一种快速检测向日葵地块土壤中黄萎病菌微菌核的新方法

由大丽轮枝孢菌侵染引起的向日葵黄萎病是一种重要的土传病害，微菌核是该病害主要的初侵染来源。目前，土壤中大丽轮枝孢菌微菌核的定量检测方法多操作步骤复杂繁琐，如利用PCR方法进行检测，对仪器设备和操作人员的素质都有较高的要求，而常规的土壤梯度稀释湿筛法的实验周期长且检测效率低，因此，建立一种快速定量检测土壤中大丽轮枝孢菌微菌核的方法，对于向日葵黄萎病的预报预测和防控非常重要。为了能够快速的定量检测土壤中微菌核的数量，以期探明不同耕作方式地块中土壤微菌核数量的差异，本实验建立了一套操作相对简单，实验周期较短的微菌核快速分离和定量检测的方法，即采样器—干筛法。该方法将微生物采样器和选择性培养基相结合，基于微生物采样器的撞击法原理，使土壤微生物粒子加速撞击到选择性培养基的培养皿表面，经培养后可见单菌落形成。利用该方法对内蒙古巴彦淖尔市不同的向日葵黄萎病发病地块中采集到的土壤样本中微菌核进行了定量检测，结果表明：两年向日葵连作地（样地1）土壤中微菌核的数量最多，平均每克土样中含有微菌核32.80个；与非寄主作物玉米轮作地块（样地2）土样中微菌核的数量最少，平均每克土样中含有微菌核11.80个，与寄主作物打籽葫芦轮作地块（样地3）微菌核数量介于二者之间。利用该方法能够明显区分不同地块土壤中微菌核的数量。通过和荧光定量PCR检测的结果进行相关性分析发现，该方法能够准确检测土壤中大丽轮枝孢菌微菌核的数量。

A new method for rapid and quantitative detection of Verticillium dahliae microsclerotia in sunflower soil

Verticillium wilt is a plant vascular disease caused by soilborne fungus Verticillium dahliae which severely limits sunflower production. Molecular detection method of the pathogen requires high sensitivity and poor repeatability, while conventional dilution plate coating method takes a long time. It is important to establish a method for rapid and quantitative detection of microsclerotia in the prediction and control of sunflower verticillium wilt. To quantitatively detect microsclerotia number in soil and find out difference among farming systems, a sampler-dry sieve method, for rapidly and quantitatively detection of soil microsclerotia was summarized. It combines a microbial sampler with a selective medium, and based on the impact method principle of the microbial sampler. And soil microbial particles are accelerated to impact petri dish surface of the selective medium, and single colony formation could be seen after culturing. This method was used to quantitatively detect microsclerotia in soil samples collected from different sunflower verticillium wilt disease sites in Bayannur City, Inner Mongolia. Results showed that in soil of the two-year sunflower continuous cropping field （plot 1）, the number of micro sclerotia was the most （32.80 per gram of soil）. In the plot of rotation with the non-host crop maize （plot 2）, the number of microsclerotia was the least, containing 11.80 per gram of soil. The number of microsclerotia in the rotation plot （plot 3） was between the other two plots. It indicated that the number of microsclerotia colonized in soil could be clearly distinguished among different plots by using this method.