Fungus Inoculation and Disease Evaluation

For fungus inoculation, seedlings of G. hirsutum cv. YZ-1 that underwent VIGS treatment, and 3-week-old WT or transgenic tobacco plants were each gently uprooted, the roots were first rinsed in water and subsequently dipped into the spore suspension of 1×10⁵ conidia·ml⁻¹ for 1min. The plants were then replanted in fresh soil to monitor disease development. The fungal recovery assay in cotton was performed according to the method of Fradin et al. (2009) (link). Stem sections immediately above cotyledons were taken from cotton seedlings 10 days after V. dahliae inoculation and surface sterilized. The stem sections were subsequently cut into 5–8mm slices and incubated at 25 °C on potato dextrose agar. Cotton seedlings infiltrated with binary vectors pTRV-RNA1 and pTRV-RNA2 were used as the vector control, and the plants of WT and transgenic tobacco inoculated with water were used as mock controls. The disease index (DI) of Verticillium wilt was calculated according to the following formula:
Cotton leaves were classified in one of five levels of severity of disease symptoms during fungal invasion, and n denotes the disease level from 0 to 4 (Xu et al., 2012a). The resistance of tobacco to V. dahliae was evaluated by determining the extent of stunting (leaf size, weight of the plant) as well as the degree of damage to infected leaves (leaf chlorosis/wilting). The same formula above was adopted for disease index calculations in tobacco.
B. cinerea was incubated on potato dextrose agar medium for 4 days and then put onto leaves in concentric circles using a punch. A fungus with identical virulence was inoculated for 72h onto the second leaf in vitro, collected from the top of cotton seedlings. Growth of B. cinerea was estimated by the size of the necrotic lesions on the leaves.