Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode(Ditylenchus destructor) populations from Chinese medicinal herbs

The potato rot nematode(Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2–D3of 28S-rDNA genes of D. destructor were compared and analyzed. Nine types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. Ten haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes(A–C) were found in 7 populations isolated from potato,sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes(A–G) according to Subbotin' system. These unique haplotypes were different from haplotypes A–G, and we named them as haplotypes H–N. The present results showed that a total of 14 haplotypes(A–N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITSrDNA and D2–D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing haplotype A from sweet potato and the other containing haplotypes B–N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.

狄氏瓦螨(Varroa destructor)的研究进展

狄氏瓦螨(Varroa destructor)是严重危害西方蜜蜂(Apismellifera)的一种寄生螨,近几年对狄氏瓦螨的研究有了新的进展,本文就血统进化、生理机制、携带病原菌和最新的防治方法等领域作一综述。

瓦螨(Varroa destructor)寄生密度与蜜蜂卷翅病毒动态复制相关性研究

病毒复制动态变化对揭示病毒病致病的机理具有重要意义。本研究以蜜蜂卷翅病毒(deformed wing virus,简写为DWV)为模型,使用荧光反转录定量PCR方法检测DWV病毒隐性感染蜂群在体壁寄生螨侵袭压力下工蜂体内病毒基因表达量的变化动态,以揭示外界生物压力与隐性感染蜜蜂体内病毒动态复制的相关性。结果显示:弱群势蜜蜂体内DWV基因表达量与引入瓦螨密度呈显著正相关(第3天:R2=0.82;第7天:R2=0.99;双向方差分析F=5.059,P=0.0171);在瓦螨密度增至30%,处理后第7天时DWV浓度达最大值;强群势的蜜蜂群体引入瓦螨后,蜜蜂体内DWV基因表达量在处理后第3天表现出与弱群相似的变化规律(R2=0.88;双向方差分析F=11.74,P=0.001 3),但在处理后第7天时,供试蜜蜂体内DWV浓度整体下降,且不同瓦螨处理水平试验组间的DWV浓度差异不明显(R2=0.66)。结果显示,强群势蜂群的工蜂可有效抑制病毒拷贝量的持续增长,从而表现较强抗病性,而弱群势蜜蜂因病毒动态复制的不可逆激增而导致病毒病暴发。该研究结果在一定程度上解释了弱群势蜜蜂高病毒病易感性和年周损失的原因。

马铃薯茎线虫Ditylenchus destructor Thorne,1945的描述

1995～ 2 0 0 2年期间 ,在辽宁省、吉林省、河北省、山东省的多种植物的根际土壤里发现马铃薯茎线虫Dity lenchusdestructorThorne,1 945 。

Rapid Molecular Identification of Tribolium destructor

In this study, a rapid molecular identification method of Tribolium destructor was established with PCR and PCR-RFLP （polymerase chain reaction-restriction fragment length polymorphism） technology. According to the results, （ 1 ） with PCR method, specific primers were designed based on CO1 gene of T. destructor for PCR amplification, and electrophoresis detection confirmed that PCR method could be used to rapidly and accurately identify T. destructor; （2） with PCR-RFLP method, two pairs of degenerate primers were used to amplify CO1 gene of Tribolium species, PCR products were digested with HindIII and detected by electrophoresis, results indicated that PCR-RFLP method could also be used for rapid identification of T. destructor in quarantine practice.

Relationship of Secreted Salivary Protein Variants to Virulence in Hessian Fly (<i>Mayetiola destructor</i>(Say))

Salivary proteins are the initial contact between sedentary insect pests and their host plants. It is expected that one or more salivary proteins mediate the interaction between Hessian fly and wheat, in which a feeding site is established to the benefit of the fly. A survey of 52 loci annotated as insect secreted salivary proteins was conducted in 384 individuals evenly distributed among eight biotypes of Hessian fly (B, C, D, E, GP, L, O, and vH9). Amplicons were sequenced with Illumina, and sequence reads were aligned to the reference sequences from which primers had been designed. Positions of consistent base variation (998 in all) were identified and tabulated by biotype. No varying position was associated with biotype-wide virulence to any one of wheat resistance genes H3, H5, H6, H7/H8, H9, H11, H13, and H26. The multiplate pooling strategy utilized in this study is an effective, affordable way to reveal the genotype of hundreds of individuals at tens of genetic loci.

Preliminary Researches Regarding the Effectiveness of the Formic Acid Treatment on Varroa(Varroa destructor) Found in the Artificially Decapped Bee Brood

The objective of the study was to establish the effect of formic acid on varroa(Varroa destructor),inside the capped brood cells,artificially decapped.The experiments were carried out in 2017-2018 on honeybee colonies infested with varroa(V.destructor),in a research apiary belonging to the Institute for Beekeeping Research and Development in Bucharest.The decapping method in the present researches used the decapping fork to scrape the capped comb,without affecting the brood,in order to open it for an effective treatment.The combined treatment method was applied on honeybee colonies as a whole,as well as on brood combs,without bees,put in a special treatment box.The researches were focused on establishing the mortality level of various stages of varroa in artificially decapped brood,in normal colony and separately,as well as to make observations on the effect of formic acid on viability of capped bee brood,artificially decapped.The results show a high mortality of varroa,especially the protonymphs and deutonymphs stages(over 80%).The main conclusion is that the brood decapping method combined with formic acid treatment could be a useful technique to control varroa infestation,both in brood and honeybees,shortening strongly the treatment duration as compared to the usual treatments,increasing the efficacy of treatment by cutting the life cycle of varroa in brood.

Wheat Yield in a Tolerant Winter Wheat Line Infested by Hessian Fly (<i>Mayetiola destructor</i>)

The Hessian fly, Mayetiola destructor, is a harmful pest of wheat (Triticum aestivum). Pioneer variety 25R78 is putatively tolerant, meaning that the plant can survive successful Hessian fly infestation with reduced growth effects. To understand if Hessian fly-tolerance in wheat results in reduced yield effects and to analyze the economic feasibility of tolerant wheat as a Hessian fly control method, this study focused on analyzing the effect of infestation on tolerant wheat yield. This study analyzed tolerant Pioneer variety 25R78, resistant Pioneer brand variety 25R32, and susceptible Pioneer brand variety 25R47 through harvest. Treated plants were infested using a plastic cover and allowing 1 - 2 female flies to lay eggs for two hours. We measured head, fertile head number and tiller number. Seeds were analyzed by measuring total seed number and weight, as well as average seed number and weight. Tolerant and resistant plants showed no significant effects on yield in comparison to susceptible wheat. The infested tolerant plants were comparable in yield to infested resistant plants. Therefore, we propose that tolerance incorporated into wheat varieties will lower selection pressure on Hessian fly populations and increase the durability of these wheat lines.

腐烂茎线虫(Ditylenchus destructor Thorne,1945)生物学研究进展

腐烂茎线虫(Ditylenchus destructor Thorne,1945)是世界上重要的植物病原线虫和我国全国农业植物检疫性有害生物,为害甘薯、马铃薯、大蒜等多种地下结实作物,在许多杂草和真菌上也能生长和繁殖。腐烂茎线虫喜凉怕热、喜湿怕干,主要通过寄主作物的无性繁殖材料传播和扩散。在不同作物上,它的田间持续存活、侵染和损害表现存在明显差异,但相关原因和机制不甚明确。本文综述了腐烂茎线虫的地理分布、寄主范围、生殖发育、侵染循环、环境适应能力及其存活寄生相关分子机制,指出了需要深入探讨的主要问题,有望为这种危险性有害生物的预警防控研究和实践提供新的思路和有用参考。

噻唑膦与硅肥联合使用对甘薯茎线虫病的防治效果

为了有效控制甘薯茎线虫病的危害,通过盆栽试验和田间试验分别研究了不同种类硅肥及噻唑膦对甘薯茎线虫病的防控效果。室内试验结果表明,单独施用硅肥对甘薯茎线虫病防治效果较差,但10%噻唑膦颗粒剂与不同种类硅肥联合使用后可显著降低甘薯种薯及薯苗受线虫侵染率,其效果优于单独使用等量的10%噻唑膦颗粒剂。田间施用噻唑膦后再增施硅肥,可使噻唑膦防治效果显著增高,甘薯受害率降低,甘薯产量提高。其中硅肥途保佳15 L/hm^(2)与10%噻唑膦颗粒剂18 kg/hm^(2)或22.5 kg/hm^(2)联合使用后,对茎线虫病的防治效果均达到87%以上,显著高于10%噻唑膦颗粒剂22.5 kg/hm^(2)处理。联合施用硅肥可以在减少噻唑膦用量情况下保持对茎线虫病较高防效,具有较好的推广应用前景。

基于MaxEnt模型的腐烂茎线虫在内蒙古地区适生区预测

【目的】为保障马铃薯产业健康发展,加强腐烂茎线虫的检疫防控工作,对其在内蒙古地区的适生区进行预测分析。【方法】根据腐烂茎线虫在我国最新的分布和环境数据,通过MaxEnt生态位模型和ArcGIS软件,预测当前和未来气候条件下腐烂茎线虫在内蒙古地区的适生区。【结果】MaxEnt模型的AUC平均值为0.929,预测结果准确可靠。预测结果显示:当前气候条件下腐烂茎线虫在内蒙古地区的适生区面积为41.8万km^(2),占全区面积的35.31%。研究表明,在2021—2040年和2041—2060年,腐烂茎线虫在内蒙古地区的总适生区面积将不断扩大;其中在SSP1_2.6气候情景下,在赤峰市、通辽市和兴安盟将出现腐烂茎线虫的高适生区;在SSP3_7.0气候情景下,在鄂尔多斯市、赤峰市、通辽市将出现腐烂茎线虫高适生区。利用Jackknife刀切法计算得到影响腐烂茎线虫分布的主导环境因子为温度季节性变化方差、最湿月降雨量、最冷季平均温、昼夜温差月均温、最湿季平均温。【结论】腐烂茎线虫在内蒙古地区有进一步扩散的风险,建议在未来气候情景下出现腐烂茎线虫高度适生区的盟市,应加强调运检疫和疫情监测,防止腐烂茎线虫进一步扩散。

Genes important for survival or reproduction in Varroa destructor identified by RNAi

The Varroa mite,(Varroa destructor),is the worst threat to honey bee health worldwide.To explore the possibility of using RNA interference to control this pest, we determined the effects of knocking down various genes on Varroa mite survival and reproduction.Double-stranded RNA (dsRNA)of six candidate genes (Da,Pros26S,RpL8, RpL11,RpPO and RpS13)were synthesized and each injected into Varroa mites,then mite survival and reproduction were assessed.Injection of dsRNA for Da (Daughterless)and Pros26S (Gene for proteasome 26S subunit adenosine triphosphatase)caused a significant reduction in mite survival,with 3.57%±1.94% and 30.03%±11.43% mites surviving at 72 h post-inj ection (hpi),respectively.Control mites injected with green fluorescent protein (GFP)-dsRNA showed survival rates of 81.95%±5.03% and 82.36 ±2.81%,respectively. Injections of dsRNA for four other genes (RpL8,RpL11,RpPO and RpS13)did not affect survival significantly,enabling us to assess their effect on Varroa mite reproduction.The number of female offspring per mite was significantly reduced for mites injected with dsRNA of each of these four genes compared to their GFP-dsRNA controls.Knockdown of the target genes was verified by real-time polymerase chain reaction for two genes important for reproduction (RpL8,RpL11)and one gene important for survival (Pros26S). In conclusion,through RNA interference,we have discovered two genes important for mite survival and four genes important for mite reproduction.These genes could be explored as possible targets for the control of Varroa destructor in the future.

商洛市甘薯茎线虫病初步调查和防治措施研究

首次在商洛市3个田块中调查发现甘薯腐烂茎线虫病感染,感染率分别为5.80%、5.93%和4.98%,病情指数分别为2.72、3.28和2.74。实验室形态学观察和病原线虫,rDNA-ITS区PCR检测进一步确认了甘薯腐烂茎线虫的存在。跨地区购买易感作物种苗、易感作物轮作、种薯带病原线虫可能是商洛市发生甘薯腐烂茎线虫病的主要原因。生产中应提高认识,调整茬口;选择抗病品种,提倡高剪苗技术;加强检疫,严禁从疫区购买种苗;合理使用杀线虫农药。积极采取预防措施来遏制甘薯腐烂茎线虫病在商洛市的传播和蔓延。

Genes encoding a group of related small secreted proteins from the gut of Hessian fly larvae [Mayetiola destructor （Say）]

A group of related genes has been isolated and characterized from the gut of Hessian fly larvae [Mayetiola destructor （Say）]. Members in this group appear to encode proteins with secretary signal peptides at the N-terminals. The mature putative proteins are small, acidic proteins with calculated molecular masses of 14.5 to 15.3 kDa, and isoelectric points from 4.56 to 4.88. Northern blot analysis revealed that these genes are expressed predominantly in the gut of Hessian fly larvae and pupae. Two related genes, GIOK1 and GIOK2, were isolated as tandem repeats. Both genes contain three exons and two introns. The intron/exon boundaries were conserved in terms of amino acid encoding, suggesting that they arose by gene duplication. The fact that the frequency of this group of clones in a gut cDNA library higher than that of total cDNA clones encoding digestive enzymes suggested that this group of proteins may perform an important function in the gut physiology of this insect. However, the exact functions of these proteins are as yet known since no sequence similarity could be identified between these proteins and any known sequences in public databases using standard methods.

Association of Varroa destructor females in multiply infested cells of the honeybee Apis mellifera

The genetic diversity of Varroa destructor (Anderson &Trueman)is limited outside its natural range due to population bottlenecks and its propensity to inbreed.In light of the arms race between V.destructor and its honeybee (Apis mellifera L.)host, any mechanism enhancing population admixture of the mite may be favored.One way that admixture can occur is when two genetically dissimilar mites coinvade a brood cell, with the progeny of the foundresses admixing.We determined the relatedness of 393 pairs of V.destructor foundresses,each pair collected from a single bee brood cell (n =five colonies).We used six microsatellites to identify the genotypes of mites coinvading a cell and calculated the frequency of pairs with different or the same genotypes.We found no deviation from random coinvasion,but the frequency of cells infested by mites with different genotypes was high.This rate of recombination,coupled with a high transmission rate of mites,homogenized the allelic pool of mites within the apiary.

Varroa destructor mite infestations in capped brood cells of honeybee workers affect emergence development and adult foraging ability

Given recent higher declines in managed Apis mellifera honeybees,which are the most commonly managed bee species around the world(Garibaldi et al.2013),numerous health threats involved in the losses have been received great attention.To date,Varroa destructor mite infestation has become the biggest challenge in commercial beekeeping,which is considered as a thorn stuck in the throat of beekeepers.The Varroa mite threatens bee health by absorbing the fat body and hemolymph of immature and mature bees(Ramsey et al.2019).Varroa mite infestation during the development of worker bees was found to reduce birth weight,influence water content and protein level in hemolymph of newly emerged worker bees,decrease flight performance of drones,change gene expression patterns related to immune system of honeybees,and result in honeybee colony losses(Duay et al.2002;Bowen-Walker and Gunn 2010;Zhang et al.2010;Annoscia et al.2012;Dooremalen et al.2013;Locke 2016).The Varroa mite,as an ectoparasite of the honeybee,prefers living in the sealed brood cells(Bogdanov 2006).Thus,bees are more likely to become hosts of mites in the pupal stage.In this study,we systematically investigated the current impacts of V.destructor infestations on the development from capped larvae to emerged bees.

Changing climate patterns risk the spread of Varroa destructor infestation of African honey bees in Tanzania

Background:Climate change creates opportune conditions that favour the spread of pests and diseases outside their known active range.Modelling climate change scenarios is oftentimes useful tool to assess the climate analogues to unveil the potential risk of spreading suitability conditions for pests and diseases and hence allows development of appropriate responses to address the impending challenge.In the current study,we modelled the impact of climate change on the distribution of Varroa destructor,a parasitic mite that attacks all life forms of honey bees and remains a significant threat to their survival and productivity of bee products in Tanzania and elsewhere.Methods:The data about the presence of V.destructor were collected in eight regions of Tanzania selected in consideration of several factors including potentials for beekeeping activities,elevation(highlands vs.lowlands)and differences in climatic conditions.A total of 19 bioclimatic datasets covering the entire country were used for developing climate scenarios of mid-century 2055 and late-century 2085 for both rcp4.5 and rcp8.5.We thereafter modelled the current and future risk distribution of V.destructor using MaxEnt.Results:The results indicated a model performance of AUC=0.85,with mean diurnal range in temperature(Bio2,43.9%),mean temperature(Bio1,20.6%)and mean annual rainfall(Bio12,11.7%)as the important variables.Future risk projections indicated mixed responses of the potential risk of spreads of V.destructor,exhibiting both decrease and increases in the mid-century 2055 and late-century 2085 on different sites.Overall,there is a general decline of highly suitable areas of V.destructor in mid-and late-century across all scenarios(rcp4.5 and rcp8.5).The moderately suitable areas indicated a mixed response in mid-century with decline(under rcp4.5)and increase(under rcp8.5)and consistent increase in late century.The marginally suitable areas show a decline in mid-century and increase in late-century.Our results suggest that the climate change will continue to significantly affect the distribution and risks spread of V.destructor in Tanzania.The suitability range of V.destructor will shift where highly suitable areas will be diminishing to the advantage of the honey bees’populations,but increase of moderately suitable sites indicates an expansion to new areas.The late century projections show the increased risks due to surge in the moderate and marginal suitability which means expansion in the areas where V.destructor will operate Conclusion:The current and predicted areas of habitat suitability for V.destructor’s host provides information useful for beekeeping stakeholders in Tanzania to consider the impending risks and allow adequate interventions to address challenges facing honey bees and the beekeeping industry.We recommend further studies on understanding the severity of V.destructor in health and stability of the honey bees in Tanzania.This will provide a better picture on how the country will need to monitor and reduce the risks associated with the increase of V.destructor activities as triggered by climate change.The loss of honey bees’colonies and its subsequent impact in bees’products production and pollination effect have both ecological and economic implications that need to have prioritization by the stakeholders in the country to address the challenge of spreading V.destructor.

腐烂茎线虫分子鉴定及在不同寄主上繁殖力分析

腐烂茎线虫危害马铃薯在我国多地已有发生,为了明确腐烂茎线虫的生物型及在不同寄主上的繁殖能力,本研究采用贝曼漏斗法对线虫进行分离,根据国标GB/T 29577-2013描述方法对分离到线虫群体进行分子鉴定。从NCBI选取马铃薯和甘薯的腐烂茎线虫ITS序列,进行系统发育分析,利用胡萝卜愈伤组织进行培养,用玉米、马铃薯、高粱、大豆和小麦测定腐烂茎线虫在不同寄主上的繁殖能力。结果显示:分离到的线虫为腐烂茎线虫B型,来自甘薯和马铃薯寄主腐烂茎线虫明显分为2支,推断腐烂茎线虫的进化受寄主的影响。该线虫群体在参试寄主上都能生长,在小麦上繁殖系数最低,其次为大豆。种植小麦和大豆可能对腐烂茎线虫有抑制作用。

一种消杀蜜蜂狄斯瓦螨方法的研究

狄斯瓦螨(Varroa destructor)是危害西方蜜蜂的主要寄生螨之一。为了提高养蜂生产实践中消杀狄斯瓦螨的能力,试验组采用本团队研发的“一种规模化养蜂用高效杀螨装置”,放置在底箱或继箱上,每群自动喷洒药剂90~120 s;对照组采用传统杀螨剂喷洒法,比较两组的落螨数量、治螨时间以及对蜂群的影响。结果表明,在治螨20 min、40 min、1 h、2 h和24 h后,试验组落螨数量均显著高于对照组(P<0.05);试验组治螨时间明显少于对照组;试验组和对照组均对蜂群无不良的影响。使用“一种规模化养蜂用高效杀螨装置”比传统杀螨剂喷洒法消杀瓦螨见效快、效果好,且方便快捷,节约劳动成本。

杜鹃属植物内生真菌的分离及对赤拟谷盗和马铃薯腐烂茎线虫的防治作用

本文以千里香杜鹃、头花杜鹃、烈香杜鹃、陇蜀杜鹃4种杜鹃属植物为材料,通过组织分离法分离其内生真菌,测定内生真菌粗提物的抗虫活性,筛选出具有良好抗虫活性的菌株,并对这些菌株进行形态学及分子生物学鉴定,以此初步筛选出对赤拟谷盗和马铃薯腐烂茎线虫具有较好抗虫活性的内生真菌菌株。结果表明:从4种杜鹃属植物中分离得到22株内生真菌菌株,共发酵产生44种粗提物,其中LS-Y-2菌丝体粗提物对赤拟谷盗具有一定触杀活性(LD_(50)=69.31μg/adult);LS-Y-2菌液粗提物、TH-Z-1菌液粗提物和LS-Y-4菌丝体粗提物对马铃薯腐烂茎线虫具有较好杀虫活性,其LC_(50)分别为0.06、0.07、0.18 mg/m L;LS-Y-2菌液粗提物和LS-Y-4菌液粗提物在质量浓度为2 mg/mL时,对赤拟谷盗的拒食率为84.28%和73.12%。通过形态学和分子生物学鉴定发现,LS-Y-2和LS-Y-4属于链格孢属真菌、TH-Z-1属于栓菌属真菌。综上,链格孢属真菌LS-Y-2、LS-Y-4和栓菌属真菌TH-Z-1对赤拟谷盗和马铃薯腐烂茎线虫具有一定防治作用。