于蔗种茎热水处理脱除宿根矮化病菌及其对再生苗的影响

50℃2h热水处理黑皮果蔗、黄皮果蔗和粤糖93-159双芽段种茎，结果表明：黑皮果蔗全部丧失出苗能力；黄皮果蔗出苗时间延迟，但最终出苗率与对照无差异；而粤糖93-159出苗率和出苗速度均不受影响。两个品种的热处理再生苗生长早期株高和茎径均显著低于对照，但随着生长时间的增加，这种差异逐渐缩小。采用高灵敏度的巢式PCR技术检测再生植株体内的宿根矮化病菌，在再生苗生长前期的120d内所有经热处理植株均呈阴性，其后部分植株呈阳性，至播种200d时，黄史果蔗阳性率为50％，粤糖93-159阳性率为100％。试验还发现，种茎热水处理能增强再生植株中后期抗旱能力。本研究指出，虽然种茎热水处理能减少生长早期再生苗体内宿根矮化病菌含量但难以实现所有种茎完全脱菌。在进行脱菌效果评价时，要对生长中后期的再生苗进行检验；生产上采用该措施防病，要充分考虑不同品种耐热性能的差异。     

广东果蔗宿根矮化病菌检测

为探明广东果蔗宿根矮化病发生情况,为果蔗健康种苗生产及推广应用提供科学依据,本研究采用常规PCR与巢式PCR技术分别对广东韶关和广州南沙果蔗产区的主栽品种‘黑皮果蔗’(‘Badila’)及华南农业大学甘蔗育种基地新引进的果蔗品种‘内江蜜蔗’、‘甜城21号’、‘甜城22号’和‘甜城99号’等进行宿根矮化病菌的检测。结果表明,巢式PCR检测的阳性检出率达88.6%,明显高于常规PCR检测(40.4%);广东韶关果蔗产区‘黑皮果蔗’宿根矮化病阳性率为86.8%;广州南沙果蔗产区‘黑皮果蔗’宿根矮化病阳性率为92.7%;华南农业大学甘蔗育种基地新引进的果蔗品种除‘甜城22号’外,其他3个品种‘内江蜜蔗’、‘甜城21号’和‘甜城99号’均感染宿根矮化病菌。甘蔗宿根矮化病已在广东主要果蔗产区普遍发生,健康种苗研究与应用十分必要。     

粤北地区果蔗细菌性病害PCR分子检测

为了解粤北地区果蔗主产区甘蔗宿根矮化病、赤条病和白条病病害发生和为害,本研究通过PCR技术对粤北果蔗产地植株蔗茎样品进行分子检测。结果表明,果蔗宿根矮化病菌总检出率为41.5%,各采样地块均检测出宿根矮化病病原菌,地块检出率达到100%,不同地块的检出率为10%~82.8%。赤条病和白条病病原菌未检出。粤北地区果蔗细菌性病害以甘蔗宿根矮化病为主。     

应用PCR技术检测甘蔗各部位宿根矮化病病菌

甘蔗宿根矮化病(ratoon stunting disease,RSD)是中国甘蔗产区一种主要病害。为保证甘蔗健康种苗生产,应用实验室建立的甘蔗宿根矮化病菌PCR检测技术对甘蔗品种粤糖00-236、新台糖22宿根蔗和其健康种苗各部位进行检测。检测结果表明,两品种宿根蔗的老根、新根、老叶、新叶、叶脉、蔗汁都检测到RSD;茎尖和腋芽部位均未检测出RSD。因此,采取腋芽或茎尖部位进行甘蔗组培生产健康种苗,可有效地除去甘蔗RSD病菌。     

广东甘蔗黄叶病田间调查及病原病毒的分子检测

 广东省粤北和粤西蔗区多个县市的田间甘蔗上观察到甘蔗黄叶病(Sugarcane yellow leaf disease,SYLD)典型症状,目前该病仅局部分布,但部分田块病株率为5%~80%,发病品种有青皮果蔗、黑皮果蔗、新台糖系列品种、粤糖79/177和粤糖93/159等。采集发病田间显症叶片、无症叶片和在病叶上取食的甘蔗绵蚜(Ceratovacuna lanigera)样品,抽提总RNA,以基于甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV) CP基因序列的特异引物进行一步RT-PCR和巢式PCR扩增,并对扩增产物进行核苷酸序列测定和BLAST比对。结果显示,RT-PCR及巢式PCR产物核苷酸序列与分离自巴西的SCYLV B1株系相应区段同一率为100%;一步RT-PCR可从约70%的显症叶片样品中检测到SCYLV,而病田中的无症叶片样品以及在病叶上取食的单头甘蔗绵蚜样品需经巢式PCR扩增方可检测到SCYLV,阳性率分别为1%~5%和83%。本研究表明,广东省栽培甘蔗已受到SCYLV侵染,甘蔗绵蚜携带SCYLV。     

黄皮甲醇提取物的抑真菌作用

采用生长速率法测定了黄皮植株甲醇提取物对香蕉炭疽病菌等12种植物病原真菌的生物活性．研究结果表明：黄皮植株甲醇提取物,对香蕉炭疽病菌和辣椒炭疽病菌等有一定的活性;在10mg／mL的浓度下,黄皮果核、树皮、枝叶和花索的甲醇提取物,对香蕉炭疽病菌菌丝生长的抑制率分别为88．83％、84．61％、72．23％和50．11％;黄皮果核甲醇提取物对香蕉炭疽病菌的EC50为4．34mg／mL。黄皮果核甲醇提取物的石油醚、氯仿、乙酸乙酯和正丁醇萃取组分对香蕉炭疽病菌的活性研究表明,氯仿层的活性最高,抑制率为86．11％。黄皮果核甲醇提取物的氯仿萃取物对香蕉炭疽病菌的EC50为2．37mg／mL。     

果蔗脱毒种苗甘蔗花叶病、黄叶病和宿根矮化病分子检测

为监测2016-2017年种植的果蔗脱毒种苗脱毒效果,分别采集广州市南沙区和增城区、湛江市麻章区及华南农业大学甘蔗育种基地共83份果蔗脱毒种苗样本,进行甘蔗花叶病毒(SCMV)、高粱花叶病毒(SrMV)和甘蔗黄叶病毒(SCYLV)RT-PCR检测。结果表明SCMV的阳性样本数为3个,阳性检出率3.61%;SrMV的阳性样本数为0;SCYLV的阳性样本数为78个,阳性检出率93.98%。采用常规PCR和巢式PCR技术对采集于广州市增城区和华南农业大学甘蔗育种基地的30份果蔗脱毒种苗样本进行宿根矮化病菌(Lxx)检测,常规PCR检测阳性样本数为0,巢式PCR检测疑似阳性样本数为8,疑似阳性检出率26.67%。本研究采用茎尖组织培养脱毒技术培育的果蔗脱毒种苗能有效脱除果蔗种苗内的SCMV、SrMV和Lxx,但SCYLV的脱除效果有待进一步研究。     

柳州和来宾蔗区甘蔗花叶病发生情况和品种抗性分析

2016年通过对广西柳州和来宾蔗区甘蔗花叶病发生情况调查和当地主栽品种的田间抗性的分析评价,明确了不同种植区域、不同品种及不同种植期感抗花叶病有一定差异。柳州和来宾蔗区新植蔗发病率为0.00~56.72%,宿根蔗发病率为0.00~93.51%。宿根蔗感病程度比新植蔗重。柳州蔗区发病率较高,平均为19.36%,来宾蔗区发病率较低,平均为8.12%。品种间存在显著的抗性差异。ROC16表现为免疫,桂柳2号、桂糖40和粤糖00-236表现为高抗,桂糖21和粤糖93-159表现为中抗,ROC22和台优表现为感病,柳城05-136表现为高感。系统聚类分析结果与品种的田间抗性评价结果一致。     

氟乐灵处理后棉苗对枯萎病菌的抗侵入及抗扩展特性

 以1μg/g土的氟乐灵播前土壤处理,棉花出苗后移栽到无氟乐灵的土壤中并接种棉花枯萎病菌(Fusarium axysporum f.sp.vasinfectum)。结果表明氟乐灵处理明显降低了棉苗枯萎病的株发病率和病情指数,提高了棉苗对枯萎病的抗病性。氟乐灵处理组棉苗根部和茎部枯萎病菌侵染率均低于对照组,特别是上部茎片段中处理组侵染率降低更为明显。氟乐灵处理组棉苗根部和茎部的菌量明显降低,而且处理组茎部菌量的增长明显滞后于对照。以上结果表明,病菌对氟乐灵处理棉苗根部的侵入和病菌在棉苗体内的纵向扩展及增殖受到了阻抑,因而氟乐灵处理后棉苗对枯萎病菌的抗性表现了抗侵入和抗扩展的特性。试验还证明,氟乐灵处理并接种病菌的棉苗茎部组织中类萜烯醛的含量明显提高;并产生一种能抑制病菌大型分生孢子萌发的真菌毒性物质。这些抑菌物质的产生和积累可能与抗扩展特性有关。     

氟乐灵处理后棉苗对枯萎病菌的抗侵入及抗扩展特性

以1μg/g土的氟乐灵播前土壤处理,棉花出苗后移栽到无氟乐灵的土壤中并接种棉花枯萎病菌(Fusarium axysporum f. sp. vasinfectum)。结果表明氟乐灵处理明显降低了棉苗枯萎病的株发病率和病情指数,提高了棉苗对枯萎病的抗病性。氟乐灵处理组棉苗根部和茎部枯萎病菌侵染率均低于对照组,特别是上部茎片段中处理组侵染率降低更为明显。氟乐灵处理组棉苗根部和茎部的菌量明显降低,而且处理组茎部菌量的增长明显滞后于对照。以上结果表明,病菌对氟乐灵处理棉苗根部的侵入和病菌在棉苗体内的纵向扩展及增殖受到了阻抑,因而氟乐灵处理后棉苗对枯萎病菌的抗性表现了抗侵入和抗扩展的特性。试验还证明,氟乐灵处理并接种病菌的棉苗茎部组织中类萜烯醛的含量明显提高;并产生一种能抑制病菌大型分生孢子萌发的真菌毒性物质。这些抑菌物质的产生和积累可能与抗扩展特性有关。     

宿根矮化病菌对甘蔗品质及茎、叶超微结构的影响

 甘蔗宿根矮化病(Ratoon Stunting Disease, RSD)是由Leifsonia xyli subsp.xyli (Lxx)引起的,是目前世界所有植蔗地区危害性极大的病害之一。本实验以甘蔗品种新台糖22号(ROC22)健康植株为对照,感染RSD植株为处理,观察和测定RSD侵染甘蔗引起的蔗株农艺性状、蔗糖分以及茎、叶超微结构的变化。结果表明:(1)感染RSD种茎的出苗率比对照减少2.94个百分点;株高比对照下降28.85 cm;茎径比对照减少0.28 cm;节间长度比对照减短3.50 cm;单茎重比对照低0.36 kg。(2)感染RSD植株的蔗糖分低于对照0.9个百分点(绝对值)。(3)利用透射电镜技术对感染RSD植株茎、叶细胞超微结构进行观察表明,叶片叶肉细胞、维管束鞘细胞及茎细胞内的细胞器及细胞核都发生了明显的病理变化。与健康叶片相比,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离。线粒体形态异常,有的肿大、内嵴模糊,严重者内嵴消失并空泡化,仅剩未被消解的残骸;细胞核形态变为不规则,核膜破裂,染色质分布不均匀,呈降解状态。在感染RSD甘蔗茎维管束导管细胞内积累有大量的电子致密物质,细胞壁有不同程度的溶解和断裂,这可能和RSD病原细菌侵染有关。以上结果表明:RSD侵染甘蔗后,可能导致光合效率下降,对水分和营养物质的运输能力降低,从而导致甘蔗品质和产量的降低。     

A stable Leifsonia xyli subsp. xyli GFP‐tagged strain reveals a new colonization niche in sugarcane tissues

Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically important diseases of sugarcane worldwide. Because knowledge on the interaction of Lxx with its host at the microscopic level is limited, the development of tools to monitor Lxx during the colonization process could shed new light on the processes that control disease development. In this investigation, a transformation protocol was optimized and a mutant Lxx strain engineered that stably expressed the gfp gene in sugarcane tissues. In vitro, the growth of the mutant did not differ from that of the wild type. Also, plants inoculated with both strains showed comparable growth and development when analysed 180 days after inoculation (dai). Fluorescence microscopy of roots, stalks, meristems and leaf tissues of Lxx‐GFP‐inoculated plants was performed at 180 dai. In the leaves, Lxx‐tagged cells were observed within the xylem vessels as has been described before but, in addition, they were found in a new niche within the host tissues, in the mesophyll and in the bundle sheath cells surrounding the vascular system. This finding indicates that Lxx is able to move from the xylem to the parenchyma of the leaf cells. This first report of an Lxx mutant expressing a heterologous gene revealed that colonization of sugarcane by this pathogen is not limited to the xylem vessels as commonly reported.     

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli . This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis , nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.     

The growth of a plant-parasitic bacterium,Clavibacter xyli subsp. cynodontis,is enhanced by xylem fluid components

Clavibacter xyli subspecies cynodontis is a gram-positive coryneform bacterium, originally isolated from Bermuda grass. The bacterium is xylem-limited, slow-growing and nutritionally fastidious. We found that extracts of Bermuda grass and maize, as well as xylem fluid collected from decapitated maize plants, enhanced the growth of the bacteria in vitro. The growth was enhanced even with 1% (v/v) maize xylem sap in a rich culture medium, giving both an increased growth rate and a higher stationary phase cell density. The relative increase in growth was inversely correlated with the size of bacterial inoculum. When liquid medium was stored for more than 2 months, the bacteria did not grow at all unless the medium was conditioned with a plant exudate. We fractionated maize xylem fluid by ultrafiltration and chromatographic methods and tested all the fractions for bacterial growth induction. The xylem sap was found to contain two distinct growth-enhancing compounds: a small hydrophilic compound and a proteinaceous compound of more than 10 kDa.     

厚朴病虫害种类的初步调查

采用标准地法和线路调查法,对湖北恩施市新塘乡双河厚朴基地的厚朴病虫害进行了系统调查,记录主要虫害13种,其中叶部害虫9种,枝干害虫2种,根部害虫2种。厚朴主要病害5种。藤壶蚧、厚朴枝角叶蜂和厚朴新丽斑蚜为湖北省首次报道,小绿叶蝉为厚朴新寄主记录种。同时记录了藤壶蚧的天敌6种,其中寄生小蜂2种,瓢虫4种;厚朴新丽斑蚜的天敌昆虫8种。对藤壶蚧、厚朴枝角叶蜂和厚朴苗木根腐病等重要病虫害的发生规律进行了初步调查,同时提出了防治建议。     

Mode of action of etoxazole

The mode of action of the 2,4-diphenyl-1,3-oxazoline acaricide/insecticide etoxazole has been argued to be moulting inhibition, but experimental results supporting this hypothesis are lacking. This study investigated the effect of etoxazole on chitin biosynthesis in the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Etoxazole induced moulting defects in fall armyworm larvae similar, if not identical, to those caused by benzoylphenylureas, a well-known class of insecticidal chitin biosynthesis inhibitors. Furthermore, in contrast to untreated larvae, the chitin content in the integuments of larvae several days after treatment did not differ from that in freshly ecdysed individuals, thus suggesting strong chitin biosynthesis inhibition in vivo. A more detailed investigation of the inhibitory potential by incubating cultured integument pieces from larvae of S. frugiperda with [14C]N-acetyl-D-glucosamine, a radiolabelled chitin precursor, revealed I50 values of 2.95 and 0.071 microM for etoxazole and triflumuron respectively. The incorporation of radiolabel into potassium hydroxide-resistant material was inhibited by etoxazole in a dose-dependent manner. Based on these results, it is concluded that the acaricidal and insecticidal mode of action of etoxazole is chitin biosynthesis inhibition.     

青稞种子带菌检测及杀菌剂消毒处理效果

为了研究青稞种子外部和内部携带真菌情况,比较不同杀菌剂对青稞种子的带菌消毒效果和对幼苗生长的影响,为青稞种子播前包衣处理和种传真菌病害防控提供依据,采用离体平皿法对云南迪庆‘云青1号’、‘云青2号’和‘短白青稞’3个主栽品种进行带菌检测,并对种子进行拌种或浸种处理测定6种杀菌剂对种子消毒效果,分析杀菌剂对种子发芽和幼苗生长的影响。结果表明:供试青稞种子表面携带的优势菌群为青霉(Penicilliumspp.)、镰刀菌(Fusariumspp.);种子内部寄藏的真菌主要为镰刀菌、核腔菌(Pyrenophoraspp.)、附球菌(Epicoccumspp.)、丝核菌(Rhizoctoniaspp.)、链格孢(Alternariaspp.)和木霉(Trichoderma spp.)。青稞不同品种的种子表面及内部携带的真菌种类差异较大。致病性测定表明,镰刀菌对种子萌发和幼苗生长影响最大,后期出现幼苗坏死现象。45%咪鲜胺EW、75%百菌清WP、50%福美双WP对青稞种子携带真菌均有显著抑制作用和消毒效果,50%福美双WP消毒效果最优,达100%;45%咪鲜胺EW、75%百菌清WP、50%福美双WP处理对青稞种子发芽和幼苗生长均无显著影响。     

Investigation of the Mode of Action of Nematode Neuropeptides

The neuropeptide AF2 has a complex set of actions on the dorsal muscle strip of Ascaris suum, including a potentiation of the acetylcholine-stimulated muscle contraction. Caffeine at 100 μm and 5 mm inhibited this potentiation, as did 100 μm theophylline in two out of six studies. The cyclic-AMP-potentiating compounds IBMX, dibutyryl cAMP and forskolin had no effect on the AF2-induced potentiation of the acetylcholine-stimulated muscle contraction. These preliminary data suggest that the potentiating action of AF2 is not mediated by a cAMP pathway.