定殖烟草根结线虫卵和雌虫机会真菌的多样性

机会真菌对根结线虫的生物防治具有巨大潜力。本研究于2010—2011年针对来源于中国7个省份的156份烟草根结线虫样本,分别分离根结线虫游离卵、卵块和雌虫上定殖的机会真菌。应用18S rDNA-ITS片段测序结果结合菌株的形态学观察和培养性状鉴定定殖游离卵、卵块和雌虫上的真菌。根据分离到菌株的培养性状、形态学和rDNA-ITS序列分析,对定殖于南方根结线虫或北方根结线虫的9 839个游离卵、408个卵块和284个雌虫的真菌菌株,明确鉴定出9属13个种,其中定殖根结线虫新记录种5个,即长梗木霉、芬芳镰刀菌、渐狭蜡蚧菌、虫草棒束孢和交枝顶孢。淡紫紫孢菌Purpureocillium lilacinum(原名:淡紫拟青霉)为优势种,分布广泛,在云南、安徽、湖北、贵州和山东省均被分离到。该菌在南方根结线虫和北方根结线虫的游离卵、卵块和雌虫平均分离率分别为0.49%、24.00%和16.90%,说明其地理和生态适应性广泛。不同地区来源的烟草根结线虫卵和雌虫上定殖的机会真菌种类存在明显差异。目前尚无渐狭蜡蚧菌和虫草棒束孢作为线虫病原物的报道,有待于进一步深入研究其对根结线虫的致病性。     

昆虫病原线虫共生细菌对南方根结线虫卵孵化的影响

为探讨不同种(品系)昆虫病原线虫共生细菌的不同稀释倍数对南方根结线虫Meloidogyne incognita卵孵化的影响,采用培养皿法进行室内生测,定期调查根结线虫卵孵化情况。结果表明,4种(品系)昆虫病原线虫(斯氏属两个种和嗜菌异小杆两个品系)共生细菌的10×稀释液,6 d时Hb-NJ共生菌使M.incognita的卵孵化率最低,为8.6%,以TSY培养液为对照,其孵化率为29.1%,Hb-NJ共生菌对M.incognita卵孵化的抑制作用最强,相对抑制率为70.6%,其它3种昆虫病原线虫Hb-IGA、Sc-All和Sf-IGA共生细菌的孵化率为分别为24%、22.6%和25.2%,其相对抑制率是17.2%、22.3%和13.4%;8 d时线虫Hb-NJ、Hb-IGA和Sc-All共生菌的相对抑制率分别为67.1%、39.3%和41.7%,10 d时4种(品系)昆虫病原线虫共生菌对M.incognita的孵化率和抑制率均无显著性差异(p0.05)。4种(品系)昆虫病原线虫共生菌的20×和50×稀释液对M.incognita卵孵化没有明显的抑制作用。基于以上结果,Hb-NJ的共生细菌可以通过抑制南方根结线虫的卵孵化导致线虫群体下降而达到防治线虫的目的,因此是一种潜在的生物杀线虫剂。     

黄瓜砧木对南方根结线虫生长发育的影响及其与根系分泌物的关系

黄瓜是设施主栽蔬菜,长期连作导致根结线虫病发生和危害加重;嫁接是提高黄瓜对根结线虫抗性的有效手段,但不同砧木品种间存在差异。为探明嫁接提高黄瓜抗根结线虫能力的机制,试验选用不同抗性的砧木品种‘云南黑籽南瓜’(低感)和‘甜砧南瓜’(高感),以‘新泰密刺’自根黄瓜为对照,采用人工接种,研究了黄瓜砧木对南方根结线虫生长和繁殖的影响,并探讨了根系分泌物与线虫卵孵化的关系。结果表明:线虫侵染初期,3个品种根内的二龄幼虫(J2)数差异显著,‘云南黑籽南瓜’最少,‘甜砧南瓜’次之,‘新泰密刺’最多;侵入根内的J2均能正常发育为成虫,根内线虫总数以‘云南黑籽南瓜’最少,‘新泰密刺’最多;根际J2数量以‘新泰密刺’最多,‘云南黑籽南瓜’最少;发生二次侵染前,根际卵粒数呈现出相同的特点。黄瓜及其砧木根系分泌物明显影响根结线虫卵的孵化,孵化率以‘云南黑籽南瓜’最低,‘甜砧南瓜’次之,‘新泰密刺’最高;接种根结线虫后,根系分泌物影响下的卵孵化率较未接种时明显增加,‘新泰密刺’增幅最大,‘云南黑籽南瓜’增幅最小;随着生育时间延长,三者根系分泌物对卵孵化的促进作用逐渐增强。低感砧木根系分泌物对南方根结线虫卵孵化具有明显的抑制作用,可显著降低线虫在其根内及根际的发育,可能是抗线虫的机制之一。     

用捕食性单齿线虫防治番茄根结线虫

用捕食性单齿线虫可以明显抑制由南方根结线虫引起的番茄根结症状。如果向土壤添加印度楝和蓖麻叶，则对根结症状的发展有更好的控制效果。     

广西根结线虫病害的病原鉴定

从广西不同地区采集的28种植物根结线虫病的病原,按病原线虫的主要形态学特征和寄主反应的鉴定方法,对每种病原进行了分类鉴定。其中穗状鸡冠花、胡椒、蕹菜、胡萝卜,芭蕉芋、鸡蛋果、莞荽(香菜)、苋苯、荷包豆、吉庆果、黄麻、黄穗鸡冠等根结线虫病的病原都是属南方根结线虫1号小种;风仙花、辣椒、蕹菜、番茄、独头鸡冠花、红穗鸡冠、罗汉果、黄麻、芹菜根结线虫有一种是南方根结线虫1号小种,另一种是爪哇根结线虫;甜叶菊、姜根结线虫病的病原有一种是南方根结线虫1号小种,另一种是花生根结线虫1号小种;吉庆果、四季海棠、茄、棕竹、柳树的根结线虫病的病原是爪哇根结线虫;罗汉果、丝瓜、红麻根结线虫病的病原有一种是爪哇根结线虫,另一种是花生根结线虫1号小种;花生根结线虫病的病原是花生根结线虫1号小种。发现二个新种:即水稻根结线虫病的病原是林氏根结线虫;柑桔根结线虫病的病原是孔氏根结线虫。     

航天搭载淡紫拟青霉的生物学效应

为探讨航天诱变对淡紫拟青霉的生物学效应的影响,利用淡紫拟青霉山东菌株搭载"神舟八号"宇宙飞船进行诱变处理。对筛选得到的30个淡紫拟青霉航天诱变菌株进行了形态、色素、菌丝及孢子形态、菌落生长速度、菌丝干重和产孢量等生物学检测,并测定了各诱变菌株对南方根结线虫卵和松材线虫卵的寄生率。结果表明,淡紫拟青霉航天诱变菌株各项生物学特性与原始菌株存在分化。航天诱变后的菌株菌落形态、色素和菌丝结构与原始菌株差异明显;菌落生长速度、菌丝干重和产孢量的负突变率均高于正突变率;菌株对松材线虫卵的致病力较低,寄生率最高的菌株Sd-m-20的寄生率仅为18.0%,菌株对根结线虫卵致病力较高,菌株Sd-m-9,Sd-m-11,Sd-m-16,Sd-m-22和Sd-m-26对根结线虫卵的寄生率分别提高了14.48%、14.90%、12.35%、7.66%和17.45%,显著高于原始菌株的寄生率(78.3%)。航天处理对淡紫拟青霉诱变效果显著,其优良突变菌株可能用于淡紫拟青霉对南方根结线虫的生物防治。     

沼液淹没土壤抑制根结线虫及对土壤线虫群落的影响

为探索沼液抑制根结线虫的效果,本研究通过盆栽试验,以番茄为试供作物,对比了种植前沼液淹没土壤(BSS)、种植期间浇灌沼液(BS)和加热(HE)3种方法对根结线虫的防控效果。结果表明,与不采取任何措施的对照(CK)处理相比, BSS处理抑制根结线虫效果最为明显,防效高达97.1%,根结指数分别比HE和BS处理降低96.9%和92.9%。HE处理尽管在处理土壤后显著降低了根结线虫数量,但在最后破坏性取样时(结束试验)出现反弹,根结线虫数量甚至高于CK处理。对于土壤线虫群落,CK处理中以植食性线虫为主(81.8%);两个沼液处理中食细菌线虫占优势(平均78.3%),且其中的杂食捕食性线虫在土壤前处理后消失,在试验结束时又重新出现,但所占比例依然非常低。沼液淹水方式的高效防控效果揭示了利用沼液防控根结线虫的关键期在于线虫入侵到植物根部之前的幼虫期。然而,在盆栽系统中,沼液淹水的方式也对作物生长表现出了一定的抑制趋势。高量沼液施用防控病害的同时引发的植物毒害作用以及环境污染风险,需要进一步开展田间研究。     

黄秋葵对南方根结线虫抗性的转录组分析

为了解黄秋葵抗南方根结线虫相关基因组学,利用Illumina Hi-seq TM2500高通量测序技术研究受南方根结线虫侵染后黄秋葵种质12C2转录组基因的差异表达。结果表明,接种南方根结线虫18 h后,从接种和未接种的黄秋葵种质12C2根尖中共获得71.49 Gb有效数据,Q30碱基百分比均达到94.0%以上。共获得2 318个差异表达基因(DEGs),包括1 156个上调基因,1 162个下调基因,其中功能注释基因2 202个。根据unigene库序列进行GO、KOG和KEGG注释,细胞壁代谢相关基因——内切葡聚糖酶基因家族、聚半乳糖醛酸酶基因家族、葡聚糖内-1,3-β葡糖苷酶基因家族和果胶裂解酶基因家族下调表达,植物激素代谢相关基因——生长素响应蛋白基因、生长素流入运输载体基因下调表达,茉莉酸合成酶基因上调表达,调控相关基因表达的WRKY和MYB转录因子基因家族上调表达,参与植物细胞的膜联蛋白基因家族上调表达,植物细胞周期蛋白基因家族下调表达。本研究结果为开展黄秋葵抗南方根结线虫基因组学和分子生物学研究奠定了基础。     

对根结线虫高毒力芽胞杆菌的鉴定及其活性测定

为筛选高效安全的杀虫资源,从海底淤泥中分离到一株对根结线虫(Meloidogyne spp.)高毒力的芽胞杆菌YBf-10,PCR扩增16S rRAN基因,经测序、序列比对分析和系统进化树构建,发现其与坚强芽胞杆菌(Bacillus firmus)Z1-7菌株16S rDNA同源性为99%,初步确定所分离的菌株为坚强芽胞杆菌。将该菌株培养至芽胞成熟,离心取上清,10倍稀释后进行生物测定,发现其对北方根结线虫二龄幼虫有很高的毒力。处理24h校正死亡率达到50%以上,72h达到100%。对根结线虫虫卵进行毒力测定表明,作用48h后能显著抑制虫卵孵化达到80%以上。在培养过程中分不同时段取样,并用所取样品上清进行生物测定,发现从稳定期开始表现出了对北方根结线虫的毒力,在整个稳定期毒力持续增强,直到衰亡期后期,毒力达到最高,表明坚强芽胞杆菌所产生的杀线虫活性物质主要是在稳定期合成的。将发酵上清80℃处理30min毒力无明显变化,通过饱和硫酸铵沉淀上清中蛋白,该蛋白对线虫无明显毒力,但是去蛋白后的上清对线虫仍然具有与未经处理上清相似的杀线虫活性,表明坚强芽胞杆菌产生的杀线虫活性物质是一种非蛋白类的小分子化合物。研究结果提示,本研究所分离的坚强芽胞杆菌在稳定期能够大量合成对线虫具有毒杀活性的小分子化合物,对根结线虫表现出极高毒力,为利用该菌株开发植物寄生线虫生防制剂提供了杀虫资源。     

香蕉根结线虫卵的分离与孵化试验初报

采用直接解剖分离法和粉碎筛分法分离香蕉根结线虫,并利用滤液和蒸馏水(对照)在室温和26℃恒温培养箱中培养,研究香蕉根结线虫卵的分离和孵化情况。结果表明:1)粉碎筛分法具有简单、快速的特点,能较快获取所需线虫卵的悬浮液;2)26℃恒温培养卵孵化率比室温条件下孵化率高,且用滤液培养的卵孵化率高于蒸馏水培养的孵化率。3)在26℃恒温条件下加入滤液和蒸馏水培养10 d,卵的孵化率分别为84.21%和58.36%;室温条件下培养10 d,线虫的孵化率分别为43.22%和27.55%。4)粉碎筛分法是一种较简单的从香蕉根中分离得到根结线虫卵的方法,且在26℃用滤液培养10 d能使多数线虫卵孵化。     

基于种蛋图像血线特征和深度置信网络的早期鸡胚雌雄识别

为了实现孵化早期鸡胚雌雄识别,构建了机器视觉采集系统,在LED光源下获取180枚鸡种蛋孵化第4天的图像。首先对鸡种蛋图像进行RGB分量提取、中值滤波、感兴趣区域提取等预处理,然后利用限制对比度自适应直方图均衡化、形态学处理、最大类间方差阈值分割和八连通域去噪等方法凸显血线纹理,并通过方向梯度直方图(histogram of oriented gradient,HOG)提取图像的全信息特征和利用灰度共生矩阵提取能量、对比度、相关性、熵、均匀度等5个特征,对HOG全信息特征采用主成分分析(principal component analysis,PCA)降维,最后利用全信息特征和PCA降维特征-灰度共生矩阵特征组合的简化特征,分别构建支持向量机(support vector machine,SVM)、反向传递(back propagation,BP)神经网络、深度置信网络(deep belief networks,DBN)3种鸡胚雌雄识别模型,并比较不同模型的识别准确率。试验中,全信息特征比简化特征构建的模型识别准确率高,基于简化特征的BP、SVM、DBN模型测试集识别综合准确率分别为51.67%、60%和58.33%,基于全信息特征的BP、SVM、DBN模型测试集识别综合准确率分别为58.33%、63.33%和83.33%。其中,基于全信息特征的DBN模型识别准确率最高,达到83.33%。结果表明机器视觉技术为孵化早期鸡胚雌雄识别提供了一种可行方法。     

不同成熟液对猪卵母细胞体外成熟和体外受精后早期胚胎发育的影响

本研究探讨了不同成熟液[改良TCM-199液(mTCM)和改良NCSU-23液(mNCSU)]对猪卵母细胞体外成熟和体外受精后早期胚胎发育的影响，以期提高其受精和胚胎发育潜能。试验表明：(1)猪卵母细胞的核成熟与其周围的卵丘细胞扩展没有必然的联系，卵丘细胞扩散或成放射状不宜作为猪卵母细胞核成熟的标准，只有排出第一极体才能作为猪卵母细胞核成熟的标志；(2)利用mTCM 10％猪卵泡液和mNCSU 10％猪卵泡液的猪卵母细胞体外培养核成熟效果优于mNCSU 10％胎牛血清，但这三组成熟液对猪卵母细胞的卵丘细胞扩散和体外受精后的早期胚胎发育无显著影响。     

骆驼蓬种子提取物对斜纹夜蛾的生物活性效应

用甲醇、乙醇、丙酮、氯仿、乙酸乙酯、苯、石油醚等7种溶剂对骆驼蓬种子进行冷浸提取。提取率分别为23．91％。18．68％．17．80％，18．49％，14．31％．15．13％，17．96％。7种提取物中．以种子乙醇提取物(ESPH)对斜纹夜蛾3龄幼虫的非选择性拒食活性最高。24h拒食率为70．15％。采用酸水总生物碱提取法对ESPH进行初步分离。得到氯仿、正丁醇、水及总生物碱萃取物。生物测定结果表明．总生物碱萃取物对斜纹夜蛾幼虫的生长发育抑制作用最为强烈，其次为氯仿萃取物。     

Effects of low ph exposure of adult salmonids on gametogenesis and embryo development

Mature male and female rainbow trout were treated with low pH (Av. 4.5) sulfuric acid water for 1 or 2 weeks. Percentage of eyed embryos in eggs from control, 1-week-treated and 2-week-treated females were 100%, 80% and 0%, respectively. Low pH exposure of male trout induced 11% deformation in embryos fertilized with their sperm. In order to clarify the physiological mechanism of the effects of low pH exposure, sex hormone levels were compared. In female fish, plasma sex hormones levels showed no difference among the groups, but egg contents of 1720-dihydroxy-4-pregnen-3-one, which induces oocyte maturation, were significantly lower in low pH exposed groups. Acidified male fish showed higher plasma levels of the spermiation inducing hormone, 11-ketotestosterone. Effects on eggs were manifested under pH lower than 5. Under continual rearing of land-locked sockeye salmon in acid water, the effects on the oocyte were observed in those adult females which were exposed to acidic condition for more than 1 week before the timing of ovulation. These results suggest that low pH affects oocyte maturational events occurring just 1 week before ovulation, and that acid stress affects the endocrinological mechanisms of final maturation in fish gametocytes. Thus, acidification of the aquatic environment may reduce the reproductive activity of fish at rather low acidity levels.     

Life history traits and food supplementation affect productivity in a translocated population of the endangered Hihi (Stitchbird, Notiomystis cincta)

We studied the effect of food supplementation, female age and clutch order on productivity in a translocated species. Food supplementation increased clutch size from 3.9 to 4.4 (average) eggs per nesting attempt, and more than doubled fledging and recruitment success. Supplemented females started a second clutch 9 days sooner after fledging first clutch chicks than unfed females. During second clutches, supplemented females incubated the eggs for a shorter period of time (15.2 days vs. 16.8). Older females laid larger clutches (4.6 vs. 3.7 eggs) than yearlings and incubated second clutch eggs for a shorter period (15.4 vs. 16.6 days). Females laid more eggs in first clutches (4.2 vs. 3.8 eggs), and those eggs took approximately 30% longer to lay than eggs in second clutches. The successful maintenance of hihi populations on the available islands may be dependent on the permanent provision of supplemental food at nest sites.     

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Boron and zinc inhibit embryonic development,hatching and reproduction of Meloidogyne incognita

Nematodes have difficult control and complex handling, but considering the physiological and biochemical changes that micronutrients promoting in plants, there is possibility that the supply with these chemicals increases the resistance of plants against nematodes. Thus, the present study aimed to assess the effect of the application of boron and zinc on the reproduction of Meloidogyne incognita, embryonic development and juvenile nematode hatching. Nematode reproduction was evaluated in tomato plant inoculated with 2000 eggs and treated in the aerial part with boron or zinc at the following doses: 0, 1/2, 1×, 2× and 4× the manufacturer's recommendation (100 and 30?g/L, respectively), with the plants assessed 60 days after inoculation. For assessment of embryonic development and juvenile hatching, 1?mL nematode suspension was placed in Petri dishes containing 9?mL of the same doses of boron and zinc, and assessment occurred four and eight days after incubation. Results obtained showed that boron controlled nematode population at the dose of 400?g/L and promoted juvenile hatching when used at maximum dosage on the eighth day. Zinc reduced the number of galls and the number of eggs at the dose of 60?g/L, but did not exhibit direct effect on nematode.     

Population ecology of the Chinese grouse, Bonasa sewerzowi, in a fragmented landscape

We report the first population ecology study of the endangered Chinese grouse (Bonasa sewerzowi). The study population was stable during 1995-2000. Annual rates of survival were 0.64 for adults and only 0.17 for chicks to 13 weeks of age. Reproductive parameters were 6.1 eggs/clutch, 63% nest success, and overall 3.6 chicks hatched/female/year. The spring sex ratio was 64% males, with 44% of the males unmated in spring. Local people found an estimated 10-29% of the nests and took the eggs for food. Only about 9% of the reserve containing the study area is mature mixed forest, the preferred habitat. Inadequate brood habitat may have influenced chick survival and the skewed adult sex ratio may be a result of habitat fragmentation. We recommend protecting the remaining mixed forest, creating more brood habitat, and stopping the taking of eggs.     

Identification and characterization of the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) in hen egg

Using zymography and mass spectrometry, we identified for the first time the precursor of chicken matrix metalloprotease 2 (pro-MMP-2) as a complex with TIMP-2 (tissue inhibitor of metalloproteinases) in egg white and yolk. Real-time polymerase chain reaction confirmed that MMP-2 and its inhibitors TIMP-2 and TIMP-3 were expressed all along the oviduct and in the liver of laying hens. We also demonstrated that the processing of pro-MMP-2 into mature MMP-2 by serine proteases does not occur in vivo, although purified pro-MMP-2 undergoes proteolytic maturation by these proteases in vitro. Moreover, the relative pro-MMP-2 activity assessed by gelatin zymography was shown to decrease in egg white during the storage of unfertilized or fertilized eggs. However, the mature form of 62 kDa MMP-2 could not be detected. The fact that MMP-2 is found as a proform in fresh eggs suggests that the activity of this metalloprotease is regulated under specific conditions during embryonic development.     

90-day oral toxicity study of a grape seed extract (IH636) in rats

To assess the safety of grape seed extract with less than 5.5% catechin monomers (IH636), 4 groups of male and female Sprague-Dawley rats were provided grape seed extract in the diet at levels of 0 (control), 0.5, 1.0, or 2.0% for a period of 90 days. All animals survived the duration of the study, and no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings were observed. A significant increase in food consumption was observed in male and female rats provided the grape seed extract diets compared to that of the control rats, especially in male rats consuming 2.0% grape seed extract. This effect was not accompanied by increases in body weight gains. Grape seed extract appeared to increase the insoluble fraction of the diet. Male rats in the high-dose group exhibited decreased serum iron levels and decreased serum iron/total iron binding capacity ratio compared to those of the controls, although all values were within historical ranges for Sprague-Dawley rats. In conclusion, administration of the grape seed extract IH636 to male and female Sprague-Dawley rats in the feed at levels of 0.5, 1.0, or 2.0% for 90 days did not induce any significant toxicological effects.