Arbuscular Mycorrhizal Group, Department Biofertilizers and Plant Nutrition, Instituto Nacional de Ciencias Agrícolas (INCA) Gaveta Postal No 1 San José de Las Lajas, 32700, Mayabeque, Cuba.
Department of Botany, Center for Life Science and Health, Federal University of Rio de Janeiro State (UNIRIO), Rio de Janeiro, RJ, 22290-255, Brazil.
Folia Microbiol (Praha). 2021 Jun;66(3):371-384. doi: 10.1007/s12223-020-00844-y. Epub 2021 Feb 3.
Crop inoculation with Glomus cubense isolate (INCAM-4, DAOM-241198) promotes yield in banana, cassava, forages, and others. Yield improvements range from 20 to 80% depending on crops, nutrient supply, and edaphoclimatic conditions. However, it is difficult to connect yield effects with G. cubense abundance in roots due to the lack of an adequate methodology to trace this taxon in the field. It is necessary to establish an accurate evaluation framework of its contribution to root colonization separated from native arbuscular mycorrhizal fungi (AMF). A taxon-discriminating primer set was designed based on the ITS nrDNA marker and two molecular approaches were optimized and validated (endpoint PCR and quantitative real-time PCR) to trace and quantify the G. cubense isolate in root and soil samples under greenhouse and environmental conditions. The detection limit and specificity assays were performed by both approaches. Different 18 AMF taxa were used for endpoint PCR specificity assay, showing that primers specifically amplified the INCAM-4 isolate yielding a 370 bp-PCR product. In the greenhouse, Urochloa brizantha plants inoculated with three isolates (Rhizophagus irregularis, R. clarus, and G. cubense) and environmental root and soil samples were successfully traced and quantified by qPCR. The AMF root colonization reached 41-70% and the spore number 4-128 per g of soil. This study demonstrates for the first time the feasibility to trace and quantify the G. cubense isolate using a taxon-discriminating ITS marker in roots and soils. The validated approaches reveal their potential to be used for the quality control of other mycorrhizal inoculants and their relative quantification in agroecosystems.
根内接种丛枝菌根真菌（Glomus cubense）隔离物（INCAM-4、DAOM-241198）可促进香蕉、木薯、饲料等作物的产量。产量的提高幅度因作物、养分供应和土壤气候条件而异，范围在 20%至 80%之间。然而，由于缺乏在田间追踪该分类群的适当方法，因此很难将产量效应与根内丛枝菌根真菌的丰度联系起来。有必要建立一个准确的评价框架，将其对根定植的贡献与本地丛枝菌根真菌（AMF）区分开来。本研究基于 ITS nrDNA 标记设计了一套分类群区分引物，并对两种分子方法进行了优化和验证（终点 PCR 和定量实时 PCR），以在温室和环境条件下追踪和定量根和土壤样本中的 G. cubense 分离株。两种方法均进行了检测限和特异性试验。通过终点 PCR 特异性试验，使用了 18 种不同的 AMF 分类群，结果表明，引物特异性扩增了 INCAM-4 分离株，产生了 370 bp-PCR 产物。在温室中，用三种分离株（R. irregularis、R. clarus 和 G. cubense）接种 Urochloa brizantha 植物，以及环境根和土壤样本，通过 qPCR 成功地进行了追踪和定量。AMF 根定植率达到 41-70%，土壤孢子数为 4-128 个/g。本研究首次证明，在根和土壤中使用分类群区分 ITS 标记追踪和定量 G. cubense 分离株是可行的。验证方法表明，它们具有在农业生态系统中用于其他菌根接种剂的质量控制及其相对定量的潜力。
