物检疫处理的目的。
菠萝洁粉蚧的的天敌有香港角毛瓢虫H. hisamatsui、长管小瓢虫S.(P.) tenuis、细毛小瓢虫S.(P.) syoitii、台毛艳瓢虫P. taoi、丽草蛉C. formosa及瘿蚊(Aphidoletes
Mealybugs belonging to Hemiptera, Pseudococcidae, are one of the most economically damaging groups of insects on food crops and ornamental plants. Pink pineapple mealybug (PPM), Dysmicoccus brevipes (Cockerell), and gray pineapple mealybug (GPM), Dysmicoccus neobrevipes Beardsley,(Hemiptera:Pseudococcidae) are widespread plant-sucking insect and paid more and more attention by people for their role in promoting Mealybug Wilt of Pineapple (MWP), the most widely distributed and devastating disease of pineapple. Vector control operation is the mast controlling measure of most virus diseases, and that's the same to Mealybug Wilt of Pineapple.
The studies on biology, ecology and population evolution will play an important decisive role for a pest efficient controlling. Currently, few sthdies on D. brevipes&D. neobrevipes focus on the ecology and the population genetics and evolution, largely because the difficulty in mass rearing and the lack of technique of quick and accurate identification. For the efficient controlling of Mealybug Wilt of Pineapple, the distribution and damagement of D. brevipes&D. neobrevipes in south China were surveyed. And the molecular identification of mealybugs, genetic relationship among different geographic populations of D. brevipes was researched in this study. Finally, the geographic origin and the integrated control techniques were disscused based on the studies of the genetic differentiation, biology and ecology of D. brevipes. The main results are as follows:
1. The occurrence, damaging and identification of mealybugs in south China
The occurrence and damaging of mealybugs in south China were investigated, and the molecular identification of12species of mealybug were studied based on DNA sequences analyzing. There are10species of mealybug most important in south China, including Dysmicoccus brevipes, Dysmicoccus neobrevipes, Planococcus minor, Planococcus citri, Pseudococcus comstock, Pseudococcus baliteus, Saccharicoccus sacchari, Phenacoccus solenopsis, Oracella acuta, and Ferrisia virgata. Alignment of sequenced18S&28S showed that they were highly conserved within intraspecific taxa but that significant genetic distances existed between interspecific taxa. Comparison and analysis of the genetic distance led to the clear conclusion that18S&28S is suitable only for species-level identification. The mtDNA COI genes of P. minor and P. citri had been sequenced, for rDNA18S&28S failed to distinguish them from each other by molecular identification. The genetic distance between the collections of P. baliteus from two different hosts was approximately0.014. These data suggest that P. baliteus might be a species complex which includes at least two cryptic lineages or sibling species.
2. The genetic differentiation and the geographic origin of D. brevipes in China
We found that the pink pineapple mealybug, D. brevipes was the main mealybug on pineapple in China, and only one pineapple orchard at Ledong of Hainan was found damaged by gray pineapple mealybug, D. neobrevipes. Pink pineapple mealybugs were collected from10counties of5provinces in China, and mealybug samples in Hawaii were obtained. Partial ITS2, COI&Cytb sequences were sequenced for each sample. Four haplotypes were found in China, one from mainland China, three from Hainan island. Phylogenetic analyses suggest that the most pink pineapple mealybugs in mainland China were likely closely related to populations of mealybugs in Philippines, and the source of D. brevipes in Hawaii was very likely the mealybug lineage living predominantly in Brazil and Thailand. However mealybugs from Wanning, Hainan Pronvince, China, represent a different lineages that were clearly diverged from other populations. There is no conclusive evidence for a recent introduction of D. brevipes into Wanning. Possibly the lineage present in Wanning was introduced from an as yet unsampled native lineage in Oceania or Europe. Also, it seems reasonable to contend that the Wanning population had been established long before the introductions of the early20th century.
3. The biology and ecology of D. brevipes
A mass rearing program was developed for the pink pineapple mealybug. In this program, squash was used to rear PPM and vermiculite was used to remove the honeydew from the squash surface. More than2000PPMs per individual squash were produced after a month100mature PPM adults inoculated.The normal means of reproduction of PPM is parthenogernesis, and rare male adult of PPM was found in China. The females of pink pineapple mealybug, D. brevipes, have three larval instars prior to the adult stage, and5-7generations per year. The lifespans of the female are45.4and205.0days at27℃and16℃respectively, and the the population trend index range from0.68to39.86at different constant temperature. The population trend is incremental in most months one year, and the population trend index is57.36at temperature gradients of21～26℃. It may show that the variable temperature is more suitable to D. brevipes population growth than constant temperature.
The pink pineapple mealybug, D. brevipes is generally cryptic in habitat, and is normally found gathering in crowds and groups on the base of the leaf or stem or on the roots of the plant, or in the grooves of the fruit, often under the soil. The spatial distribution pattern of the natural population of D. brevipes is aggregation distribution. The natural population of PPM develope fastest from October to December, and the fastigium of PPM emerged in the first days of Januuary in south China. Temperature and rainfall are the critical factor of population developping of PPM.
4. The sustainable controlling techniques of pink pineapple mealybug
The toxicity of several kinds of chemical insecticides to the second instar larvae, and female adult of pink pineapple mealybug, D.brevipes, was respectively determined in laboratory conditions. The results showed that the toxicity of acetamiprid, chlorpyrifos and methidathion to the pink pineapple mealybug was evidently higher than that of other tested insecticides. And test in field indicate that the complex pesticides, imidacloprid complex with buprofezin, is efficient to control the population of D.brevipes, and pesticide application with suckers soaking is more efficient than spraying to control the population of D.brevipes.
Here we obtained several efficient pesticides of controlling Dysmicoccus brevipes by field testament too. It's hardly found any D. brevipes on pineapple plant treaded by pineapple suckers soaking in4months.
We determined the residue of some pesticides in pineapple fruit which hand been sprayed insecticide in different intervals, by using GC-FPD, GC-MS and HPLC. There is a tendency that shorter interval of pesticide application lead to higher residue, and lower residue when interval is longer. We found not any residue in pineapple fruits of suckers soaking with pesticides before planting. The results show that there are evidently different for the residue of different pesticide. Omethoate and imidacloprid are more residues than any other pesticides, and methidathion is hardly any residue relatively.
The population of Dysmicoccus brevipes theated with ant bait in field is less than that of check plot, and the incidence of Mealybug Wilt of Pineapple (MWP) is2.38%, notable less than that of check plot. Heat treatment of sucker is an effective control measure of D. brevipes, owing to D. brevipes in all stages can't survive the process of5hours in49℃and sucker can survive the process of10hours in49℃.
There are more than7species of natural enemies of D. brevipes in Leizhou Peninsula, and the main natural enemies are Chrysopa formosa Brauer, Horniolus hisamatsui Miyatake, Scymnus (Pullus) tenuis Yang and Aphidoletes sp., and H. hisamatsui&S.(P.) syoitii are reported the first time in Gunagdong province. The functional and interfering response of Horniolus hisamatsui Miyatake to D. brevipes was carried out in condition of laboratory. The result showed that their functional response is fit to Holling Ⅱ type. The predating number increased with the density of the D. brevipes and the maximal predation numbers of1st,2nd,3rd,4th instar larva and adult of H, hisamatsui were11.53,26.11,61.73,70.42, and74.07per day respectively. The searching efficiency decreased as the beetle density increased.
目前,由于受到粉蚧分类、鉴定及饲养技术的限制,关于我国南方粉蚧系统发育及生物学方面的研究较少,而关于菠萝洁粉蚧生物学、生态学及种群遗传与进化的研究更是鲜有报道。研究南方常见粉蚧的核糖体及线粒体基因序列是南方粉蚧分子鉴定的基础。研究菠萝洁粉蚧D. brevipes、新菠萝灰粉蚧D. neobrevipes遗传分化、生物学、生态学及控制技术,不仅可以为我国南方粉蚧的研究提供参考,更是菠萝重要病害——菠萝凋萎病有效控制的前提。
本学位论文围绕菠萝粉蚧的遗传结构及综合防治技术这一主题,明确了菠萝洁粉蚧D. brevipes、新菠萝灰粉蚧D. neobrevipes在我国的分布和危害；对包括菠萝洁粉蚧、新菠萝灰粉蚧在内的我国12种粉蚧的分子鉴定进行了初步研究；并在我国菠萝洁粉蚧不同地理种群的遗传结构及其与夏威夷、巴西、菲律宾、泰国菠萝洁粉蚧亲缘关系分析及生物学、生态学研究的基础上,对我国菠萝洁粉蚧的起源和综合防治技术进行了探讨。主要内容如下：
1.我国热区常见粉蚧调查的及分子鉴定
采用形态学和核糖体18S、28S及线粒体COI基因片段序列比对相结合的方法对我国热区采集的粉蚧进行鉴定,初步明确了菠萝洁粉蚧、新菠萝灰粉蚧在我国的分布和危害。菠萝洁粉蚧在我国海南、广东、广西、云南和福建5省区均有分布,主要为害菠萝根部、叶片和果实。新菠萝灰粉蚧目前主要分布在海南和广东,主要为害剑麻叶片的基部,在海南乐东发现可为害菠萝的叶片和果实,但一般不为害根部；此外,还在粉蕉上发现新菠萝灰粉蚧的为害。
对菠萝洁粉蚧、新菠萝灰粉蚧等9种粉蚧(14个种群)核糖体基因18S、28S进行测序,结合GenBank中同源序列,对不同粉蚧的18S、28S序列进行了多重排比和聚类分析。序列分析结果表明,18S、28S适合于粉蚧种级的分子鉴定,榕树粉蚧Pseudococcus baliteus Lit两个种群核糖体基因18S、28S序列遗传距离约0.014,该蚧虫可能是至少含有两个隐存谱系或姊妹种的复合种。臀纹粉蚧属Planococcus的3种粉蚧柑橘臀纹粉蚧Planococcus citri (Risso)、大洋臀纹粉蚧Planococcus minor (Maskell)、无花果臀纹粉蚧Planococcus ficus (Signoret)的核糖体基因18S、28S序列具有高度相似性,但线粒体COI基因序列有33个变异位点,可作为3个近缘种分子鉴定的依据。
2.我国菠萝洁粉蚧遗传分化和原产地分析
对我国菠萝产区10个县市14份菠萝洁粉蚧标本及夏威夷1份菠萝洁粉蚧标本的核糖体ITS2、线粒体COI、Cytb基因部分片段序列进行了测序,结合巴西、泰国、菲律宾的菠萝洁粉蚧标Genebank中同源序列对我国菠萝洁粉蚧遗传结构及原产地进行了分析。结果表明,菠萝洁粉蚧D. brevipes以孤雌生殖为主,其遗传变异较小。我国菠萝洁粉蚧D. brevipes不同地理种群可以分为3个不同的枝系,即：以曲界(Qujie)为代表的大陆枝系、以海南琼海(Qionghai)为代表的琼海枝系和以海南万宁(Wanning)为代表的万宁枝系。夏威夷种群与巴西、泰国种群可能拥有最近的亲缘关系,其次是夏威夷种群与菲律宾种群,然后是夏威夷种群与我国大陆种群和海南琼海种群,而我国海南万宁种群与其它各种群均具有较远的亲缘关系,我国海南万宁种群可能是菠萝洁粉蚧的一个隐存谱系。
我国菠萝洁粉蚧各单倍型的分布揭示了我国菠萝洁粉蚧的扩散路径,我国大陆菠萝洁粉蚧、海南琼海菠萝洁粉蚧很可能来自菲律宾,这与19世纪末期20世纪初我国从南洋大量引进菠萝种苗的史实相符；我国菠萝洁粉蚧海南万宁谱系可能来源于夏威夷、巴西、泰国、菲律宾之外的某个区域,亦或者我国菠萝洁粉蚧海南万宁谱系早在上世纪初从南洋大量引进菠萝种植品种前就已经在我国海南万宁地区定殖。我国菠萝洁粉蚧单倍型的分布揭示了我国菠萝洁粉蚧的扩散路径,其扩散路径与菠萝种苗调运路径一致。
3.菠萝洁粉蚧饲养技术及生物学生态学研究
采用南瓜和蛭石相结合的方法,成功建立了菠萝洁粉蚧实验种群。饲养1个月后菠萝洁粉蚧增长系数达20倍以上。菠萝洁粉蚧在我国以孤雌生殖为主,鲜见雄虫；菠萝洁粉蚧为不完全变态昆虫,若虫3龄,年发生5-7代。不同温度(恒温、梯度温度)对菠萝洁粉蚧的生长发育和种群数量均有一定的影响。供试温度下,南瓜饲养菠萝洁粉蚧雌虫世代历期介于42.40-205.00天之间,高于29℃及低于21℃时菠萝洁粉蚧生长发育受到一定的抑制；菠萝洁粉蚧种群趋势指数分析表明,菠萝洁粉蚧最适恒温为24℃(I=39.86),最适梯度温度为21～26℃(I=57.36),梯度温度比恒温更有利于菠萝洁粉蚧的生长发育和种群的增长。
菠萝洁粉蚧喜欢在集聚在隐蔽环境处生活,常集聚在根部或者叶腋及果实凹陷处为害,菠萝洁粉蚧田间分布呈聚集分布,自然种群年发生动态呈单峰型,雷州半岛地区10～12月份是菠萝洁粉蚧自然种群数量增长最快的时期,菠萝洁粉蚧自然种群数量高峰期位于12月下旬至于1月上旬。气温和降雨量是影响菠萝洁粉蚧自然种群数量的关键因素。
4.菠萝洁粉蚧的综合防治技术
就8种化学农药对菠萝洁粉蚧雌性成虫及2龄若虫的毒力进行了测定,各药剂对菠萝洁粉蚧的致死浓度表现出一定的差异。从LC50来看,各药剂对菠萝洁粉蚧成虫毒力大小依次为：啶虫脒>毒死蜱>杀扑磷>氧化乐果>噻嗪酮>乐果>吡虫啉>哒螨灵,对菠萝洁粉蚧2龄若虫毒力大小依次为：啶虫脒>毒死蜱>杀扑磷>噻嗪酮>氧化乐果>吡虫啉>乐果>哒螨灵。而对菠萝洁粉蚧成虫LC90浓度所对应的稀释倍数从高到底依次是：48%毒死蜱EC(1286.73×)、3%啶虫脒EC(843.17×)、40%杀扑磷EC(750.20×)、70%吡虫啉WG(647.19×)、40%氧化乐果EC(606.52×)、40%乐果EC(350.66×)、25%噻嗪酮WP(275.15×)、15%哒螨灵EC(31.79×),对菠萝洁粉蚧2龄若虫LC90浓度所对应的稀释倍数从高到底依次是：48%毒死蜱EC(2739.88×)、40%杀扑磷EC(1991.04×)、3%啶虫脒EC(1252.61×)、70%吡虫啉WG(1021.51×)、25%噻嗪酮WP(639.58×)、40%氧化乐果EC(475.93×)、40%乐果EC(309.86×)、15%哒螨灵EC(59.85×)。吡虫·噻嗪酮等对菠萝洁粉蚧具有良好的防治效果,种苗浸泡处理效果尤为明显。
几种常用的杀虫杀螨剂对菠萝洁粉蚧的防治效果及其在菠萝上的安全间隔期的研究表明：种苗浸泡处理比田间喷雾处理对菠萝洁粉蚧的防治效果好,吡虫·噻嗪酮、吡虫啉、杀扑磷(速扑杀)等对菠萝洁粉蚧具有良好的防治效果,吡虫·噻嗪酮、吡虫啉、杀扑磷等药剂浸泡处理的菠萝植株种植4个月后未发现菠萝洁粉蚧为害,对菠萝洁粉蚧的防治效果达100%；氧化乐果、噻嗪酮、炔螨特、吡虫啉等药剂在菠萝上的降解速度慢,安全间隔期长,杀扑磷、毒死蜱、哒螨灵等药剂在菠萝上降解速度快,安全间隔期短。菠萝种前进行种苗浸泡处理,收获时果实均未检出农药残留。
在田间用蚂蚁毒饵诱杀蚂蚁可以降低菠萝洁粉蚧的种群数量,且蚂蚁毒饵诱杀蚂蚁的地块菠萝凋萎病发病率明显低于对照区。49℃处理2h后各龄菠萝洁粉蚧大量死亡,处理4～5h后各龄菠萝洁粉蚧全部死亡。经过45℃处理24h及49℃处理12h后的菠萝吸芽种植后能够正常生长,因此通过菠萝吸芽(腋芽)高温(49℃)处理可以达到菠萝洁粉蚧植物检疫处理的目的。
菠萝洁粉蚧的的天敌有香港角毛瓢虫H. hisamatsui、长管小瓢虫S.(P.) tenuis、细毛小瓢虫S.(P.) syoitii、台毛艳瓢虫P. taoi、丽草蛉C. formosa及瘿蚊(Aphidoletes
Mealybugs belonging to Hemiptera, Pseudococcidae, are one of the most economically damaging groups of insects on food crops and ornamental plants. Pink pineapple mealybug (PPM), Dysmicoccus brevipes (Cockerell), and gray pineapple mealybug (GPM), Dysmicoccus neobrevipes Beardsley,(Hemiptera:Pseudococcidae) are widespread plant-sucking insect and paid more and more attention by people for their role in promoting Mealybug Wilt of Pineapple (MWP), the most widely distributed and devastating disease of pineapple. Vector control operation is the mast controlling measure of most virus diseases, and that's the same to Mealybug Wilt of Pineapple.
The studies on biology, ecology and population evolution will play an important decisive role for a pest efficient controlling. Currently, few sthdies on D. brevipes&D. neobrevipes focus on the ecology and the population genetics and evolution, largely because the difficulty in mass rearing and the lack of technique of quick and accurate identification. For the efficient controlling of Mealybug Wilt of Pineapple, the distribution and damagement of D. brevipes&D. neobrevipes in south China were surveyed. And the molecular identification of mealybugs, genetic relationship among different geographic populations of D. brevipes was researched in this study. Finally, the geographic origin and the integrated control techniques were disscused based on the studies of the genetic differentiation, biology and ecology of D. brevipes. The main results are as follows:
1. The occurrence, damaging and identification of mealybugs in south China
The occurrence and damaging of mealybugs in south China were investigated, and the molecular identification of12species of mealybug were studied based on DNA sequences analyzing. There are10species of mealybug most important in south China, including Dysmicoccus brevipes, Dysmicoccus neobrevipes, Planococcus minor, Planococcus citri, Pseudococcus comstock, Pseudococcus baliteus, Saccharicoccus sacchari, Phenacoccus solenopsis, Oracella acuta, and Ferrisia virgata. Alignment of sequenced18S&28S showed that they were highly conserved within intraspecific taxa but that significant genetic distances existed between interspecific taxa. Comparison and analysis of the genetic distance led to the clear conclusion that18S&28S is suitable only for species-level identification. The mtDNA COI genes of P. minor and P. citri had been sequenced, for rDNA18S&28S failed to distinguish them from each other by molecular identification. The genetic distance between the collections of P. baliteus from two different hosts was approximately0.014. These data suggest that P. baliteus might be a species complex which includes at least two cryptic lineages or sibling species.
2. The genetic differentiation and the geographic origin of D. brevipes in China
We found that the pink pineapple mealybug, D. brevipes was the main mealybug on pineapple in China, and only one pineapple orchard at Ledong of Hainan was found damaged by gray pineapple mealybug, D. neobrevipes. Pink pineapple mealybugs were collected from10counties of5provinces in China, and mealybug samples in Hawaii were obtained. Partial ITS2, COI&Cytb sequences were sequenced for each sample. Four haplotypes were found in China, one from mainland China, three from Hainan island. Phylogenetic analyses suggest that the most pink pineapple mealybugs in mainland China were likely closely related to populations of mealybugs in Philippines, and the source of D. brevipes in Hawaii was very likely the mealybug lineage living predominantly in Brazil and Thailand. However mealybugs from Wanning, Hainan Pronvince, China, represent a different lineages that were clearly diverged from other populations. There is no conclusive evidence for a recent introduction of D. brevipes into Wanning. Possibly the lineage present in Wanning was introduced from an as yet unsampled native lineage in Oceania or Europe. Also, it seems reasonable to contend that the Wanning population had been established long before the introductions of the early20th century.
3. The biology and ecology of D. brevipes
A mass rearing program was developed for the pink pineapple mealybug. In this program, squash was used to rear PPM and vermiculite was used to remove the honeydew from the squash surface. More than2000PPMs per individual squash were produced after a month100mature PPM adults inoculated.The normal means of reproduction of PPM is parthenogernesis, and rare male adult of PPM was found in China. The females of pink pineapple mealybug, D. brevipes, have three larval instars prior to the adult stage, and5-7generations per year. The lifespans of the female are45.4and205.0days at27℃and16℃respectively, and the the population trend index range from0.68to39.86at different constant temperature. The population trend is incremental in most months one year, and the population trend index is57.36at temperature gradients of21～26℃. It may show that the variable temperature is more suitable to D. brevipes population growth than constant temperature.
The pink pineapple mealybug, D. brevipes is generally cryptic in habitat, and is normally found gathering in crowds and groups on the base of the leaf or stem or on the roots of the plant, or in the grooves of the fruit, often under the soil. The spatial distribution pattern of the natural population of D. brevipes is aggregation distribution. The natural population of PPM develope fastest from October to December, and the fastigium of PPM emerged in the first days of Januuary in south China. Temperature and rainfall are the critical factor of population developping of PPM.
4. The sustainable controlling techniques of pink pineapple mealybug
The toxicity of several kinds of chemical insecticides to the second instar larvae, and female adult of pink pineapple mealybug, D.brevipes, was respectively determined in laboratory conditions. The results showed that the toxicity of acetamiprid, chlorpyrifos and methidathion to the pink pineapple mealybug was evidently higher than that of other tested insecticides. And test in field indicate that the complex pesticides, imidacloprid complex with buprofezin, is efficient to control the population of D.brevipes, and pesticide application with suckers soaking is more efficient than spraying to control the population of D.brevipes.
Here we obtained several efficient pesticides of controlling Dysmicoccus brevipes by field testament too. It's hardly found any D. brevipes on pineapple plant treaded by pineapple suckers soaking in4months.
We determined the residue of some pesticides in pineapple fruit which hand been sprayed insecticide in different intervals, by using GC-FPD, GC-MS and HPLC. There is a tendency that shorter interval of pesticide application lead to higher residue, and lower residue when interval is longer. We found not any residue in pineapple fruits of suckers soaking with pesticides before planting. The results show that there are evidently different for the residue of different pesticide. Omethoate and imidacloprid are more residues than any other pesticides, and methidathion is hardly any residue relatively.
The population of Dysmicoccus brevipes theated with ant bait in field is less than that of check plot, and the incidence of Mealybug Wilt of Pineapple (MWP) is2.38%, notable less than that of check plot. Heat treatment of sucker is an effective control measure of D. brevipes, owing to D. brevipes in all stages can't survive the process of5hours in49℃and sucker can survive the process of10hours in49℃.
There are more than7species of natural enemies of D. brevipes in Leizhou Peninsula, and the main natural enemies are Chrysopa formosa Brauer, Horniolus hisamatsui Miyatake, Scymnus (Pullus) tenuis Yang and Aphidoletes sp., and H. hisamatsui&S.(P.) syoitii are reported the first time in Gunagdong province. The functional and interfering response of Horniolus hisamatsui Miyatake to D. brevipes was carried out in condition of laboratory. The result showed that their functional response is fit to Holling Ⅱ type. The predating number increased with the density of the D. brevipes and the maximal predation numbers of1st,2nd,3rd,4th instar larva and adult of H, hisamatsui were11.53,26.11,61.73,70.42, and74.07per day respectively. The searching efficiency decreased as the beetle density increased.
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