The Colonization Process of Sunflower by a Green Fluorescent Protein-Tagged Isolate of Verticillium dahliae and its Seed Transmission

Abstract

Sunflower Verticillium wilt is a widespread and destructive disease caused by the soilborne pathogen Verticillium dahliae. To better understand the process of infection and seed transmission of the fungus, sunflower roots were inoculated with a V. dahliae strain (VdBM9-6) labeled with green fluorescent protein (GFP) and monitored microscopically. After 24 to 96 h postinoculation (hpi), conidia germinated and developed into mycelium on root hairs, elongation zones, and caps of lateral roots. Mycelium colonized vascular bundles of lateral roots and taproots at 7 days postinoculation (dpi). At 10 weeks postinoculation (wpi), the epidermal cells, cortical tissues, and vascular elements of stem, petiole, and leaf veins were colonized by mycelium. By 12 wpi, strong GFP signals were detected not only on different tissues of inflorescence but also on testa of seed and a small fraction of pollen grains. A GFP signal was not observed on cotyledon tissues in the seed. Additionally, the colonization of V. dahliae on testa was also confirmed with MNP-10 selection medium, indicating that the testa of seed is the main carrier for the long distance transmission of sunflower yellow wilt.

Fungicide Treatments to Control Seed-borne Fungi of Sunflower Seeds

“…This result indicated that sunflower seed coats are the major carriers for long-distance transmission of seed-borne pathogens. Verticillium dahliae was detected in almost all the sampled sunflower varieties collected (84%), which suggests that seed-to-seed transmission and long-distance transmission of the fungi is carried out by the seed coat, as seen in spinach production [12]. The presence of multiple fungi on the seed coats supports the fact that seed contamination is the main machinery for the long-distance transmission of seed-borne fungal diseases [7,8,11,31].…”

“…Results from previous research on the contamination process of sunflower verticillium wilt using GFP (green fluorescent protein) -labeled V. dahliae showed that the seed coat was one of the main tissues where V. dahliae inoculum infect and accumulate [12]. This finding led us to find out the rate of contamination among commercial sunflower varieties with emphasis on their seed coats.…”

“…Seed soaking was used as a method for pretreating the seeds collected from the verticillium wilt-infected field. Seeds were soaked in formulated flusilazole fungicide solution with varying intervals (6,12,18, and 24 h). The seeds were soaked in 100 mL of formulated flusilazole fungicide.…”

“…The virulent Verticillium dahliae Gn3 strain was isolated from a diseased LD 5009 sunflower plant in Gannan, Heilongjiang Province of China, and identified using Koch's postulate as V. dahliae. Its GenBank accession number is HQ 441164 [12], this was used as a test strain to determine the efficacy of the fungicides based on its moderate virulent and typical morphology of V. dahliae.…”

“…were not recorded in this study [6]. V. dahliae can colonize the pericarp and testa of certain safflower seeds [7] and also the pericarp and endosperm of lettuce seed [8], hence the possibility of seeds being the main dispersal agents for long-distance transmission of V. dahliae [7,[9][10][11][12]. A. alternata, Rhizopus stolonifer, and Fusarium spp.…”