Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi

Arabidopsis thaliana (Arabidopsis) Col-0 was inoculated with Phytophthora cinnamomi to assess the interaction and defence responses involved. Pathogen ingress and asexual reproduction occurred on root tissue but not leaf tissue. The colonisation of root tissue did not cause disease symptoms or plant death, indicating that Arabidopsis Col-0 was tolerant of the infection. The induction of several plant defence responses including the expression of defence-related genes were found, with differences displayed between inoculated root and leaf tissue. Arabidopsis defence-related gene mutant/over-expressing lines were also inoculated with P. cinnamomi but none of the lines tested exhibited a marked increase in susceptibility to the pathogen.     

毛乌素沙地樟子松人工林土壤微生物数量变化

对毛乌素沙地榆林沙区樟子松人工固沙林地土壤微生物进行测定和分析,结果表明:不同林龄樟子松林地细菌、放线菌和真菌数量差异显著,细菌和真菌数量随林龄增大不断增加;放线菌数量在流沙地(CK)、7～23a生林地这一时期内随着林龄增大显著增加,23a生林地达到最大值,31 a生林地出现明显回落.三大微生物均随土层深度加深而减少,在沙丘部位的分布表现为:丘间地＞背风坡＞迎风坡＞丘顶.在流沙地上栽植樟子松人工林后,土壤微生物数量增加,土壤肥力水平提高.     

北京市生菜链格孢根腐病病原菌鉴定

为明确北京市生菜链格孢根腐病的病原菌种类,采用常规组织分离法分离获得病原菌,依据柯赫氏法则对病原菌进行致病力检测,并利用分子生物学技术结合形态学鉴定确定病原菌分类地位。结果显示,从生菜病样组织中分离到2种病原菌共18株,形态学鉴定结果为芸薹链格孢Alternaria brassicae和万寿菊链格孢A. tagetica,分离比例分别为55.6%和44.4%,且二者均能单独侵染生菜根部,前者致病力较后者强,亦能复合侵染。对致病菌株进行GAPDH基因的PCR扩增和测序,并建立了基于GAPDH基因序列的系统发育树,聚类分析结果与形态学鉴定结果一致,因此证实北京市生菜链格孢根腐病是由芸薹链格孢和万寿菊链格孢复合侵染所致。     

Enhancing resistance of transgenic carrot to fungal pathogens by the expression of Pseudomonas fluorescence microbial factor 3 (MF3) gene

Microbial factor 3 (MF3) gene from a plant-growth promoting rhizobacteria Pseudomonas fluorescence was introduced into carrot genome using Agrobacterium-mediated transformation. The obtained transgenic plants expressing MF3 protein were grown in a glasshouse and evaluated for their resistance to phytopathogens. The assays were performed by the assessment of disease symptoms developing on the detached leaflets and petioles inoculated by three fungal pathogens. The results showed that transgenic plants had significantly enhanced resistance to Alternaria dauci, Alternaria radicina and Botrytis cinerea, on average, by 20–40% in comparison to the non-transformed control plants. This is the first report of enhancing plant resistance by expressing the bacterial protein homologous to the family of FK506-binding proteins.     

短翅蚜小蜂对桃蚜的取食和寄生功能反应

为评估短翅蚜小蜂对桃蚜的搜寻及控害潜能,采用叶碟法研究了短翅蚜小蜂对甘蓝和辣椒2种植物上桃蚜2龄若虫的取食与寄生行为。结果表明,短翅蚜小蜂对甘蓝桃蚜和辣椒桃蚜的取食和寄生功能反应均符合Holling II及Holling III型方程。短翅蚜小蜂的取食和寄生搜寻效应均随蚜虫密度的增加而降低,当桃蚜密度小于35头时,短翅蚜小蜂对甘蓝桃蚜的取食搜寻效应大于辣椒桃蚜,而当桃蚜密度大于35头时,短翅蚜小蜂对辣椒桃蚜的取食搜寻效应大于甘蓝桃蚜;短翅蚜小蜂在所有密度下对辣椒桃蚜的寄生搜寻效应均大于甘蓝桃蚜。短翅蚜小蜂取食甘蓝桃蚜的瞬间攻击率a'为0.3289,处理时间T_h为0.2597,均大于辣椒桃蚜,寄生甘蓝桃蚜的瞬时攻击率a'为0.8213,小于辣椒桃蚜,而处理时间T_h为0.0275,大于寄生辣椒桃蚜;短翅蚜小蜂对甘蓝桃蚜的理论最大取食量和寄生量均小于辣椒桃蚜。研究表明,短翅蚜小蜂对不同密度的桃蚜均有一定的控制能力,且对辣椒桃蚜的控制作用大于甘蓝桃蚜。     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

注射大肠杆菌后草地贪夜蛾高龄幼虫的免疫应激反应

为采用生物技术防控草地贪夜蛾Spodoptera frugiperda的扩散为害,对草地贪夜蛾5龄和6龄幼虫注射浓度为1×109CFU/mL的大肠杆菌Escherichia coli菌液,并以注射等量磷酸盐缓冲液(phosphate buffer solution,PBS)和未做任何处理(CK)为对照,24 h后测定幼虫体重、集结数和酚氧化酶(phenoloxidase,PO)活性。结果显示,注射大肠杆菌菌液24 h后,草地贪夜蛾5龄和6龄幼虫体重均受到抑制,其体重分别为0.170 g和0.411 g,均显著低于CK的0.181 g和0.484 g;注射大肠杆菌菌液24 h后,草地贪夜蛾5龄和6龄幼虫集结数分别为135.0、338.4个索引集结数(the indexed nodules,INs),前者极显著低于后者,且均显著高于CK和PBS处理,分别为0.4、10.2个INs和0.3、10.9个INs;注射大肠杆菌菌液24 h后,草地贪夜蛾5龄幼虫PO活性为0.156 ABS·min-1·mg-1,显著高于CK和PBS处理,分别为0.046 ABS·min-1·mg-1和0.066 ABS·min-1·mg-1,但草地贪夜蛾6龄幼虫的PO活性为0.050 ABS·min-1·mg-1,显著低于CK和PBS处理,分别为0.066 ABS·min-1·mg-1和0.069 ABS·min-1·mg-1,且草地贪夜蛾5龄幼虫的PO活性显著高于6龄幼虫的PO活性。表明细菌侵染后草地贪夜蛾不同高龄幼虫的免疫应激反应存在差异,而这种差异可能受幼虫生长发育及细胞免疫和体液免疫功能之间权衡现象的影响。     

Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species

An inoculation method for Exobasidium splendidum and Exobasidium vaccinii was developed on the dwarf shrub Vaccinium vitis-idaea. Using inoculated ramets, we investigated whether there are differences between V. vitis-idaea populations in the susceptibility to Exobasidium infections and whether the defence reaction of V. vitis-idaea is visible at a molecular level. Sixteen V. vitis-idaea clones from four populations were propagated in tissue cultures and the ramets were inoculated with E. splendidum or E. vaccinii fungi. The expression of three flavonoid biosynthetic genes (chalcone synthase, dihydroflavonol 4-reductase and anthocyanidin synthase) and the accumulation of flavonoids and hydroxycinnamic acids were determined in response to E. splendidum infection. A pathogenesis-related (PR 4) gene was isolated and its expression was studied in host ramet leaves. To our knowledge, this was the first successful artificial infection reported with E. splendidum. Disease frequencies of the inoculated ramets were between 32% and 47% for E. splendidum and 33% for E. vaccinii, but below 10% in uninoculated control ramets. There were no differences in disease frequencies between V. vitis-idaea populations. Both symptomatic leaves and healthy leaves of diseased ramets showed activation of flavonoid biosynthesis at the gene level, whereas expression of PR 4 was observed only in symptomatic leaves. The increase of flavonoid biosynthesis in healthy leaves of diseased ramets may represent a general response to stress or a role in defence against the pathogen E. splendidium. Ability of V. vitis-idaea to defend chemically against Exobasidium fungi and the heterogeneity of genotypes, age, size, and growth rates in host plant populations might be reasons for the low infection incidence of Exobasidia in nature.     

两种葡萄溃疡病菌双重PCR检测方法的建立与应用

为快速准确地鉴定出2种不同的葡萄溃疡病菌——葡萄座腔菌Botryosphaeria dothidea和小新壳梭孢Neofusicoccum parvum,根据Gen Bank中已报道的引起我国葡萄溃疡病的6个主要种的延伸因子-1序列及β-微管蛋白序列分别设计了葡萄座腔菌和小新壳梭孢的特异性引物B.d-F/B.d-R及N.p-F/N.p-R,建立了双重PCR检测方法,并对田间病样进行检测。结果表明,引物B.d-F/B.d-R和N.p-F/N.p-R可以分别在葡萄座腔菌和小新壳梭孢中扩增到324 bp和212 bp的特异性条带,检测灵敏度均为10 pg。双重PCR检测体系中Taq DNA聚合酶的最佳用量为0.05 U/μL,引物B.d-F/B.d-R和N.p-F/N.p-R的最佳终浓度均为0.2μmol/L,最适退火温度和退火时间分别为58℃和30 s。对田间葡萄病样的检测结果与室内病样常规病原菌分离结果一致。表明该双重PCR检测方法能够同时检测葡萄座腔菌和小新壳梭孢,有助于快速、灵敏地检测葡萄苗木的带菌情况。     

三重PCR检测茄子青枯病菌、黄萎病菌和白绢病菌

为快速、准确对田间茄子发病植株和土壤中3种土传病害的病原菌青枯病菌Ralstonia solanacearum、黄萎病菌Verticillium dahliae和白绢病菌Sclerotium rolfsii进行检测,筛选这3种病菌的特异性引物,建立这3种病菌的三重PCR检测体系,对其退火温度、引物浓度、10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶进行优化,对优化后体系的特异性和灵敏度进行检测,并利用优化体系对田间采集的病害样品和土壤样品进行检测。结果表明,在优化后的50μL三重PCR检测体系中,RS-1-F/RS-3-R、dllz1/dllz2和SRITSF/SRITSR引物的最佳浓度分别为0.16、0.16和0.28μmol/L,10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶体积分别为6、5和1μL,检测体系的最佳退火温度为54℃。按照优化后的反应条件能分别扩增出青枯病菌、黄萎病菌和白绢病菌3种病菌的特异条带,分别为716、350和500 bp,其他对照病菌无条带。该体系对病菌DNA检测灵敏度达到0.1 ng/μL。该...     

蠋蝽对棉铃虫幼虫的捕食功能反应

为明确蠋蝽Arma custos对棉铃虫Helicoverpa armigera幼虫的生物防控潜能,在室内条件下测定蠋蝽5龄若虫和雌、雄成虫对棉铃虫3龄幼虫的捕食能力以及不同蠋蝽密度对自身捕食作用的影响。结果显示:蠋蝽5龄若虫和雌、雄成虫对棉铃虫3龄幼虫的捕食功能反应均符合HollingⅡ模型。蠋蝽5龄若虫对棉铃虫3龄幼虫的瞬时攻击率、处理时间和日最大捕食量分别为0.829、0.024 d和41.667头;蠋蝽雌成虫对棉铃虫3龄幼虫的瞬时攻击率、处理时间和日最大捕食量分别为0.796、0.020 d和50.000头;蠋蝽雄成虫对棉铃虫3龄幼虫的瞬时攻击率、处理时间和日最大捕食量分别为0.752、0.021 d和47.619头。蠋蝽5龄若虫和雌、雄成虫对棉铃虫3龄幼虫的搜寻效应均随猎物密度的增加而逐渐降低。当棉铃虫幼虫密度固定时,单头蠋蝽的日捕食量随蠋蝽密度的增加而逐渐降低,蠋蝽对棉铃虫的捕食作用受到自身密度的干扰,5龄若虫和雌、雄成虫的干扰系数分别为0.548、0.702和0.772。表明蠋蝽5龄若虫及雌、雄成虫均对棉铃虫3龄幼虫具有一定的防控能力,且成虫对棉铃虫幼虫的防控能力大于5龄...     

六种植物精油对褐飞虱和稻虱缨小蜂行为的影响

为研发基于植物精油的褐飞虱Nilaparvata lugens驱避剂和稻虱缨小蜂Anagrus nilaparvatae引诱剂,利用嗅觉仪测试了6种植物精油在不同浓度下对褐飞虱和稻虱缨小蜂的行为反应。结果表明,6种植物精油对褐飞虱和稻虱缨小蜂行为的影响具有种类差异性,不同植物精油对昆虫的行为影响有较大差异。此外,褐飞虱和稻虱缨小蜂对植物精油的嗅觉反应还与精油浓度有关,精油浓度范围在100～5 000 mg/L时,山楂油、白木香油、金银花油、肉桂油和鱼腥草油对褐飞虱雌成虫表现出明显的趋避效果,其中山楂油和金银花油的驱避效果最好,二者在100、500、1 000和5 000 mg/L这4个浓度下均能显著驱避褐飞虱。0.1 mg/L和1 mg/L浓度的山楂油、0.1 mg/L浓度的白木香油和1 mg/L浓度的丁香花蕾油对稻虱缨小蜂雌成虫有较强的吸引力。较高浓度10 mg/L的鱼腥草油对稻虱缨小蜂有明显的驱避性。表明植物精油可以明显地影响褐飞虱和稻虱缨小蜂的行为,可作为行为调控物在"推-拉"策略中防控褐飞虱。     

在小檗碱作用下水稻细菌性条斑病菌生理及转录反应分析

为探究小檗碱对水稻细菌性条斑病菌Xanthomonas oryzae pv. oryzicola的抑菌机制,测定小檗碱对水稻细菌性条斑病菌GX13菌株的毒力、致病因子、细胞结构和能量代谢的影响,并利用Illumina-HiSeq高通量测序技术分析小檗碱对水稻细菌性条斑病菌菌体基因表达的影响。结果表明:经15μg/mL小檗碱处理后,GX13菌株侵染水稻品种玉丝占后引起水稻细菌性条斑病的病情指数为6.70,低于对照组;每1×108CFU菌体胞外多糖产量为1.58μg,显著低于对照的2.42μg;纤维素酶、蛋白酶、淀粉酶等胞外酶活性均低于对照组;与能量代谢相关的苹果酸脱氢酶(MDH)活性为1.42 U/g prot,低于对照组,而丙酮酸和三磷酸腺苷含量升高,分别为1.71μmol/mL和379.49μmol/g prot,均高于对照组。同时,细菌的生物膜形成受到抑制,细胞结构受到破坏。转录反应分析发现,与II型分泌系统相关的xpsD、xpsF和xpsM等基因、与III型分泌系统相关的hrcC、hrcJ、hrcN和hrcQ等基因、与胞外多糖分泌相关的gumF和gumI基因,以及与细胞结构与功能...     

河北省甘薯镰孢菌腐烂与溃疡病的病原鉴定

为明确河北省甘薯镰孢菌腐烂与溃疡病的症状特点和病原种类,在不同种植区采集储藏期和育秧期病样,描述其症状特征;对病原菌进行分离纯化,采用柯赫氏法则回接验证,依据病原菌的形态特征和基因序列确定病原菌种类。结果表明,在储藏期甘薯发病可导致薯块表面腐烂的占总病薯的59.09%,端部腐烂的占40.91%;薯块带有褐色轮纹病斑的占61.36%,病斑没有轮纹或者轮纹不明显的占38.64%;发病初期薯块内部病斑浅、黑褐色的占27.27%,发病后期薯块内部形成空腔、布满白色菌丝的占72.73%;病斑带有苦味的占59.09%,病斑没有苦味或苦味不明显的占40.91%;在育秧田发病导致薯秧溃疡,表现为主茎基部呈点片发生黑色或者褐色病斑,部分有开裂。分离的病原菌能够同时侵染薯块和薯秧;病原菌单瓶梗产孢,大型分生孢子稍弯,两端钝圆,多数3~7个分隔,顶细胞钝圆,基细胞足跟较明显。其rDNA-ITS、EF-1α、β-tubulin基因序列与茄镰孢菌Fusarium solani的同源性分别为97%、99%和98%。初步确定甘薯镰孢菌腐烂与溃疡病的病原菌为茄镰孢菌F.solani。     

以豌豆修尾蚜为猎物的大草蛉两性生命表和捕食率

为明确大草蛉Chrysopa pallens (Rambur)的发育、存活、繁殖和捕食能力,利用年龄-龄期两性生命表方法,在25 ℃、光周期L 15:D 9条件下研究了以豌豆修尾蚜Megoura japonica (Matsumura)为猎物的大草蛉的生命表及其捕食率。结果表明:大草蛉雌成虫个体的繁殖力为683.6±55.99粒,种群的内禀增长率、周限增长率、净增殖率和平均世代周期分别为0.1247/d、1.133/d、333.64粒/个体和46.5 d;大草蛉幼虫、雌成虫和雄成虫个体的平均捕食率分别为145.25、1 192.47和669.08头。若考虑存活率,个体的净捕食率为1 026.26头;从猎物种群到捕食者后代的转化率为3.08;种群个体的稳定捕食率和周限捕食率分别为5.49和6.22头。表明以豌豆修尾蚜为猎物时,大草蛉表现出较好的个体发育、种群增长和捕食特性,豌豆修尾蚜可作为大草蛉人工繁殖选择的猎物之一。     

麦冬主要病害病原菌巢式多重PCR检测方法的建立

为准确快速地诊断浙江省慈溪市麦冬常见真菌病害——炭疽病和黑斑病,基于巢式多重PCR技术,针对核糖体内转录间隔区(ITS)序列分别设计特异性引物,并优化巢式多重PCR反应条件,建立可同时快速准确检测炭疽病病原菌山麦冬炭疽菌Colletotrichum liriopes和黑斑病病原菌互隔交链孢菌Alternaria alternata的方法。结果表明,建立的巢式多重PCR检测体系特异性好,同时检测2种病原菌的灵敏度高达100 pg DNA/μL,对10个田间病样进行检测,有5个病样检测到2种病原菌,3个病样检测到其中1种病原菌,2个样品未检测到目标病原菌,验证了该体系的可行性与准确性。表明所建立的巢氏多重PCR技术可用于快速准确检测麦冬2种主要病害的病原菌。     

辣木枝枯病病原菌鉴定及其生物学特性

为明确海南省辣木枝枯病病原菌及其生物学特性,采用组织分离法获得菌株MO157,通过柯赫氏法则、形态学特征及分子生物学技术对该菌株进行鉴定,并测定了其菌丝生长的最佳条件。结果表明,菌株MO157回接10 d后,辣木茎干表面周围呈黄褐色,随后发病部位表现出水渍状病斑,与自然发病辣木的病状相符;菌落近圆形,菌丝初期呈白色绒毛状,后期呈浅黄色,分生孢子顶胞钩状,3~5个隔膜,厚垣孢子呈球形,在菌丝间串生;菌株MO157的ITS序列与Gen Bank中木贼镰刀菌Fusarium equiseti的相似性为100%,结合形态特征与分子鉴定最终将其确定为木贼镰刀菌(Gen Bank登陆号:KX197955)。该菌菌丝生长适宜温度25~30℃,最适温度28℃;pH 6~8时菌丝生长速率增快,pH 7时最适菌丝生长;PDA培养基和PSA培养基最适合该菌生长,以蔗糖为碳源时利用率最高;以牛肉浸膏为氮源时利用率最高;该菌致死温度为65℃,10 min;光照对菌丝生长影响较小,不同处理间菌落直径无显著差异。     

Systemic aspects of host–pathogen interactions in Austrian pine (Pinus nigra): A proteomics approach

The molecular basis of systemic resistance induced in Austrian pine by two canker pathogens was investigated using a proteomics approach. Protein from phloem located 50 cm above the induction site was extracted 26 days after inoculation. Nineteen proteins shown to be up/down-regulated by the pathogens were selected for sequencing. Fifteen proteins were assigned putative functions, revealing that several represented small heat shock proteins. No PR-proteins were identified among the others, which included a proteasome endopeptidase, two oxygen-evolving enhancer proteins, a nucleoside diphosphate kinase, a fructose bisphosphaste aldolase, a ribulose bisphosphate carboxylase, a phosphoserine aminotransferase, and a formate dehydrogenase.     

Use of some plant extracts to control Biomphalaria alexandrina snails with emphasis on some biological effects

The present work was carried out to evaluate, the molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of some plant species against Biomphalaria alexandrina snails. Preliminary screening tests on 10 plant species showed that the highest molluscicidal potency was recorded for Euphorbia splendens, Atriplex stylosa and Guayacum officinalis. Exposure of B. alexandrina snails to plant’s methanol extracts led to a significant reduction in their survival and growth rates. In addition, newly hatched snails were susceptible to plant’s methanol extracts than elder ones. LC₂₅ of methanol extract from these plants caused a considerable reduction in the infectivity of Schistosoma mansoni miracidia to the snails. It caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results, also, revealed that the glucose concentration in treated snails was increased in haemolymph, while soft tissue glycogen decreased. The activities of glycogen phosphorylase, succinate dehydrogenase (SDH), glucose-6-phosphatase and acetylcholinesterase (AchE) in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with plants methanol extract, while glucose-6-phosphate dehydrogenase (G-6-PD) activity increased (P < 0.001). It was concluded that the application of LC₂₅ of methanol extracts of E. splendens, A. stylosa and G. officinalis may be helpful in snail control as it interferes with the snails’ biology and physiology.