Multi-gene sequence analysis and phenotypic diversity of Phaeoacremonium species isolated from grapevines in Spain

Esca and Petri disease, caused by Phaeoa-cremonium, and other grapevine decline diseases, such as eutypa dieback, are responsible for serious economic losses to the wine industry worldwide. In the present work, 175 isolates of Phaeoacremonium spp. were collected (mostly in the region of Castilla y León, Spain) from vines either asymptomatic or symptomatic for grapevine decline diseases. The sequences of DNA fragments of 30 isolates were analysed. Amplicons of ITS, and partial gene of the β-tubulin, actin, calmodulin and translation elongation factor 1-α [EF1-α], were obtained by PCR. This is the first time that any exon of the EF1-α gene or exons 6–7 of the β-tubulin gene of seven Phaeoacremonium spp. have been amplified. Amplicons of the calmodulin and EF1-α exons were the most informative in terms of species differentiation: only 52.6 % and 56.6 % similarity across six and seven species was respectively seen. Phylogenetic analysis of combined DNA sequences revealed five Phaeoacremonium species isolated from the sampled plants; P. aleophilum was the most common. Four other species were found, however only one isolate each. P. mortoniae and P. iranianum are here reported for the first time on grapevines in the region of Castilla y León and the neighbouring region of La Rioja respectively. In total, 29 nucleotide differences among the studied gene fragments were seen across 23 isolates of P. aleophilum. Network analysis showed these Spanish P. aleophilum isolates to fall into two main groups of genotypes. P. aleophilum genotypes belonging to group 1 were mostly isolated from young grapevines affected by Petri disease but genotypes included in group 2 were isolated from adult plants showing esca or eutypa dieback symptoms.     

Role of weed hosts and the western flower thrips, Frankliniella occidentalis, in epidemiology of Tomato spotted wilt virus in the Çukurova region of Turkey

Surveys of thrips and Tomato spotted wilt virus (TSWV) on weeds were conducted in the eastern Mediterranean region of Turkey during the years 2004–2006. Thrips species were collected by vigorously shaking weedy plants into a white container for 15 sec. Plant material collected during field surveys and plants which were used for mechanical inoculation of TSWV, were tested by DAS–ELISA. The weed species Ranunculus muricatus, Melilotus officinalis, Sinapis arvensis and Portulaca oleracea were used for the virus transmission experiments in an enclosed high plastic tunnel and in cage experiments. Western flower thrips, Frankliniella occidentlis (Pergande) (Thysanoptera: Thripidae), was the most common thrips, inhabiting 80 of the 82 weed species sampled. Adults of F. occidentalis and thrips larvae were significantly more abundant on S. arvensis than on the other weed species sampled (P<0.05). Adult and larval thrips showed peak densities on most weeds in April or May. Summer annual weeds were not good hosts for reproduction of the thrips. A total of 90 samples from 17 plant species belonging to 11 plant families were ELISA positive for TSWV. No TSWV was detected on 65 weed species belonging to 26 plant families. High numbers of plant samples infected by TSWV were obtained in P. oleracea (21 samples) and in R. muricatus (15 samples). In field surveys symptoms of TSWV were detected on only R. muricatus. Incidence of the TSWV on weeds ranged between 5% and 25%. Transmission rates of TSWV by F. occidentalis to the weeds ranged from 33% to 83% in the pepper plastic tunnel and cage experiments.     

猪肉源金黄色葡萄球菌毒力基因检测与耐药性分析

【目的】了解陕西关中地区猪肉中金黄色葡萄球菌(Staphylococcus aureus)的污染状况、耐药性及其毒素基因的分布。【方法】采集陕西关中6个地区的猪肉165份,按国标GB/T 4789.10-2010的方法,对其中的金黄色葡萄球菌进行分离,采用PCR方法对该菌进行确证并对其相关基因(如nuc、mecA、PVL、SEs和ETs)进行检测,最后采用琼脂稀释法检测金黄色葡萄球菌对11种抗菌药物的耐药性。另外,在BP平板中分别添加头孢西丁(4μg/mL)和苯唑西林(4μg/mL),分离耐甲氧西林金黄色葡萄球菌(MRSA)。【结果】165份样品的金黄色葡萄球菌污染率为33.33%(55/165);从中分离出103株金黄色葡萄球菌,但未检测出MRSA,这些菌对甲氧苄啶的耐药性最强,耐药率为100%;其次对红霉素和四环素的耐药率较高,分别为57.28%和34.95%;对苯唑西林、庆大霉素、氯霉素、环丙沙星的耐药率分别为2.91%,10.68%,2.91%和3.88%;所有菌株对头孢西丁、头孢哌酮、万古霉素、阿米卡星均敏感,同时得到21种耐药谱,多重耐药率达20.39%。猪肉金黄色葡萄球菌中杀白细胞素基因(Panton-valentine leu-kocidin,PVL)的检出率为34.95%,肠毒素基因(SEs)中sej的检出率最高,为98.06%,然后依次为sea(50.49%)、see(34.95%)、sed(31.07%)、sec(13.59%)、seh(8.74%)、sei(8.74%)、seg(6.80%)和seb(1.94%);同时得到71种毒素基因型,以sea+sej(11.65%)最为流行,分布地区不尽相同,其次为PVL+sea+see+sej(9.71%),耐红霉素的金黄色葡萄球菌含的毒素基因类型比较复杂,sej基因检出率高达98.68%。在BP平板中分别添加头孢西丁和苯唑西林,均未检测出MRSA。【结论】猪肉存在金黄色葡萄球菌的污染,其污染菌株存在多重耐药性并携带较多毒素基因,提示应加强猪肉金黄色葡萄球菌的监测。在BP平板中分别添加头孢西丁(4μg/mL)和苯唑西林(4μg/mL)筛选MRSA的方法不一定可靠,其可信度有待证明。     

红花槐花药培养最适取材时期及选蕾标准

【目的】确定红花槐花药离体培养的最适宜取样时期及其相应的花器官形态特征判断标准。【方法】采用小孢子染色压片观察、花器官形态特征观测和花药离体培养试验,研究红花槐花药小孢子发育阶段,探讨小孢子发育阶段与花器官形态特征及花药愈伤组织诱导率的关系。【结果】1)小孢子发育过程可分为四分体、单核及二细胞花粉3个时期,这3个时期之间具有明显的细胞学特征差异,其中单核时期又分为单核早中期和单核靠边期。2)小孢子单核时期(单核早中期及单核靠边期)花药适宜离体培养,其中单核靠边期愈伤组织诱导率高达(77.05±1.56)%,为红花槐花药离体培养的最佳时期。3)在单核靠边期,花蕾纵径介于10.01～12.20 mm、花冠与花萼的长度比>1/4～≤2/5。同时,小花花萼上部为红色,白色花冠从花萼中露出且顶部开始变为粉红色,花序大而松散,小花间距离较大。【结论】红花槐花药离体培养最适取材时期为小孢子单核靠边期,并可通过小孢子与花器官形态特征的对应关系进行取材。     

基于元胞自动机与智能体的水污染可视化模拟仿真

【目的】建立水污染可视化扩散模型,以刻画水污染突发事件的复杂性及满足其应急决策平台对可视化的需求。【方法】构建基于元胞自动机和智能体的三维水污染可视化扩散模型,用元胞自动机刻画研究区流域及所在区域地形,并从元胞空间、元胞状态、演化规则方面扩展普通元胞自动机模型,应用水质Agent描述流域中携带污染物的水体,在此基础上结合3S技术充分展现污染物的扩散运移过程,并将文中模型在渭河宝鸡段水污染事件中进行实例应用。【结果】基于元胞自动机和智能体的三维水污染可视化扩散模型充分展现了流域水污染扩散模拟现象的动态性、复杂性、非线性特点。模型在渭河宝鸡段水污染事件中的应用表明,静态水面元胞层和水下元胞层以及动态水质Agent的结合,简化了污染物在水面及水下不同的扩散运移现象,所建模型合理、可靠,能有效预测及模拟污染物的运移扩散过程;同时利用3S技术对流域空间信息进行了展示分析,满足突发水污染事件应急平台对事件模拟高效率、可视化的要求。【结论】所建模型可以为突发水污染事件提供一种预测和模拟方法。     

岚皋魔芋软腐病病原细菌生物多样性研究

【目的】研究岚皋魔芋软腐病病原细菌生物多样性。【方法】采用组织分离法分离纯化魔芋软腐病球茎及叶柄中的致病菌,通过魔芋球茎、胡萝卜及马铃薯块的侵染试验初步确定其致病性,再利用魔芋球茎侵染致病及盆栽致病性试验确认其致病性;经菌落与细胞形态、生理生化特性及16SrRNA序列测定确定病原菌的类型。【结果】从魔芋软腐病球茎及叶柄中共分离获得16株细菌,侵染试验表明,其中CDS1-B1、CDS1-B2、CDS2-B1、CDS2-B2、CZS-B4和CZS-B6共6株细菌可使魔芋球茎表现软腐症状,并导致盆栽魔芋发病;6株细菌的菌落与细胞形态均存在差异;在魔芋、胡萝卜及马铃薯块上的侵染能力不同;16SrRNA序列分析表明,菌株CDS1-B1、CDS1-B2、CDS2-B1及CDS2-B2均与菊欧氏杆菌(Erwinia chrysanthemi)亲缘关系最近,菌株CZS-B4与菊欧文氏菌(Dickeya dadantii)亲缘关系最近,相似度为98.8%,菌株CZS-B6与菊果胶杆菌(Pectobacterium chrysanthemi)的相似度达99.9%。【结论】导致岚皋县魔芋软腐病的致病细菌存在生物多样性,其致病性也存在差异,其中新发现的魔芋软腐病原菌菊果胶杆菌(Pecto-bacterium chrysanthemi)致病性最强,菊欧氏杆菌(Erwinia chrysanthemi)和菊欧文氏菌(Dickeya dadantii)致病性较弱。     

东北天南星茎挥发油成分GC-MS分析

用气相色谱-质谱法对东北天南星茎的挥发油进行化学成分分析。采用水蒸气蒸馏法从东北天南星茎中提取挥发油,用归一化法测定其百分含量,并用气相色谱-质谱法对化学成分进行鉴定。共鉴定了48种成分,占挥发油总量的96.637%。本方法稳定可靠具有良好的重现性适用于中药挥发油的化学成分分析。     

植物根际放线菌分离方法初探及根皮苷降解活性分析

【目的】研究不同培养基及不同土样处理方法对植物根际放线菌分离效果的影响,并从中挖掘具有开发潜力的高活性根皮苷降解放线菌株,为苹果连作障碍机制研究及微生物修复提供理论和实践指导。【方法】从吉林、河北、山东、陕西、甘肃、河南、湖北、四川、重庆、上海、江西、安徽、浙江等13个省(市)采集植物根际土样40份,自然风干处理后分别经10mmol/L的MOPS溶液及CaCO3预处理,用涂布平板法在7种不同的培养基中进行放线菌的分离培养。将分离获得的菌株点种于根皮苷筛选培养基进行活性检测。【结果】在7种培养基B、MSC、MG、MT、Chi、HSG、HV中分别分离获得100、78、108、64、79、113和88株放线菌。土样用MOPS溶液处理后所分离得到的放线菌总数明显高于用CaCO3预处理所得。分离所得630株菌株中有29株对根皮苷有降解活性。对其中41株高活性菌株及形态特殊菌株进行16SrRNA基因测序分析,发现分属于14个不同的放线菌属。【结论】7种分离培养基中HSG培养基的分离效果最好,MG培养基次之,MT培养基最差。土样处理中用MOPS溶液处理的方法优于用CaCO3预处理。根际放线菌株中有一定数量具有降解根皮苷能力的菌株。根际放线菌资源具有丰富的生物多样性。     

杧果短截结果母枝玉米素核苷含量的动态变化

【目的】测定杧果花芽分化期间结果母枝各器官内玉米素核苷(ZR)含量,探索ZR在杧果花芽分化中的作用。【方法】利用杧果花枝短截后剪口芽在当年还能再次花芽分化并开花结果的现象,应用酶联免疫吸附分析方法(ELISA)定期测定剪口叶、附近韧皮部及芽内ZR含量。【结果】短截结果母枝后,剪口叶、附近韧皮部和剪口芽内ZR含量分别于2月25、20日和3月2日降至最低,为1.73、1.52、2.0μg·g-1FW;叶片、韧皮部及剪口芽于3月12日达最大值,为6.39、8.47、10.32μg·g-1FW。2月25日剪口芽芽眼中心颜色逐渐呈蜡黄,并向芽体四周扩散;3月2～7日,芽体逐渐膨大,蜡黄色加深,芽眼呈半透明状;3月7日后绿色逐渐增加,半透明状消失。【结论】ZR在杧果花芽分化的不同阶段具有不同作用。花芽分化临界期前,ZR含量低,生长点细胞活性下降,有利于杧果植株从叶芽生理状态转化为花芽生理状态;花芽临界期后,相对高的ZR含量有利于杧果植株生长点进行花芽分化。