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。     

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。     

甘蔗宿根矮化病菌巢式PCR检测

为了探索甘蔗宿根矮化病快速检测技术,以细菌16S-23S rDNA内转录间隔区(ITS)通用引物ITS1/ITS2为第1轮引物,以甘蔗宿根矮化病菌特异引物Lxx1/Lxx2为第2轮引物,建立甘蔗宿根矮化病菌巢式PCR检测技术。巢式PCR能扩增出438 bp的目的条带,其核苷酸序列与巴西、澳大利亚及美国甘蔗宿根矮化病菌分离物核苷酸序列同源率为100%或99.8%;其检测灵敏度为10fg甘蔗宿根矮化病菌基因组DNA,较常规PCR提高100倍;样品检测结果也表明巢式PCR检测灵敏度明显优于常规PCR,可用于+1片嫩叶和心叶等微量病菌样品的检测。     

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。     

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。     

甘蔗宿根矮化病菌实时荧光定量PCR检测方法的建立

甘蔗宿根矮化病是由Leifsonia xyli subsp.xyli(Lxx)引起的一种世界性甘蔗细菌病害。根据Lxx的Pat1基因保守序列,设计并合成了一对特异性引物Pat1F(5'-GGTTCCATTGCTTACCGATT-3')/Pat1R(5'-CAAGTTTCGACAGGAACAGC-3'),和一条Taq M an探针(FAM-5'-CCACGGCTACGTCAATTCGGG-3'-TAM RA),建立了一种特异性强、灵敏度高的甘蔗宿根矮化病菌实时荧光定量PCR检测方法。结果表明,本研究建立的实时荧光定量PCR方法,对Lxx的检测最低下限为102copies·μL-1。应用实时荧光定量PCR与常规PCR方法对14个甘蔗品种进行Lxx检测,阳性检出率分别为86%和43%,表明实时荧光定量PCR比常规PCR检测方法具有更高的灵敏度。研究结果为甘蔗宿根矮化病的诊断、田间发生动态监测、脱毒健康种苗检测及品种/材料交换检疫检测提供了新技术支撑。     

广西宜州甘蔗白条病调查及分子检测

甘蔗白条病是一种检疫性细菌病害,具有危害性大,潜伏期长的特点,由白条黄单胞菌（Xanthomonas albilineans）引起,在种植甘蔗的主要国家或地区均有发生。2020年在广西宜州蔗区发现疑似感染甘蔗白条病蔗株,为明确病原及发生情况,为其科学防控提供科学依据。本研究对不同甘蔗品种新植和宿根蔗进行发病率调查,并采集病样进行PCR检测分析。田间调查结果显示：不同品种自然发病率不同,桂糖44新植和宿根蔗均有白条病发生,且宿根蔗发病率高于新植蔗,其中新植蔗发病率为8%～18%,平均发病率为13.83%;宿根蔗发病率为18%～34%,平均发病率为24.71%。而桂糖42、桂糖55、柳城05-136等3个品种新植和宿根蔗均未发现白条病。PCR检测结果表明：有10份样品扩增出608 bp的特异性条带,阳性检出率为90.9%,所得序列与X. albilineans序列同源性高达100%,且在构建的系统发育树处于同一分支。本研究结果表明,广西宜州蔗区发现由X. albilineans引起的甘蔗白条病。     

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。     

阿维菌素及甲氨基阿维菌素苯甲酸盐的三氟乙酰胺衍生化产物稳定性研究

采用衍生化液相色谱-荧光检测法,研究了阿维菌素及甲氨基阿维菌素苯甲酸盐（甲维盐）在甲基咪唑存在下与三氟乙酸酐衍生化反应后样品溶液放置时间及保存温度对检测结果的影响。研究表明:在常温下,阿维菌素衍生化反应溶液放置180 min后、甲维盐衍生化反应溶液放置360 min后,两者衍生化产物的峰面积即急剧下降;衍生化反应20 min后,样品于4 ℃下保存（冷藏）,阿维菌素在 2 d内、甲维盐在3 d内,两者衍生化产物的检测结果保持稳定;而在-20 ℃下保存（冷冻）,5 d内可保证阿维菌素和甲维盐衍生化后检测结果的准确性。因此在运用衍生化液相色谱荧光检测法时,应严格控制好衍生化反应后样品溶液的放置时间及保存温度,以保证检测结果的准确性。     

甘蔗重要病害分子检测技术

快速有效地对甘蔗重要病害病原进行诊断检测,明确监测病害的病原是科学有效防控甘蔗病害的基础和关键。云南省农业科学院甘蔗研究所通过探索研究、改进创新、优化建立了甘蔗黑穗病、锈病、白条病、宿根矮化病、赤条病、花叶病、斐济病、黄叶病、杆状病毒病和白叶病等10种重要病害13种病原的分子快速检测技术,为甘蔗病害的有效诊断和防控、脱毒健康种苗检测及引种检疫提供了技术支撑。     

云南勐海蔗区草地贪夜蛾种群动态与性诱捕器诱集效果评价

为准确掌握甘蔗田草地贪夜蛾发生为害情况及种群动态,筛选适合当地种群监测的性诱捕器,通过田间人工调查不同种植方式甘蔗田草地贪夜蛾幼虫发生为害情况,采用2种类型草地贪夜蛾性诱捕器进行田间诱捕试验,探讨甘蔗田草地贪夜蛾性诱捕器的使用技术。结果表明：不论新植蔗田还是宿根蔗田,均有草地贪夜蛾发生为害,被害株率呈先缓慢上升后下降趋势,6月中下旬到7月上旬达到高峰,最高可达88.40%。甘蔗田草地贪夜蛾幼虫数量不多,宿根蔗田草地贪夜蛾幼虫数量较新植蔗田多,5月下旬到6月上旬达到高峰。监测期间不同种植方式甘蔗田草地贪夜蛾共出现5个诱集高峰期,新植蔗田草地贪夜蛾诱集高峰较宿根蔗田迟一个月出现。船形诱捕器和桶形诱捕器均可诱捕到草地贪夜蛾成虫,船形诱捕器田间诱集效果明显,但两者周均诱集量差异不显著。综合考虑各种因素,不论新植蔗田还是宿根蔗田,应加强甘蔗苗期草地贪夜蛾虫情普查和早期监测预警,在开展草地贪夜蛾监测时建议使用桶形诱捕器,在实际防治中结合甘蔗绵蚜防控采用植保无人机科学施药,防早防小,以达到最好的防治效果。     

广西主要蔗区螟虫种类调查与分析

甘蔗螟虫是甘蔗生产中的重要害虫?为掌握目前广西主要蔗区苗期螟虫的主要种类和发生情况, 本研究于2019年4月-5月, 对广西主要蔗区苗期螟害种类及其为害率进行了田间实地调查, 并分析了螟害种类结构的变化?结果表明:在宿根蔗地, 甘蔗苗期黄螟为害最严重, 造成的枯心苗率为5.59%; 其次为条螟, 造成花叶苗率为5.29%; 黄螟为害造成的宿根蔗枯心苗率以桂西南和桂南两大蔗区相对较高, 其平均值分别为7.34%和6.76%; 条螟为害造成的宿根蔗花叶苗率以桂西南蔗区最高, 平均值达7.82%, 桂中和桂南蔗区其次, 其平均值分别为4.59%和4.46%?同一蔗区, 黄螟和条螟在宿根蔗地发生均比新植蔗地严重, 在宿根蔗地平均枯心苗率分别为7.13%和7.99%, 而在新植蔗地分别为2.31%和3.43%?以黄螟枯心苗率为防治指标的达标防治蔗地样点数占比为52.06%, 条螟花叶苗率为防治指标的达标防治蔗地样点数占比为56.16%?广西蔗区甘蔗苗期的主要螟虫为黄螟和条螟, 尤其是黄螟已上升为主要害虫, 表明广西蔗区的优势螟虫种类结构已发生明显变化; 广西南部和西南蔗区的苗期螟害重于北部蔗区; 宿根甘蔗地的螟害重于新植蔗地, 宿根蔗地是螟害防治的重点?     

小麦白粉菌无性阶段生物学特性研究初报

 小麦白粉菌分生孢子萌发的温度范围为-2~34℃,最适为10~17℃。分生孢子离体后在2℃ 5天,7~17℃ 3天,20℃ 2天都还有侵染力。分生孢子在0~100%的湿度下都可以萌发,湿度愈大萌发率愈高,尤以在水面萌发率最高。分生孢子侵入寄主的湿度必须在65%以上,湿度愈大发病愈重。分生孢子在pH2.2~12.4的范围内都可以萌发,以pH4.2~7.7最适。紫外光对分生孢子有强裂的杀伤作用。在直射阳光下分生孢子的寿命比散光和黑暗条件下短。分生孢子荫发必须要有充足的氧气。光照能促使分生孢子的形成。在自然条件下,分生孢子形成有昼夜变化的规律,白天形成的孢子数比夜间形成的孢子数约大3倍。分生孢子传播的有效距离力15公里以上,传播的有效高度大于979公尺。     

温度、湿度和光照对苹果炭疽叶枯病菌(Glomerella cingulata)产孢的影响

苹果炭疽叶枯病(Glomerella cingulata)是我国苹果上新发现的一种病害,为了了解病原菌的产孢条件和产孢动态,为病害的预测预报与防控提供依据。本研究在人工控制条件下,测试了温度、湿度和光照对苹果炭疽叶枯病菌产生分生孢子和子囊孢子的影响。结果表明,苹果炭疽叶枯病新形成的病叶润湿后,在15℃~30℃下保湿培养2~6 d后可产生大量橘黄色的分生孢子堆,其中30℃下产孢量最大,产孢速度最快,仅需2 d时间。炭疽叶枯病菌在新形成的病叶上于15℃~30℃下培养20~30 d可形成子囊孢子,最适温度为25℃,子囊孢子的形成需要高湿环境或叶片润湿。炭疽叶枯病菌的单孢分离菌株于15℃~25℃下,在马铃薯葡萄糖琼脂培养基(PDA)上培养20~30 d也可形成子囊孢子,最适产孢温度25℃。紫外光、黑光和日光都能促进子囊孢子的形成。     

Latent infection of asymptomatic Hawaiian sugarcane cultivars with 16SrI and 16SrXI phytoplasmas

Leaves from sugarcane were collected at the Hawaiian sugarcane breeding station and from recent and previous Hawaiian plantation fields and tested for phytoplasma by nested PCR, quantitative PCR and partly by the 16S/23S internal spacer sequence. Phytoplasmas were found in samples of 10 of the 11 tested cultivars from the station and identified as strains 16SrI phytoplasma (aster yellows phytoplasma) and 16SrXI phytoplasma (rice yellow dwarf phytoplasma). Hot water treatment could partially eliminate the phytoplasmas, but sugarcane plants in the Hawaiian plantations, which routinely use hot-water-treated seed cane cuttings, were nevertheless infected by 16SrXI phytoplasma. Samples from abandoned sugarcane plantations contained 16SrI phytoplasma or 16SrXI phytoplasma. The titre of phytoplasma was very low in all cases, i.e., at or below the detection threshold of quantitative PCR, and no difference in phytoplasma infection was observed between healthy-looking, green plants and plants that had YLS symptoms. Apparently the Hawaiian sugarcane cultivars have some kind of phytoplasma resistance under the growth conditions in Hawaii. The latent presence of phytoplasma strains calls for awareness and rigorous treatment of sugarcane setts even in cases, where YLS was so far exclusively related to the presence of Sugarcane yellow leaf virus.     

向日葵霜霉病研究

 向日葵霜霉病菌Plasmopara halstedii (Farl.) Berl.&de Toni在5-25℃条件下均可侵染寄主,以10-15℃为侵染适温。对不向苗龄幼苗进行人工接种,3-14天的幼苗均被侵染,3天幼苗为最感病,发病率随植株苗龄增大而递减。病菌侵入寄主后,在恒温15℃、20℃、25℃ 4组及变温8℃和15℃、13℃和20℃、18℃和25℃、23℃和30℃条件下均可发病,恒温20℃及变温13℃和20℃发病率最高,恒温30℃及变温28℃和35℃不发病。     

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.     

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.