Pain Naomi A, Green Jonathan R, Gammie Fiona, O'Connell Richard J
School of Biological Sciences, The University of Birmingham, P.O. Box 363, Birmingham BJ5 2TT, UK.
Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, UK.
New Phytol. 1994 Jun;127(2):223-332. doi: 10.1111/j.1469-8137.1994.tb04274.x.
A method is described for isolating intracellular hyphae (IH, i.e. infection vesicles and primary hyphae) of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected leaves-of bean (Phaseolus vulgaris L.). 1H were recovered from homogenates of infected leaves after filtration through a 45 μm nylon mesh and isopyenic centrifugation on Percoll. 1H were then affinity-purified by immunomagnetic separation using Dynabeads coated with monoclonal antibody UB25, specific for IH surface glycoproteins. The method yielded 7 × 10 IH g f. wt leaf tissue, with 27% purity and 62% viability, as judged by staining with fluorescein diacetate. The Viability of isolated IH was confirmed by their ability to grow in nutrient medium and by the normal ultrastructure of their cytoplasm. The host plasma membrane and matrix layer which surround IH in planta were absent from isolated IH. Staining with lectins. Calcofluor and aniline blue showed that the walls of IH contain N-acetylgalactosamine. α-linked mannose residues and β-linked polysaccharides, including-chitin and β-1,3-glucans. Potential uses of the isolated IH are discussed.
本文描述了一种从感染菜豆（Phaseolus vulgaris L.）叶片中分离炭疽菌（Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav.）细胞内菌丝（即感染泡囊和初生菌丝）的方法。通过45μm尼龙网过滤和在Percoll上进行等密度离心后，从感染叶片的匀浆中回收细胞内菌丝。然后使用包被有针对细胞内菌丝表面糖蛋白的单克隆抗体UB25的磁珠，通过免疫磁分离对细胞内菌丝进行亲和纯化。通过用荧光素二乙酸酯染色判断，该方法每克叶片组织可获得7×10个细胞内菌丝，纯度为27%，活力为62%。分离得到的细胞内菌丝在营养培养基中生长的能力以及其细胞质正常的超微结构证实了其活力。分离得到的细胞内菌丝中不存在植物体内围绕细胞内菌丝的宿主质膜和基质层。用凝集素、荧光增白剂和苯胺蓝染色表明，细胞内菌丝的细胞壁含有N-乙酰半乳糖胺、α-连接的甘露糖残基和β-连接的多糖，包括几丁质和β-1,3-葡聚糖。文中还讨论了分离得到的细胞内菌丝的潜在用途。
