为明确腰果角盲蝽Helopeltis theivora气味结合蛋白3(odorant binding protein 3,OBP3)功能及其嗅觉感受机制,利用PCR技术结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆其cDNA全长序列,利用多个生物信息学软件对其进行序列分析,并通过实时荧光定量PCR(real-time quantitative PCR,qPCR)技术检测其在腰果角盲蝽成虫不同组织中的表达量.结果显示,腰果角盲蝽HtheOBP3基因(GenBank登录号为QHI06949)开放阅读框为474bp,编码157个氨基酸残基,预测蛋白分子量约为17.15 kD,等电点为5.14,无信号肽和跨膜结构,蛋白氨基酸序列中具有6个保守半胱氨酸残基和性信息素结合蛋白-普通气味结合蛋白(pheromone bind-ing protein-general odorant binding protein,PBP-GOBP)家族的保守结构域.HtheOBP3 蛋白具有 6 个a-螺旋和3对二硫键,其中5个a-螺旋形成1个结合口袋.腰果角盲蝽HtheOBP3与其他20种半翅目昆虫OBP的6个保守半胱氨酸位点完全一致,腰果角盲蝽HtheOBP3的氨基酸序列与薇甘菊颈盲蝽-Pachypeltis micranthus PmicOBP4的氨基酸序列一致性最高,为55.56％.在37种不同昆虫的OBP蛋白中,腰果角盲蝽HtheOBP3与6种半翅目昆虫OBP聚为一个分支,其中与薇甘菊颈盲蝽PmicOBP4亲缘关系最近.腰果角盲蝽成虫各组织中HtheOBP3均有表达,其中在触角中表达量最高,其次为足.表明腰果角盲蝽HtheOBP3是典型的气味结合蛋白,其可能兼具嗅觉和非嗅觉感受等生理功能.

为筛选黄翅绢野螟(Diaphania caesalis)逆境胁迫下稳定表达的内参基因,使该虫抗逆基因的定量表达分析标准化.本研究根据黄翅绢野螟成虫转录组注释结果,利用qPCR技术分析了9个分别编码ACT、β-TUB、GAPDH、G6PDH、RPS3a、RPL13a、EF1α、EIF4A、/3-ACT的候选内参基因在温度及药剂胁迫下mRNA的表达水平,并通过geNorm、NormFinder、BestKeeper和RefFinder软件(算法)分析各基因的稳定性.结果表明:温度胁迫下,geNorm分析表明ACT和RPS3a为表达最稳定的基因,NormFinder和BestKeeper分析结果表明GAPDH和EIF4a的表达最稳定.药剂胁迫下,geNorm和NormFinder分析结果均表明EIF4a和ACT为最稳定的内参基因,而BestKeeper分析结果表明GAPDH和RPS3a为最稳定内参基因.全样品条件下,geNorm和NormFinder分析结果相似,表明EF1α和EIF4a为最稳定的内参基因,BestKeeper表明GAPDH和RPL13a为最稳定的内参基因.根据geNorm软件成对变异值Vn/Vn+1＜0.15的原则,确定3种条件下对靶标基因表达进行标准化时需引入的内参基因的数目均为2个.利用在线分析软件RefFinder综合评价上述3个分析结果,最终确定温度胁迫下最合适的内参基因为EIF4A和GAPDH,药剂胁迫下最适内参基因为ACT和EIF4A,全样品下最适内参基因为EF1α和EIF4A.本研究结果为利用qPCR技术分析黄翅绢野螟温敏及抗药性相关基因表达差异提供了稳定的内参基因,进而为基因的功能分析奠定了基础.

The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

生防菌株Bacillus velezensis Z对胡椒瘟病等多种植物病害具有良好的生防效果;全基因组测序能够为其分子机理研究和开发应用提供信息基础.本研究开展该菌株全基因组测序,并进行比较基因组学和抑菌次生代谢产物合成基因簇预测研究.结果表明:B.velezensis Z菌株的基因组中含有1条4054780 bp大小的环形染色体DNA和1个17122 bp大小的环形质粒,总基因组的GC含量为46.24％,共编码基因4173个;包含27个rRNA,86个tRNA基因,34个sRNA;含有串联重复序列179个,其中13个微卫星DNA,138个小卫星DNA;通过比较基因组学分析,结果发现该菌株与贝莱斯芽孢杆菌模式菌株FZB42同源性极高,与枯草芽孢杆菌模式菌株168之间具有一定的遗传距离;同时发现B.velezensis Z基因组中共编码次生代谢产物合成基因簇13个,其中8个与表面活性素(surfactin)、泛革素(fengycin)、溶杆菌素(bacilysin)、macrolactin H、bacillaene、difficidin、plantazolicin、amylocyclicin等已知基因簇完全相似或高度相似,其他5个基因簇皆功能未知.总之,本研究揭示了B.velezensis Z的全基因组遗传信息,明确其与贝莱斯芽孢杆菌和枯草芽孢杆菌的比较基因组学关系,预测了抑菌产物合成编码基因簇,为该生防菌株及其抑菌产物的机理研究和开发应用奠定基础.

通过室内选择性试验研究了茶角盲蝽(Helopeltis theivora Waterhouse)对26份可可种质果实的取食偏好性,以具有相对抗性差异的2-7和STS-17种质为材料,采用HS-SPME/GC-MS鉴定分析上述2份特异种质果实挥发物的组分和含量,并利用"Y"型嗅觉仪测定了该虫对2份特异种质及其挥发物的行为反应.结果表明:室内选择性试验显示2-7种质表现出对茶角盲蝽相对易感,STS-17、BGL44-64和15-9种质表现出对该虫的相对抗性,其中STS-17果实上的取食斑数量最少."Y"型选择试验显示茶角盲蝽对2-7种质的选择性显著高于STS-17,对STS-17和空白对照无选择性差异,进一步验证了二者的特异性.2-7和STS-17种质果实中共检测出6类18种挥发物,但二者挥发物组分与含量差异较大,二者共有成分为1-己醇、反-α-香柠檬烯和六甲基环三硅氧烷.茶角盲蝽对不同浓度的挥发物有不同趋性,β-石竹烯表现为在高浓度(100 mg/mL)下驱避、中浓度(10 mg/mL)下引诱、低浓度(1 mg/mL)下无影响;六甲基环三硅氧烷则在低浓度下有引诱作用;而丁酸顺式-3-己烯酯和八甲基环四硅氧烷在高浓度下驱避、其余浓度下无影响.综合分析认为,STS-17为茶角盲蝽相对抗性种质,2-7为相对易感种质,挥发物成分在茶角盲蝽寄主选择行为中具有导向作用.

Coffee (Coffea arabica L. and Coffea canephora Pierre ex A. Froehner) is one of the most important global commodities, with over 10 million hectares planted in 80 countries in tropical and subtropical regions (Aristizábal et al. 2016; Escobar-Ramı́rez et al. 2019; FAO 2015). The total value of the coffee industry during 2012 was estimated to be US$173 billion (ICO 2015). One of the major constraints on coffee production throughout the world is the damage caused by the coffee berry borer (CBB), Hypothenemus hampei (Ferrari, 1867) (Vega et al. 2009). The CBB causes enormous economic losses (Jaramillo et al. 2006; Wegbe et al. 2003). This pest poses a threat to coffee production due to its cryptic life habits inside the coffee berry, which makes management difficult (Vega et al. 2015). The beetle is indigenous to Africa (Damon 2000; Le Pelley 1968; Vega et al. 2009, 2019). To date, CBB has been found in every coffee-producing region, except China and Nepal, with the most recent introductions to Puerto Rico in 2007, Hawaii in 2010, and Papua New Guinea in 2017 (Burbano et al. 2011; Coffee Industry Corporation Ltd 2017; Duque and Baker 2003; Hamilton et al. 2019; Jaramillo et al. 2011; Vega et al. 2009). In China, the area of coffee plantations was estimated to be about 120,000 ha, mostly distributed in Yunnan and Hainan (Liu 2016). Hainan was one of the earliest areas to grow coffee in China because of its ideal climate, and cultivation began in the early 20th century (Chen 2010). Moreover, the volcanic soil on the island is very suitable for cultivating high-quality coffee. Robusta coffee is the main variety in Hainan, distributed mainly in Wanning, Chengmai, Baisha, and Qiongzhong. The only recorded bark beetle (Scolytinae) pest of coffee on Hainan Island is the black twig borer, Xylosandrus compactus (Eichhoff). It was first discovered in Wanning in the 1970s, where it was reported to cause 20–30% reduction in coffee per year (Lin et al. 1994). During June 2019, about 40 representatives of suspected CBB adults were captured in bottle traps (100% ethanol; Vega et al. 2015) in a farm near Xinglong, Wanning (18.7240°N, 110.2437°E) and hand-collected from coffee beans. The first few specimens were preserved in 100% ethanol and sent to Jiangxi Agriculture University, China for primary identification, then confirmed in the Forest Entomology Laboratory, School of Forest Resources and Conservation, University of Florida, USA. The specimens were confirmed to be CBB using the morphological diagnosis by Vega et al. (2015)

[目的]黄翅绢野螟Diaphania caesalis是热带木本粮食作物菠萝蜜Artocarpus heterophyllus的重要钻蛀性害虫,对热区快速发展的菠萝蜜产业威胁巨大.本研究旨在明确该虫的生物学特性及田间发生规律,为准确预测和高效治理该虫提供理论基础.[方法]在室内温度26 ±1℃、相对湿度70％±5％、光周期14L:10D条件下,以菠萝蜜叶片为食料,观察黄翅绢野螟各龄期形态特征、发育历期及繁殖能力,并在田间网室中观察该虫年生活史;通过室内选择和非选择试验,研究该虫的寄主多样性;2018年1-12月通过田间系统调查,分析该虫在海南省琼中县的种群动态规律.[结果]黄翅绢野螟卵、幼虫、预蛹、蛹和成虫的发育历期分别为4.58±0.50,21.00±1.36,2.50±0.51, 10.20±0.53和12.31±3.16d,平均世代历期为50.50±3.54 d.各龄期主要形态特征为:卵椭圆形,表面具网纹;幼虫体黄褐色,化蛹前变白色,蜕裂线呈倒“Y”形;蛹红褐色,足和翅芽长至第5腹节;成虫体麦黄色,前翅有瓜子状和塔状黄斑.雌蛾可多次产卵,单雌产卵量为147.25±84.24粒.幼虫偏好取食菠萝蜜、榴莲蜜Artocarpus champeden和面包果Artocarpus altilis 3种植物.室内和网室观察结果表明,黄翅绢野螟在海南一年发生8代.2018年1-12月田间调查结果表明,从3月开始种群数量明显增加,至7月始终保持相对稳定的水平;7月中下旬至9月为种群高峰期;10月后种群开始回落,至次年2月种群保持在较低的水平.[结论]黄翅绢野螟能以菠萝蜜叶片为食料完成继代繁殖,属寡食性;7-9月是该虫在海南的种群高峰期(第5代),因此精准防控的关键时期为上代(第4代)成虫期,即7-8月.

为探究槟榔不同混交模式对叶片养分吸收及土壤养分的影响,田间设置槟榔×菠萝蜜、槟榔×面包果、菠萝蜜、面包果、槟榔5种种植模式.结果 表明:槟榔×菠萝蜜混交能在一定程度上增加土壤pH值、有效磷和速效钾含量,对土壤养分的提升效果较槟榔×面包果混交模式更显著.槟榔×菠萝蜜对菠萝蜜叶片养分含量无显著影响,对槟榔叶片全氮、全磷、全钾含量也无显著影响.而槟榔×面包果显著降低了面包果叶片对磷素的吸收,也降低了槟榔叶片对氮、磷、钾、镁素的吸收,但增加了对钙素的吸收.土壤pH值与土壤有效磷含量呈显著正相关,土壤有机质与土壤碱解氮和有效磷含量呈显著正相关,与土壤速效钾呈极显著负相关关系,土壤有效磷含量与叶片全钾含量呈极显著负相关关系.综合分析认为,槟榔×菠萝蜜对提高土壤养分的综合效果较槟榔×面包果更佳.

香蜜17号是海南省万宁市兴隆本地菠萝蜜的实生优系.平均单果重12.5 kg,果实椭圆形,纵径35.5cm,横径23.7 cm,果皮黄绿色,平均厚1.1 cm.果苞橙红色,厚0.6 cm,可食率45.5％,质地脆,甜,香味不同于传统菠萝蜜,近似于榴莲香味,可溶性固形物含量23.5％～27.8％.嫁接苗定植2.5年可零星开花结果,早实,稳产.

游动孢子是胡椒瘟病最主要的侵染源,其产量受环境条件影响,但是具体影响关系尚不明确.本研究利用胡椒瘟病菌孢子囊诱导技术,逐个分析光照、温度、湿度、pH等单一环境因素变化对胡椒瘟病菌游动孢子产量的影响.结果 表明:光照强度在200～20000 lx时胡椒瘟病菌均可产孢,在5000 lx时产孢量最高(1.28×106个/mL),分别是2000和10000 lx的6.89倍和12.40倍;温度在20～30℃时,产孢量均能达到5.82×105个/mL以上,在25℃时产孢量最高;相对湿度在50％～99％时,产孢量均能达到2.27×105个/mL以上,其中相对湿度较低的条件下(50％和65％)游动孢子产量显著高于其他湿度条件;环境pH在6.0～8.0时,产孢量较高,pH 7.0时最高.综上所述,4种环境因素均对胡椒瘟病菌游动孢子产量具有显著影响,最适宜产孢的环境条件为光照强度5000 lx、温度25℃、相对湿度65％、pH 7.0.本研究为后续致病相关研究及田间病害防控奠定基础.

孢子囊的产生和发育过程是研究胡椒瘟病菌（Phytophthora capsici）及其病害防控的重要基础。本研究分析不同诱导条件下胡椒瘟病菌孢子囊的产生情况,并通过光学显微镜和共聚焦显微镜观察孢子囊及其内部游动孢子的发育过程。结果发现:（1）3种诱导方法均能诱导出大量孢子囊,其中在V8-A平板上光照和抹伤双重诱导法获得的孢子囊数量最多,其次是在V8-A平板上光照诱导法,获得孢子囊数量最少的是V8液体光照诱导法,但仅差异最大的2个处理间达到显著水平;（2）显微镜观察发现,孢子囊由气生菌丝顶端逐步膨大形成,初始为近球形逐渐发育成倒洋梨形,孢子囊成熟后从顶端排出大量游动孢子,偶尔可见孢子囊顶端直接发育出芽管;（3）共聚焦显微镜观察发现,首先孢子囊内部原生质体被膜结构隔裂成大约数十个独立小格,然后在每个格子中积累数倍于细胞核的DNA,最后每份细胞核DNA发育成一个游动孢子。本研究从微观角度揭示P.capsici孢子囊发育外观及内部的形态特征,为胡椒瘟病菌后续致病机制研究和胡椒瘟病田间防控提供技术基础。

可可新品系‘香可1号’是用特立尼达可可‘IT16’作母本、弗拉斯特洛可可‘IF5’作父本杂交育成.果实椭圆形,表面光滑,幼果颜色为紫红色,成熟果颜色为中等红色;平均单果重545.7g,果壳平均厚1.3cm,成熟果实平均籽粒数42粒,平均籽粒干重1.4g;果肉厚,可食率13.5％,乳白色,气味清香,酸甜多汁.在海南省万宁市,果实2月上旬成熟,果实生育期160d,稳产,具有良好的推广前景.

Jackfruit seed starch (JFSS) was modified by an improved extrusion cooking technology (IECT), and the supramolecular structure, molecular weight, debranched chain length distributions, relative crystallinity (Rc), and amylose content, were studied. During IECT, the α-1.4-glycosidic bond in amylopectin was broken, which led to decreased radius of gyration (Rg), number-average molar mass (Mn), weight-average molar mass (Mw), long chains and Rc. The medium and short chains and PI (Mw/Mn) increased, while the amylose content hardly changed. The crystalline structure of JFSS was converted from A-type to V-type. Increasing the temperature and screw speed during the treatment significantly increased the medium and short chains and Rg, while it decreased the long chains, amylose, Mn, Mw, PI, and Rc. However, the opposite effect was observed when increasing the moisture content. The in vitro digestibility of JFSS was significantly improved after IECT, due to destruction of starch supramolecular structure according to principal component analysis.

芽孢杆菌(Bacillus sp.)产生的脂肽类化合物具有广谱生防效果.针对广谱生防菌株B.subtilis VD18R19,采用酸沉淀法提取发酵液中的脂肽类化合物,并通过MALDI-TOF/TOF-MS进行鉴定,田间生防试验分析该菌株及其脂肽类化合物对胡椒花叶病的生防效果.结果 表明B.subtilis VD18R19发酵液中可以提取到脂肽类化合物,粗提物和提取物的平均产量分别为5.92、3.13 g/L;质谱结果发现,提取物中主要含表面活性素(Surfactin)和大侧柏素(Plipastatin)2种脂肽类化合物;田间试验表明,生防菌VD18R19及其脂肽类化合物均能有效防控胡椒花叶病,防效分别达79.49％和87.18％;且2处理均能促进胡椒冠幅和果枝生长,尤其生防菌VD18R19的促生效果达显著水平.说明B.subtilis VD18R19及其脂肽类化合物在防控胡椒花叶病上具有良好的应用潜力.

为筛选特定条件下实时荧光定量PCR检测体系中能稳定表达的茶刺盲蝽Helopeltis theivora内参基因,从其转录组中筛选并克隆11个候选基因Actin、β-tubulin1、RPL13A、EF1α、RPS3A、GAP-DH、18S RNA、G6PDH、EIF4A、TBP和UBQ,测定它们在不同温度及药剂胁迫下mRNA的表达水平,并通过geNorm、NormFinder、BestKeeper、Delta Ct和RefFinder软件(算法)分析各基因的稳定性.结果 表明,11个候选基因引物均具有良好的特异性和扩增效率(90.10％～96.87％).温度胁迫下,EIF4A有最小的平均变异度(mean variability,M)、稳定值(stability value,SV)和几何平均值,分别为0.200、0.096和2.060;RPS3A的平均标准偏差(average of standard deviation,STDEV)最小,为0.605;二者是表达最稳定的基因.药剂胁迫下,RPL13A的M值和STDEV最小,分别为0.123和0.660;RPS3A的SV、标准偏差(standard deviation,SD)和几何平均值均最小,分别为0.063、0.336和1.189;二者是表达最稳定的基因;Actin为最不稳定基因.全样品评价中,RPL13A(M值为0.282、几何平均值为1.565)和RPS3A(SV为0.099、STDEV为0.614)的评价值同比最小,是表达最稳定的基因;最不稳定的基因为dctin、β-tubulin1和TBP.geNorm分析结果还显示所有处理条件下最适内参基因数目均为2个.表明EIF4A和RPS3A可作为茶刺盲蝽与温度相关的抗性基因表达研究的内参基因,RPS3A和RPL13A可作为该虫抗药基因表达研究的内参基因.

Helopeltis theivora Waterhouse is a predominant sucking pest in many tropic economic crops, such as tea, cocoa and coffee. Quantitative real-time PCR (qRT-PCR) is one of the most powerful tools to analyze the gene expression level and investigate the mechanism of insect physiology at transcriptional level. Gene expression studies utilizing qRT-PCR have been applied to numerous insects so far. However, no universal reference genes could be used for H. theivora. To obtain accurate and reliable normalized data in H. theivora, twelve candidate reference genes were examined under different tissues, developmental stages and sexes by using geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms, respectively. The results revealed that the ideal reference genes differed across the treatments, and the consensus rankings generated from stability values provided by these programs suggested a combination of two genes for normalization. To be specific, RPS3A and Actin were the best suitable reference genes for tissues, RPL13A and GAPDH were suitable for developmental stages, EF1α and RPL13A were suitable for sexes, and RPL13A and RPS3A were suitable for all samples. This study represents the first systematic analysis of reference genes for qRT-PCR experiments in H. theivora, and the results can provide a credible normalization for qRT-PCR data, facilitating transcript profiling studies of functional genes in this insect.

Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.