Data_Sheet_1_2_An efficient inoculation method to evaluate virulence differentiation of field strains of sugarcane smut fungus.pdf

Sugarcane smut, caused by the fungal pathogen Sporisorium scitamineum, is a prominent threat to the sugarcane industry. The development of smut resistant varieties is the ultimate solution for controlling this disease, due to the lack of other efficient control methods. Artificial inoculation method is used to evaluate the virulence differentiation of pathogens. The mostly used artificial inoculation methods are soaking of the seed canes in the teliospore solution and injection of teliospores or haploid sporidia into the sugarcane sprouts. However, due to the infection nature of the pathogen that invades the sugarcane plant through meristem tissue of the sprout or shoot, the rate of successful infection is often low and fluctuated, resulting in low confidence of the assays. We recently reported a rapid and high-throughput inoculation method called plantlet soaking by using tissue culture-derived sugarcane plantlets as the test plants. Here, we compare different inoculation methods and report the characterization of parameters that may affect the sensitivity and efficiency of the plantlet soaking technique. The results showed that sugarcane plantlets were highly vulnerable to infection, even with the inoculum density at 6.0 × 105 basidial spores/ml, and this method could be applied to all varieties tested. Notably, varieties showing high smut resistance in the field exhibited high susceptibility when inoculated with the plantlet soaking method, suggesting that the plantlet soaking method is a good complement to the traditional methods for screening germplasms with internal resistance. In addition, this method could also be used to monitor the variation of cellular virulence of the smut pathogen strains in the field.

甘蔗黑穗病（Sugarcane smut）由真菌病原菌斯氏团黑粉菌（Sporisorium scitamineum）引发，是甘蔗产业的重大威胁。由于尚无其他高效防控手段，培育抗黑穗病品种是防治该病害的根本解决方案。当前评估病原菌毒力分化的主流手段为人工接种法（artificial inoculation method），常用的人工接种方式包括将蔗种浸泡于冬孢子（teliospore）悬浮液中，以及将冬孢子或单倍体孢子体（haploid sporidia）注入甘蔗芽内。但该病原菌通过芽或苗的分生组织（meristem tissue）侵染甘蔗植株，其侵染特性导致接种成功率普遍偏低且波动较大，致使实验结果置信度不足。本团队近期报道了一种以组织培养来源的甘蔗幼苗为试材的快速高通量接种方法（rapid and high-throughput inoculation method）——幼苗浸泡法（plantlet soaking）。本研究对比了不同接种方法，并系统表征了影响该幼苗浸泡法灵敏度与接种效率的各项参数。结果表明，即便接种体密度为6.0×10^5 担孢子（basidial spores)/mL，甘蔗幼苗仍极易被侵染，且该方法适用于所有供试品种。值得注意的是，田间表现为高抗黑穗病的品种在本方法接种下却呈现高感病性，表明幼苗浸泡法可作为传统方法的良好补充，用于筛选具有内在抗性的种质资源（germplasms）。此外，该方法还可用于监测田间黑粉菌菌株的细胞毒力变异情况。