Atamian Hagop S, Roberts Philip A, Kaloshian Isgouhi
Department of Nematology, University of California-Riverside, CA, USA.
J Vis Exp. 2012 Mar 12(61):3629. doi: 10.3791/3629.
Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28°C. Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.
根结线虫（根结线虫属）是专性植物寄生虫。它们食性极广，被认为是经济上最重要的植物寄生线虫之一。显微镜下的第二龄幼虫（J2）在卵内蜕一次皮，是感染阶段。J2从卵中孵化出来，在土壤中的一层水膜内自由移动，并定位到合适植物物种的根尖。穿透植物根部后，它们向维管束迁移，在那里建立取食位点并用口针开始取食。多细胞取食位点由几个称为“巨型细胞”的扩大的多核细胞组成，这些细胞由经历了核分裂（反复有丝分裂）但没有胞质分裂的细胞形成。相邻的中柱鞘细胞分裂并增大尺寸，形成典型的虫瘿或根结，这是根结线虫感染的特征症状。一旦开始取食，J2就会固定下来，并再经历三次蜕皮成为成虫。成年雌虫在根表面或根表面以下的凝胶状基质中产150 - 250枚卵。新的感染性J2从卵中孵化出来，开始新的循环。在26 - 28°C下，根结线虫的生命周期在4 - 6周内完成。在这里，我们介绍用根结线虫感染盆栽植物的传统方法以及两种高通量检测方法。第一种高通量方法用于种植小种子的植物，如番茄，而第二种方法用于种植大种子的植物，如豇豆和菜豆。大种子在营养补充最少的情况下能支持幼苗的长期生长。第一种高通量检测利用在托盘沙中生长的幼苗，而在第二种检测中，植物在无土的小袋中生长。幼苗生长小袋由一张15.5×12.5厘米的纸芯制成，顶部折叠形成一个2厘米深的槽，种子或幼苗放置在其中。纸芯装在一个透明塑料袋内。这些生长小袋可以在透明袋表面下直接观察线虫感染症状、根部虫瘿形成和卵块产生情况。两种方法都允许在表型分析后使用筛选出的植物进行杂交或种子生产。使用生长小袋的另一个优点是空间需求小，因为小袋存放在架子上排列的塑料悬挂文件夹中。
