杜梨离体培养技术的研究

本试验以一年生杜梨茎段为材料,建立其离体再生体系.结果表明,最适诱芽培养基为NN69+6-BA 1.0mg·L-1+NAA 0.01mg·L-1+IBA 0.01 mg·L-1,增值倍数达5.84,且芽苗生长健壮.继代培养的最适培养基为NN69+BA 0.5 mg·L-1+NAA 0.01mg·L-1,2.0 mg·L-1的GA3能明显地促进芽的增殖和伸长生长.     

果桑肥大性菌核病菌多聚半乳糖醛酸酶基因(CsPG1)的克隆及功能分析

多聚半乳糖醛酸酶(Polygalactuionasem, PG)是一种细胞壁结构蛋白,可以催化果胶分子多聚α-(1,4)-聚半乳糖醛酸的裂解,使细胞壁结构解体,导致果实软化。本文采用qRT-PCR方法,分析不同生长阶段菌丝侵染的油菜叶片中多聚半乳糖醛酸酶基因的表达模式。结果表明：(1)采用RT-PCR从果桑肥大性菌核病菌(Ciboria shiraiana)菌核中扩增多聚半乳糖醛酸酶基因cDNA,其全长为1143 bp,命名为CsPG1 (GenBank登录号为KR296662),编码380个氨基酸残,根据侵染油菜叶片的表达量,确认CsPG为基因家族的主效基因;(2)将CsPG1基因连接pET28a(+)载体并转化大肠杆菌E. coli BL21 (DE3),获得包涵体形式,但对底物(聚半乳糖醛酸)没有活性的蛋白;(3)最适CsPG1包涵体溶解的尿素浓度6 mol L^–1,采用缓慢稀释低温复性的方法对重组蛋白CsPG1进行了重折叠复性试验,经高亲和力的Ni-NTA树脂纯化后为得到单一条带,获得可溶性蛋白,复性后的多聚半乳糖醛酸酶比活力为5.02 U mg^–1;(4)生物试验表明,CsPG1蛋白能够加速嘉陵40 (Morus atropurpurea Roxb.)桑椹的成熟和软化,推测该蛋白与肥大性菌核病菌对桑椹的侵染有关。这一结果揭示了不同果桑品种对菌核病敏感性的差异,为果桑生产中菌核病的防控提供了分子证据.     

桦僵实杯盘菌

本文报道了我国新纪录盘菌—桦僵实杯盘菌Ciboria betulae(Woronin)White。它寄生于桦树种子上。文中叙述了它在新疆的分布、生活史和生境。     

桑椹菌核病菌实时荧光定量PCR检测体系的建立和应用

为及时高效地鉴别桑椹菌核病的病原菌桑实杯盘菌Ciboria shiraiana和肉阜状杯盘菌C. carunculoides,根据RPB2基因序列设计引物并验证其特异性,建立这2种病原菌的实时荧光定量PCR(real-time fluorescent quantitative PCR,q PCR)检测体系,运用该体系对抗、感果桑品种不同时间点芽(果)内的2种病原菌进行检测。结果表明,设计的2对引物特异性强,qPCR检测灵敏度均为10^-4ng/μL,均为常规PCR灵敏度的10~5倍,建立的桑实杯盘菌和肉阜状杯盘菌qPCR标准曲线的扩增效率分别为104.89%和95.30%。利用所建方法成功从采集的果桑样品中检测出病原菌主要为肉阜状杯盘菌,且感病品种中肉阜状杯盘菌的含量随着时间不断增加,在将要显症时激增。表明所建qPCR体系适用于桑椹菌核病早期无症状芽(果)的检测,可通过监测其病原菌类型和含量为该病害的预测预报及早期防治提供依据。     

Investigation and rapid detection of fungal pathogens causing mulberry sclerotial disease in south-west China

Mulberry sclerotial disease is a destructive disease that afflicts mulberry fruits throughout the world. In recent years, the impact of mulberry sclerotial disease has increased with the development of the mulberry fruit industry. Ciboria carunculoides, C. shiraiana, and Scleromitrula shiraiana are all pathogens causing mulberry sclerotial disease, but the dominant pathogen may be different in different mulberry planting areas. In this study, we investigated the causal agents of mulberry sclerotial disease in Chongqing and Sichuan, the main mulberry planting areas in south-west China. The results showed that C. carunculoides was the dominant pathogen in the investigated areas, and the disease incidence rate on some widely cultivated mulberry varieties exceeded 90%. Based on the differences in internal transcriber spacer sequences of different pathogens, we developed a method using cleaved amplified polymorphic sequences (CAPS) from digestion with the three restriction enzymes HindIII, EcoRI, and MluI for rapid detection of pathogens on mulberry fruits. Use of this method confirmed that the diseased fruits of some mulberry varieties were infected by a mixture of C. carunculoides and S. shiraiana. Long-fruit mulberry, which was considered to be resistant to sclerotial disease in the past, was also infected by C. carunculoides. Diseased fruit infected by C. shiraiana or by S. shiraiana alone were not detected. Our results suggest that C. carunculoides is probably the dominant pathogen causing mulberry sclerotial disease in south-west China. The CAPS method for rapid detection of pathogens could assist diagnosis, epidemiology, and disease monitoring.     

杜梨种子休眠与萌发过程中酶活性变化

以野生杜梨种子为试验材料,分别用200mg/L 6-BA溶液和1000mg/L GA₃溶液处理杜梨种子,以清水为对照,同时低温处理杜梨种子,用未处理的种子作对照,测定杜梨种子的萌发和种胚及种皮的过氧化物酶、α^-淀粉酶、多酚氧化酶的活性的活性,以分析种子与萌发机理,寻找打破杜梨种子休眠促进萌发的最佳方法。结果表明:种皮的多酚氧化酶活性显著高于种胚的酶活性;低温或激素单独处理,种胚和种皮的多酚氧化酶的活性均无明显变化;但低温和激素处理则能诱导种胚和种皮的α^-淀粉酶的活性,同时低温和激素处理能促进种胚和种皮的过氧化物酶活性的提高。     

对桑椹肥大性菌核病菌具有抑制活性的中草药筛选

桑椹肥大性菌核病是危害桑椹的真菌病害。为了保证果用桑椹的品质和食用安全,探讨利用中草药抑制该病病原菌的效果。采用半固体药基混合法检测36种中草药提取物体内外抑制桑椹肥大性菌核病菌的活性,结果表明,白癣皮、五倍子、诃子3种中草药提取物均有一定的抑菌作用,其中0.025 g/mL白藓皮提取液对桑椹肥大性菌核病菌的体外抑制效果较好,对菌丝的生长抑制率达86.12%,并且田间试验的防治效果显著,桑椹染病率下降至0.80%。家蚕食下分别喷施3种中草药提取液的桑叶后,对生长发育和吐丝营茧亦无不良影响。初步认为,白藓皮提取物可进一步开发为防治桑椹肥大性菌核病的新型植物源农药。     

桑树断梢病的防治效果初报

试验表明,在其它桑园管理措施一致的前提下,用70%甲基托布津WP800倍液及50%多菌灵WP1 000倍液于盛花期喷洒或在花椹期人工摘除花椹对防治桑树断梢病效果最佳。生产中建议伐条方式由春伐改为夏伐,推荐采用在花椹期摘除花椹的防治方法。