白条黄单胞菌侵染对蔗叶和蔗茎组织超微结构的影响

 本研究利用普通光学显微镜、透射电镜和扫描电镜观察白条黄单胞菌[Xanthomonas albilineans (Ashby) Dowson]侵染后菌体定殖及对甘蔗叶片和蔗茎组织超微结构的影响。结果表明,白条黄单胞菌侵染后叶片的薄壁组织细胞膜裂解,木质部生长受到抑制。叶绿体形状多近圆形,基质片层解体,被膜破坏呈松弛状态,出现大量的原生质膜。扫描电镜可见白条黄单胞菌存在于各个叶肉细胞中,同时在维管束中也发现菌体定殖,特别是木质部导管。蔗茎的维管束被白条黄单胞菌堵塞,使得蔗茎木质化、木栓化严重。本研究有助于理解白条黄单胞菌对甘蔗叶片和蔗茎细胞内细胞器损伤的形态破坏,为白条病防治提供超微形态学依据。     

杀虫单在甘蔗和土壤中的残留行为和风险评估研究

为了确保杀虫单在甘蔗上的安全科学使用,本研究分别于2015和2016年度在海南和广西开展了9%杀虫单颗粒剂在甘蔗和土壤中的残留消解试验和最终残留试验。残留消解试验结果表明,杀虫单在甘蔗植株中的半衰期为17.3～30.1 d,在土壤中的半衰期为1.7 d;最终残留试验结果表明,杀虫单在甘蔗蔗梢、蔗茎和土壤中的最终残留皆低于LOQ(0.02 mg/kg)。综上,在甘蔗苗期按照3 375 g/hm~2沟施9%杀虫单颗粒剂1次,甘蔗蔗梢和蔗茎中杀虫单的最终残留低于我国的限量标准,对人类的暴露风险较低;而且土壤中杀虫单残留对非靶标生物蚯蚓的环境风险也较低。     

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。     

甘蔗宿根矮化病菌多克隆抗体的制备

 甘蔗宿根矮化病（Ratoon stunting disase,RSD）是甘蔗生产中最为严重的细菌病害之一,常导致感病品种新植蔗减产10%～15%,宿根蔗减产20%～25%,在干旱的情况下感病品种的宿根蔗产量损失可达60%^［1］。RSD病原菌为 Leifsonia xyli subsp.xyli（Lxx）,寄生于甘蔗木质部导管内,革兰氏阳性^［2］,体外分离培养非常困难。该病害自从1944 年首次在澳大利亚昆士兰州的甘蔗品种Q28 上发现以来,在世界各产区普遍发生,现已广泛分布于各甘蔗种植区。该菌主要通过带菌种茎和砍收工具传播^［3］,已感病的蔗株又无明显的外部症状,从而导致该病害无意识地传播,造成病害蔓延,对甘蔗生产危害极大。甘蔗宿根矮化病的检测方法主要有形态学、血清学、分子生物学等方法,血清学由于具有操作简便、准确性高、灵敏度好,同时能够处理大量样品而在国外被广泛采用。目前国内所用的RSD血清全部依赖于国外进口,检测成本高,使得血清学方法无法在国内普及。为此我们分离纯化了RSD的病原菌并制备了RSD的多克隆抗体,为甘蔗宿根矮化病敏感、稳定和快捷的检测提供了必要的保证。     

受番茄花叶病毒侵染后寄主的超微病变研究

电镜观察了番茄花叶病毒 ( TOWV)侵染不同寄主的细胞超微结构变化。在 2 5℃下 TOWV侵染番茄( Lycopersiconesculentum Mill)后 ,病毒粒子在叶片的表皮、薄壁细胞、维管束组织的细胞质中形成大块结晶体和类结晶体 ,液泡膜处产生小泡结构 ,有多泡体和髓鞘样结构伸入液泡中。在 2 5℃下 TOWV侵染珊西烟 ( N icotianataba - cuml .cv.Xanthinn)后 ,除存在病毒结晶体和类结晶体外 ,还形成角层状聚集体 ,叶绿体产生周边中泡结构。在 35℃下 TOWV侵染珊西烟后 ,在细胞质中还形成高电子密度的无定型体。两种寄主中均未观察到 X体 ,细胞核…     

甜菜花叶病毒病研究之二——感病细胞超微结构观察

 对不同时期感染甜菜花叶病毒(BtMV)的叶片,进行病理变化系列观察。早期侵染(7天前)的细胞质、细胞器均无变化,随着症状发展,风轮状内含体、束状内含体、细胞核卫星体,细胞质泡囊化旁类似病毒的束状结构,20天达到了高峰,风轮状及束状内含体一直持续两个月还存在。田间样品也观察到了典型的风轮状及束状内含体。细胞核卫星体出现很少。侵染后期,细胞质减少,叶绿体比相应健株提前出现淀粉粒,噬锇颗粒等,基粒片层也提前消解,表现细胞提前衰老的特征。此外细胞内还有许多健株没有的泡状结构。一种为旁壁体,可能与细胞壁加厚有关;一种为多重泡体的叠加,可能是液泡的吞噬作用加强与外源物质(BtMV)侵入有关;另外还有一种可能是过氧化物酶体或线粒体在病毒诱导下泡囊化,其作用不清楚。     

甜菜花叶病毒病研究之二——感病细胞超微结构观察

对不同时期感染甜菜花叶病毒(BtMV)的叶片,进行病理变化系列观察。早期侵染(7天前)的细胞质、细胞器均无变化,随着症状发展,风轮状内含体、束状内含体、细胞核卫星体,细胞质泡囊化旁类似病毒的束状结构,20天达到了高峰,风轮状及束状内含体一直持续两个月还存在。田间样品也观察到了典型的风轮状及束状内含体。细胞核卫星体出现很少。侵染后期,细胞质减少,叶绿体比相应健株提前出现淀粉粒,噬锇颗粒等,基粒片层也提前消解,表现细胞提前衰老的特征。此外细胞内还有许多健株没有的泡状结构。一种为旁壁体,可能与细胞壁加厚有关;一种为多重泡体的叠加,可能是液泡的吞噬作用加强与外源物质(BtMV)侵入有关;另外还有一种可能是过氧化物酶体或线粒体在病毒诱导下泡囊化,其作用不清楚。     

百合无症病毒对百合光合生理、酶活性及叶绿体超微结构的影响

 以东方百合“西伯利亚”为试验材料,研究百合无症病毒(LSV)侵染百合对其叶片生理生化以及叶绿体超微结构的影响。检测结果表明:叶片中叶绿素a、b以及总叶绿素含量与健康对照相比分别下降了28.6%、33.3%和23.5%,净光合速率、气孔导度及胞间CO₂浓度分别下降33.3%、25%和13.8%;超氧化物歧化酶(SOD)、过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)与健康对照相比,分别增加了16.6%、29.4%、16.7%和22.2%。电镜观察发现:感病植株叶绿体膨胀变形,基质片层散乱,叶绿体内淀粉粒肿大且数目增多,从而证明LSV侵染破坏叶绿体结构,影响植株的光合作用。     

水鬼蕉感染HCRV不同发病时期的病毒粒体分布与亚细胞病变特征

采集疑似朱顶红褪绿环斑病毒(HCRV)侵染的水鬼蕉(Hymenocallis littoralis)不同发病时期的叶部组织，通过HCRV-N基因特异性引物的RT-PCR扩增克隆与测序分析，确定由HCRV侵染。进一步应用负染色、超薄切片制样及透射电子显微镜观察，结果显示：叶部无症、褪绿、黄化和坏死不同发病时期的HCRV粒体在细胞内呈现散布或聚集的特征，散布的病毒粒体包被双层膜结构，发病后期聚集的病毒粒体呈现串珠状，聚集于管状囊泡中。在侵染潜伏期，水鬼蕉细胞核、叶绿体等亚细胞结构较完整；发病早期和中期，细胞核结构完整，叶绿体基粒片层消解，线粒体增加且结构较完整；发病后期，大部分细胞坏死，液泡中残存内溶物，少部分细胞的液泡中含有串珠状病毒粒体聚集于管状囊泡，是HCRV区别于同属其他病毒的明显特征，可作为诊断鉴定的依据。     

干旱胁迫对银沙槐幼苗叶绿体和线粒体超微结构及膜脂过氧化的影响

以沙生植物银沙槐(Ammodendron argenteum)幼苗为实验材料,利用透射电镜技术,结合丙二醛(MDA)和细胞膜透性的测定,观察干旱胁迫对银沙槐子叶期幼苗的叶肉细胞及下胚轴细胞超微结构的变化,分析干旱胁迫对银沙槐幼苗细胞超微结构和膜脂过氧化的影响。结果显示:随着干旱胁迫的加剧,叶绿体变形、外被膜波浪状、断裂不完整。同时,基粒和基质类囊体膜结构模糊,基粒弯曲、膨胀、排列混乱;线粒体膜完整性低、内部嵴消失、空泡化。细胞膜透性和膜脂过氧化产物MDA含量均升高,显著高于对照(P0.05),细胞膜结构受到破坏,细胞膜脂过氧化作用增强。子叶的细胞膜透性和MDA含量显著高于下胚轴(P0.05),细胞器形态变化早于下胚轴,表明下胚轴耐旱性相对于子叶较强。子叶中线粒体受损晚于叶绿体,线粒体的耐旱性要比叶绿体强。不同组织间及同一组织不同细胞器之间对干旱胁迫反应的差异,可能是植物组织细胞应对外界不利环境的一种防御反应。干旱胁迫下超微结构的变化反映了细胞内膜系统的紊乱和伤害,而膜系统的伤害可能是脂质过氧化作用增强的结果。     

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.     

Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations

Two sugarcane cultivars (R570 and SP71-6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf-free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane-growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71-6163 were infected by SCYLV genotypes BRA-PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71-6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.     

云南甘蔗杆状病毒的分子检测及其对甘蔗品质和产量的影响

 Sugarcane bacilliform virus(SCBV) was detected by PCR from sugarcane showing chlorosis and mottle symptom from Kaiyuan, Yunnan Province.Part sequence of replicase gene of the isolate SCBV-Kaiyuan was determined.Sequence analysis indicated that the 589 bp of SCBV-Kaiyuan shared identities of 73.2%-74.0% and 83.1%-84.1% at nucleotide and amino acid levels with SCBV-Australia respectively, 66.7%-68.4% and 65.6%-67.7% with SCBV-Morocco.The quality and yield of the sugarcane infected with SCBV-Kaiyuan was also investigated.The juice extraction, sucrose content, gravity purity and average stalk weight were decreased 1.55%, 1.24%, 2.22% and 0.26 kg in plants infected with SCBV-Kaiyuan, but reducing sugar was increased by 0.21% in infected plants.     

甘蔗绵蚜为害损失研究

甘蔗绵蚜Ceratovacuna lanigera Zehntner是现今广泛分布于我国各甘蔗种植区,严重影响甘蔗产量和品质的叶部害虫。为探明现有生产水平条件下甘蔗绵蚜对甘蔗实测产量及糖分的为害损失和对新植宿根出苗的影响情况,给甘蔗绵蚜的科学有效防控提供理论依据和翔实的实测数据。本研究于2014—2016年,选择主栽品种,同田设立为害区和未为害区,调查评估绵蚜为害对新植宿根出苗的影响,甘蔗成熟期分别收砍称量和测定分析甘蔗产量及糖分含量,并计算甘蔗实测产量及糖分损失。研究结果显示,甘蔗实测产量减少2 503~4 123kg/667m~2,平均3 079kg/667m~2;产量损失率为28.5%~45.7%,平均35.9%;出汁率减少2.4%~4.13%,平均3.01%;甘蔗糖分降低5.48%~8.16%,平均6.38%;蔗汁锤度降低6.95~9.05°BX,平均7.66°BX;蔗汁重力纯度降低8.43%~19.97%,平均12.35%;而蔗汁还原糖分则增加1.01%~1.3%,平均1.21%;新植出苗率降低24.7%~27.3%,平均26.0%;宿根出苗数减少3 829~5 083株/667m~2,平均4 456株/667m~2,相对出苗损失率为57.6%~58.0%,平均57.8%。可见,目前云南蔗区甘蔗绵蚜为害造成的甘蔗产量及糖分损失十分严重,甘蔗绵蚜为害已成为现阶段严重影响甘蔗产量和品质的主要挑战之一。研究结果对加强甘蔗绵蚜的科学有效防控和支持中国蔗糖产业的可持续发展具有重要意义。     

Impact of Sugarcane yellow leaf virus on Sugarcane Yield and Juice Quality in Réunion Island

Sugarcane yellow leaf virus (SCYLV) was first detected in sugarcane of Réunion Island in 1997. A field experiment was undertaken to assess the potential impact of this virus on sugarcane production. The agronomic characteristics of SCYLV-infected plants were compared to those of virus-free plants of three sugarcane cultivars (R570, R577 and R579) which occupy more than 90% of the cultivated sugarcane area on Réunion Island. In the plant crop, significant losses in stalk weight (28%) and in sugar content (11%) were detected for cultivar R577, but not for either of the two other cultivars. In the first ratoon crop, yield reduction was detected for cultivar R577 (37%), but also for cultivar R579 (19%). Cultivar R577 also showed significant losses in sugar content (12%) due to reduced amount and quality of extracted cane juice. No yield reduction was found for cultivar R570, although stalk height and diameter were reduced in SCYLV-infected canes of this cultivar in the first ratoon crop. Leaf yellowing was observed at harvest of plant and ratoon crops when sugarcane was no longer irrigated, and 10–59% of symptomatic stalks could be attributed to the presence of SCYLV. The most severe yellowing symptoms were related to infection of sugarcane by the virus.     

Alternaria eichhorniae, a biological control agent for waterhyacinth: mycoherbicidal formulation and physiological and ultrastructural host responses

The bioherbicidal efficacy of different alginate formulations of Alternaria eichhorniae 5 (isolate Ae5), a virulent Egyptian isolate, was compared on waterhyacinth (Eichhornia crassipes). The fungus was formulated as alginate pellets containing mycelium alone, mycelium plus culture filtrate or culture filtrate alone. Each formulation was applied with and without a hydrophilic humectant (Evergreen 500). These formulations were evaluated for disease incidence (DI), and disease severity (DS). Maximum DS, but not DI, was obtained with the alginate pellets of mycelium plus culture filtrate. Alginate formulations supplemented with the hydrophilic polymer were more effective in promoting disease. Physiological changes associated with the treated waterhyacinth plants were determined 3, 6 and 9 days after treatment. Waterhyacinth plants treated with alginate pellets of mycelium plus culture filtrate of Ae5 had the lowest levels of pigments, carbohydrates and relative water content. Infection of waterhyacinth with Ae5 led to a significant increase in total phenols of leaves as compared to control. Penetration of waterhyacinth leaves by the fungus occurred only through the stomata, and the invading hyphae were located in the intercellular spaces of leaf tissues. Cytological changes noted in infected cells included changes in chloroplast, nucleus and mitochondria. Invagination of the plasma membrane, particularly at plasmodesmata was also noticed in infected cells. The associations between the infection process, the physiological disorder and the ultrastructure of infected leaves are discussed.     

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.     

江西甘蔗花叶病田间调查

2007年9-10月对江西甘蔗花叶病(sugarcane mosaic disease)进行了多点田间调查,结果表明江西甘蔗普遍发生花叶病,其中红皮甘蔗花叶病重于青皮甘蔗,两者病丛率分别为59.8%和19.2%,赣北、赣中、赣南3大地区之间的甘蔗花叶病发生轻重无明显差异。     

Weed competition and control in sugarcane

Losses of about 40% in cane yields due to natural stands of weeds were found in experiments conducted in sugarcane var. Co 527 in the year of planting at Guneid Sugarcane Research Station, Sudan. Weed competition lowered millable stalks per metre row by 32%, stalk height by 24%, stalk thickness by 15% and number of nodes per stalk by 14%. Tillering was the growth phase most affected by weed competition. Cane yields were increased as number of hand weedings increased, but four weedings were not markedly better than three. The average yield (67·04 t ha^?1) obtained from four weedings was not significantly (P= 0·05) better than that of three weedings carried out at 3, 6 and 9 weeks after cane planting. Juice analysis components were also affected by weeds and a 15% reduction in sucrose recovery was recorded. Reductions in the other components were only 4–7%. Atrazine and diuron (3·3 kg ha^?1), metribuzin (2·4 kg ha^?1) and metribuzin (1·3 kg ha^?1) in tank mixture with diuron (1·5 kg ha^?1) gave excellent residual weed control of the dominant weed species, Ipomoea cordofana Choisy., Brachiaria eruciformis (Sm.) Griseb., Corchorus fascicularis Lam., Ocimum basilicum L. and Dinebra retroflexa (Vahl) Panz., for most of the first growing season. Excellent control of weeds achieved by the herbicide treatments resulted in comparable yields to frequently-weeded cane. These herbicides were not phytotoxic to sugarcane var. Co 527.