Cloning and Characterization of a Putative Antifungal Peptide Gene (Pm-AMP1) in Pinus monticola

ABSTRACT We have been working on proteins that are involved in the defense response of western white pine (WWP) (Pinus monitcola) to the blister rust fungus Cronartium ribicola. Our objective was to identify candidate genes that could be used for improving resistance of WWP to this rust pathogen. During proteomic analysis of bark proteins extracted from WWP trees exhibiting slow-canker-growth (SCG) resistance, a 10.6-kDa peptide, termed Pm-AMP1, was found to be enriched at the receding canker margin. The cDNA encoding this peptide was cloned and characterized. A BLASTX search revealed that the Pm-AMP1 encoded by its cDNA has a 50% homology with MiAMP1, a broad-spectrum antifungal protein isolated from Macadamia integrifolia. Based on the deduced amino acid sequence, an antibody was produced against the Pm-AMP1. Immunochemical quantification of the Pm-AMP1 in bark samples of susceptible WWP trees revealed this protein to be barely detectable in the cankered tissues, but occurring in higher concentrations in healthy tissues away from canker margins. Foliage of SCG-resistant trees contained higher concentrations of the Pm-AMP1 than foliage from susceptible cankered trees. Both wounding and methyl jasmonate treatment of WWP needles induced the expression of this protein, further supporting its putative role as a defense response protein.     

Comparison between the penetration characteristics of methyl bromide and ethanedinitrile through the bark of pine (Pinus radiata D.Don) logs

BACKGROUND

Ethanedinitrile (EDN) is a fumigant being commercialized worldwide as an alternative phytosanitary treatment to methyl bromide (MB) for forest products. The penetration characteristics of MB and EDN were measured through the bark of wooden blocks (100 × 100 × 50 mm) cut from the upper (average bark thickness 5 ± 2 mm) and lower (average bark thickness 25 ± 5 mm) trunk of recently felled pine (Pinus radiata D.Don) trees. Doses of 48 g m⁻³ MB and 50 g m⁻³ EDN were applied to chambers at 10 and 20°C for 10 h.

RESULTS

Penetration of MB was influenced by the interaction between fumigation time and temperature, with concentrations increasing at a higher rate at 20°C compared with at 10°C. After 10 h, an average concentration of 8.05 ± 0.89 g m⁻³ had penetrated the bark of log sections at 20°C, whereas 5.20 ± 0.89 g m⁻³ was measured at 10°C. By contrast, the factors examined in this study did not significantly impact the penetration of EDN. Concentration × time (CT) values for MB under the bark were 35.20 ± 2.30 g h m⁻³ at 10°C and 55.85 ± 9.58 g h m⁻³ at 20°C; whereas for EDN, CT values were 19.50 ± 6.80 g h m⁻³ at 10°C and 19.08 ± 4.10 g h m⁻³ at 20°C.

CONCLUSION

MB can achieve a higher concentration under the bark of log sections during simulated fumigations, but all of the factors examined affected the ability of MB to penetrate the bark of wooden blocks. By comparison, the penetration of EDN through the bark is more consistent than MB under laboratory conditions. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.     

Economic criteria in the choice of weed-control methods for young pine (Pinus caribaea var. hundurensis Barr and Golf) plantations

The effect of three types of cover crop Calapogonium mucunoides Desv., Centrosema pubescens Benth. and Pueraria phaseoloides (Rexb.) Benth., and hand-weeding on the growth and yield of Pinus caribaea var. hundurensis Barr and Golf were investigated after 42 months of planting. The costs and benefits of the four methods are evaluated and compared. Results show no significant differences in survival percentage, but show significant differences in mean height of trees after 15 months. Survival percentage, mean height, girth and basal area of trees, measured after 42 months, showed no significant difference between treatments. However, trees grown with C. mucunoides had the highest values of all four variables measured. Economic analysis shows that cost per hectare is higher for hand-weeding while cover cropping has a higher net present value of investment. The results demonstrate that cover cropping is a more profitable alternative to hand-weeding in weed control in pine plantations, and that C. mucunoides will be the best choice of the three cover crops studied. Critères de choix économiques pour des méthodes de désherbage des jeunes plantations de pins (Pinus caribaea var hundurensis Barr and Golf) Les effets de 3 sortes de couvert végétal (Calapo-gonium mucunoides Desv., Centrosema pubescens Benth. et Pueraria phaseoloides (Rexb.) Benth. et du desherbage manuel sur la croissance et le rendement de Pinus caribaea var. hundurensis Barr et Golf ont étéétudiés après 42 mois de plantation. Les coûts et profits des quatre méthodes sont estimés et comparés. Les résultats ne montrent pas de différences significatives dans le pourcentage de survie, mais en revanche les différences sont significatives sur la hauteur moyenne des arbres après 15 mois. Le pourcentage de survie la hauteur moyenne, la surface basale et la circonférence des arbres mesurés après 42 mois n'expriment pas de différence significative entre les traitements. Cependant, les arbres élevés avec C. mucunoides ont des valeurs plus élevées pour les quatre variables envisagées. L'analyse économique montre que le coût par hectare est plus élevé pour le dèsherbage, tandis que le couvert végétal a une plus grande valeur nette d'investissement. Les résultats démontrent que le couvert végétal est une alternative plus rentable que le dèsherbage manuel dans les plantations de pins et que C. mucunoides est le meilleur choix parmi les trois espèces étudiées. L'analyse économique montre que le coût par hectare est plus élevé pour le désherbage manuel, tandis que le couvert végétal a une plus grande valeur nette d'investissement. Les résultats démontrent que le couvert végétal est une alternative plus rentable que le désherbage manuel dans les plantations de pins et que C. mucunoides est le meilleur choix parmi les 3 espèces étudiées. Ökonomische Kriterien für die Unkrautbekämpfung in Kulturen von Pinus caribaea var. hundurensis Barr & Golf Die Wirkung von Bodendeckern (Calapogonium mucunoides Desv., Centrosema pubescens Benth., Pueraria phaseoloides (Rexb.) Benth., und mechanischer Unkrautbekämpfung (Jäten) auf das Wachstum und den Zuwachs von Pinus caribaea var. hundurensis Barr & Golfwurde 42 Monate nach der Pflanzung untersucht. Die Kosten und die Wirksamkeit der 4 Varianten wurden verglichen. Während hinsichtlich des Anwachsens keine Unterschiede bestanden, ergaben sich für die mittlere Wuchshöhe der Bäume nach 15 Monaten signifikante Unterschiede. Überlebensrate, mittlere Höhe und Stammumfang waren 42 Monate nach der Pflanzung nicht significant verschieden, doch waren diese Parameter bei Calapogonium mucunoides am höchsten. Die Hektarkosten waren für die mechanische Unkrautbekämpfung höher als für die Bepflanzung mit Bodendeckern, die sich damit für Kiefernkulturen als wirtschaftlicher erwies. Von den 3 Bodendeckern ist die erstgenannte Art vorzuziehen.     

金荞麦的潜在分布区及生态特征

为了解我国金荞麦Fagopyrum dibtrys(D.Don)Hara的潜在分布区及生态特征,基于38个环境因子以及金荞麦在我国的153个地理分布记录,应用最大熵模型(MaxEnt)与地理信息系统(GIS)对金荞麦进行生态适宜性区划,并采用自然间断法对金荞麦的潜在区域进行划分。结果显示,金荞麦的潜在分布区主要位于我国西南地区东部、华中地区、华东地区和华南地区(22 N°~35N°),总面积约为250.99×10~4km~2;将金荞麦的潜在分布区划分为适生区、低适生区与边缘适生区,其中适生区主要集中在长江流域,总面积为88.00×10~4km~2。影响金荞麦地理分布的主要生态因子有最冷月最低温、最暖季降水量、高程、最干季降水量等9个因子,其中气候因子对金荞麦的地理分布影响最大。表明金荞麦应在云南、贵州、四川等省扩大种植面积,陕西、山西等省可考虑引种。     

棉铃虫小分子热激蛋白sHSP22.0基因的克隆及表达谱分析

棉铃虫Helicoverpa armigera是一种重要的农业害虫,本文旨在研究小分子热激蛋白在其生长发育过程、抵御高温及Cry1Ac杀虫蛋白中的功能.利用PCR结合RACE技术克隆了棉铃虫sHSP22.0(small heat shock pro-tein 22.0)基因,通过生物信息学软件分析了棉铃虫sHSP22....     

茶翅蝽非典型气味受体基因的克隆及其生物信息学特征和组织特异性表达分析

为明确茶翅蝽Halyomorpha halys的非典型气味受体（odorant receptor co-receptor,Orco）基因的生物信息学特征和组织特异性表达谱,采用Trizol法提取茶翅蝽成虫触角的总RNA,利用RTPCR技术克隆其Orco基因,使用生物信息学软件对茶翅蝽Orco蛋白进行跨膜区域预测、结构分析和系统发育分析,并采用实时荧光定量PCR技术测定Orco的组织表达谱。结果表明,克隆获得茶翅蝽Orco基因并命名为Hhal Orco,该基因的开放阅读框全长1 428 bp,编码475个氨基酸。Hhal Orco蛋白的二级结构中,α螺旋占34.11%,无规则卷曲占36.84%,延伸链占29.05%;其分子质量为53.49 kD,等电点为6.16,无信号肽,是非分泌蛋白;有7个跨膜区,是疏水性蛋白,疏水性系数的平均值为0.297;预测的Hhal Orco蛋白为对称亚基构成的同源四聚体。系统进化分析发现,Hhal Orco与同为半翅目的荔蝽Tessaratoma papillosa亲缘关系最近。qPCR检测结果显示,Hhal Orco基因主要在雌雄成虫的触角中表达,在成虫的头、胸、腹、足中仅有微量表达。     

油松新害虫——黑胫腮扁叶蜂的初步观察

黑胫腮扁叶蜂(Cephalcia nigrotibialis Wei)以雌虫为模式标本,描述于我国陕西(周至)和河南(内乡)。2009年在北京延庆发现它是油松上一种新的重要害虫,1年1代,以老熟幼虫在土室中越冬,越冬虫口密度平均达20.7头/m2。6月初开始化蛹,6月下旬出现成虫;成虫产卵于2年生的松针上,幼虫在小枝上做丝质的巢,咬断松针基部后取食,为害盛期为8-9月。色带诱集试验表明,5种色带均可诱到成虫,不同颜色之间差异不显著。目前在延庆将红色胶带用于黑胫腮扁叶蜂的防治。本文对各虫态进行了描述,并提供了彩色图片,讨论了该虫的分布特点和成灾原因。     

Non-target effect of herbicides: II. The influence of glyphosate on ectomycorrhizal symbiosis of red pine (Pinus resinosa) under greenhouse and field conditions

The effects of the herbicide glyphosate [N-(phosphonomethyl)glycine]on seedling growth of Pinus resinosa and on the ectomycorrhizal development of the symbiotic fungus Paxillus involutus were investigated under greenhouse and field conditions. Glyphosate at 0–54 and 3–23 kg ha^?1 did not reduce seedling growth or ectomycorrhizal development under greenhouse conditions. There was no seedling mortality due to glyphosate treatment at either rate. Under field conditions, seedling growth of P. resinosa and ectomycorrhizal development of P. involutus were not affected by the above rates. Seedlings (46–48%) failed to survive in non-glyphosate-treated plots, presumably because of weed competition, whereas all survived in glyphosate-treated plots. All the non-mycorrhizal seedlings were colonized bv indigenous mvcorrhizal fungi within 2 months after planting in both control and glyphosate-treated plots. The injection rates varied from 74–86 %.     

禾谷孢囊线虫(Heterodera avenae)纤维素结合 蛋白基因(Ha-cbp-1)的克隆和序列分析

 禾谷孢囊线虫(Heterodera avenae)是中国小麦上的重要病原线虫。纤维素结合蛋白基因是一种重要的植物线虫寄生和致病相关基因。用同源克隆的方法从禾谷孢囊线虫寄生前二龄幼虫中克隆出一种纤维素结合蛋白新基因Ha-cbp-1（GenBank 注册号GQ178086）cDNA序列。Ha-cbp-1基因cDNA包含1个开放阅读框,编码131个氨基酸残基的蛋白质,预测蛋白由1个长度为18氨基酸残基的信号肽和1个纤维素结合区域（CBD）组成。Ha-cbp-1的基因组DNA序列含有2个内含子。预测的禾谷孢囊线虫纤维素结合蛋白（HA-CBP-1）序列与大豆孢囊线虫纤维素结合蛋白（HG-CBP-1）序列有60%的同一性和76%的相似性,与甜菜孢囊线虫纤维素结合蛋白（HS-CBP-1）序列有60%的同一性和75%的相似性。本研究首次从禾谷孢囊线虫中成功克隆出CBP蛋白基因。     

The response of sugi (Cryptomeria japonica D. Don) sapwood to fungal invasion following attack by the sugi bark borer

The response of sapwood of the gymnosperm sugi, Cryptomeria japonica, to fungal invasion following attack by the sugi bark borer, Semanotus japonicus, was investigated. Norlignans—agatharesinol, hinokiresinol and sequirin-C—accumulated in the inner intermediate wood which surrounded discoloured sapwood. Hinokiresinol and two unidentified compounds showed inhibitory effects on Fusarium oxysporum, F. solani and Macrophoma sugi, which were isolated from discoloured sapwood, and Cladosporium herbarum. Most of the ray parenchyma cells in the inner intermediate wood were still alive. Spreading of fungal hyphae colonizing discoloured sapwood was inhibited at the boundary of the inner intermediate wood with the discoloured sapwood. Oil droplet-like deposits in tracheid lumina and pits of the inner intermediate wood were found to occlude many tracheids. The deposits contained lipid and phenolic compounds including norlignans such as hinokiresinol. These results suggest that the deposits form a continuous barrier at the margin of discoloured sapwood, in the wood formed prior to the infection, and prevent the fungi from spreading further.     

马铃薯甲虫热激蛋白基因Ld-hsp90的克隆及温度胁迫下的表达

马铃薯甲虫Leptinotarsa decemlineata是一种抗逆性极强的世界性检疫害虫,对温度胁迫具有很强的适应性,为进一步明确其对温度胁迫适应性的分子生态机制,采用RT-PCR及RACE技术克隆得到了1个hsp90基因的cDNA全长序列,命名为Ld-hsp90,并利用实时荧光定量PCR技术检测其在温度胁迫后相对表达量的变化。Ld-hsp90全长为2 489 bp,开放阅读框(ORF)长度为2 160 bp,编码719个氨基酸,理论等电点为4.98,分子量约为82.09 kD,5'端与3'端非编码区长度分别为113 bp和216 bp。氨基酸序列中含有HSP90家族的5个签名序列及胞质特征序列MEEVD。实时荧光定量PCR检测结果显示:38℃和44℃高温热激1 h,马铃薯甲虫成虫体内的Ld-hsp90的表达量均显著高于对照组,而0℃与-10℃低温处理1 h后的表达量与对照组均无显著差异。研究表明Ld-hsp90的上调表达在马铃薯甲虫抗高温中起着重要的作用。     

沙葱萤叶甲热激蛋白基因GdHsp10a的克隆、分子特征与表达分析

为明确热激蛋白HSP10在沙葱萤叶甲Galeruca daurica生长发育过程中的作用,采用RTPCR及RACE技术克隆沙葱萤叶甲Hsp10基因cDNA的全长序列,采用生物信息学方法分析其序列特征,并利用qPCR技术对该基因在沙葱萤叶甲不同发育阶段、成虫羽化后不同时期以及不同温度下的表达谱进行分析。结果表明,克隆获得1条新的沙葱萤叶甲Hsp10基因,命名为GdHsp10a,GenBank登录号为MG460308,cDNA序列全长为526 bp,开放阅读框为333 bp,编码蛋白含110个氨基酸,预测分子量为11.97 kD,等电点为9.74;无信号肽及跨膜结构。同源比对及系统进化分析表明,GdHSP10a与光肩星天牛Anoplophora glabripennis HSP10亲缘关系最近,氨基酸序列一致性为53.15%。qPCR结果表明,GdHsp10a在沙葱萤叶甲各发育阶段均有表达,其中在卵期和成虫期的表达量显著高于幼虫期、预蛹期及蛹期;成虫羽化后不同时期表达量差异显著,25 d时表达量最高,其次为100、10和7 d时的表达量;温度对GdHsp10a表达量有显著影响,30℃下表达量最高,35℃下表达量次之,15、20、25及40℃下表达量最低且无显著差异。表明GdHsp10a可能在沙葱萤叶甲生长发育及成虫越夏中起着多重作用。     

灭字脊虎天牛CSP基因的鉴定、表达谱及XquaCSP7基因的功能研究

基于已发表的灭字脊虎天牛Xylotrechus quadripes的触角转录组数据,采用BLAST同源搜索的方法从转录组中一共鉴定到14个CSP基因,所有CSP均具有全长序列、信号肽序列和4个保守的半胱氨酸。序列比对结果表明,灭字脊虎天牛CSP间氨基酸一致性差异较大(12%~73%),平均值为34%;具有C1-X6-C2-X18-C3-X2-C4的半胱氨酸保守模式。进化分析表明,除XquaCSP8和XquaCSP12外,其余XquaCSPs聚类到不同分支。表达谱分析表明,XquaCSP7基因在触角特异表达;XquaCSP4、CSP5、CSP12和CSP14基因在触角高表达;XquaCSP13基因在足特异表达;其余XquaCSPs基因在多个组织中有表达。结合测定结果发现,XquaCSP7与测试的21种寄主气味结合能力均较弱,荧光取代率在4.5%~13.5%之间,其中与苯乙酮的结合能力最强,荧光取代率为13.5%。研究结果可为后续灭字脊虎天牛CSP基因的功能研究奠定基础。     

烟粉虱内共生菌传毒相关groEL基因的克隆及其原核表达

 烟粉虱内共生菌groEL基因编码一种63kD的GroEL蛋白,利用一对特异性引物,通过PCR对长期培养在黄瓜上的烟粉虱体内groEL基因进行了扩增,序列测定表明其长度为1668bp,编码555个氨基酸,与GenBank中烟粉虱内共生菌groEL(AF130421)的核苷酸同源性为99.58%,仅7个核苷酸发生了变异,二者氨基酸序列同源性为99.28%,仅4个氨基酸有别。构建了一个原核表达载体,表达得到了76kD的融合蛋白