Leaf blotch and head rot caused by Botrytis cinerea on sunflower and effect of heat stress on sclerotium formation of the pathogen

Abstract:

[Background] In August and September 2022, the plant samples of leaf blotch and head rot were collected from sporadic diseased fields of confectionery sunflower (Helianthus annuus) in Lanzhou, Gansu, China. [Objective] To identify and characterize the pathogens of leaf blotch and head rot. [Methods] We isolated the pathogens by the single spore isolation method, measured the pathogenicity of the isolates based on Koch's postulates, and identified the isolates by morphological observation and molecular biological methods. Furthermore, we determined the optimum growth temperatures of the isolates by the plate culture method, and the changes of sclerotium formation characteristics of the tested isolates were observed after cultured at 30℃ (growth inhibition temperature). [Results] Twelve Botrytis isolates with different colony morphology were isolated from the diseased plant samples. The inoculation of four isolates, SKY-A to SKY-D, on detached sunflower leaves and heads induced the symptoms similar to those of natural diseases in the field, and the re-isolation rates of inoculated isolates from the infected leaves and heads were 100%. When the four isolates were cultured on PDA plates at 15℃ and 20℃ for 10–40 d, macroconidia were pale to light brown, unicellular, oval, oblong, spherical, subspherical, clavate or irregular shaped, (6.0–14.2 (–20.1)) μm×(6.0–10.4 (–14.9)) μm. The sclerotia were superficial, black, spherical, subspherical, ellipse or irregularly shaped, (0.5–11.1) mm×(0.5–5.0) mm. Type-I microsclerotia were reddish brown to dark brown, superficial or submerged, (26.9–492.5) μm×(14.9–149.3) μm, with the cell sizes of (12.7–35.0) μm×(11.9–25.3) μm. Type-II microsclerotia were nearly black, submerged, (35.8–373.1) μm×(23.9–229.9) μm, with the cell sizes of (8.2–16.4) μm×(8.2–14.9) μm. Pseudo-microsclerotia consisting of specialized appressoria were dark brown to nearly black, submerged, (32.0–447.8) μm×(19.4–358.2) μm. The optimal temperature range for mycelial growth and the temperature range for sclerotium formation were 20–25℃ and 5–25℃, respectively. After incubation at 30℃ for 7 d and then at 20℃ for 14 d, SKY-B and SKY-C originally uncapable of forming sclerotia formed sclerotia. Moreover, the mutants retained the ability of forming sclerotia in subculturing. BLASTn analysis showed that the rDNA-ITS sequences of SKY-A to SKY-D (National Microbiology Data Center Acc. No. NMDCN00038NP–NMDCN00038NS) had the similarity of 99.8%–100.0% with those of Botrytis cinerea strains in GenBank. The phylogenetic analysis based on the glyceraldehyde 3-phosphate dehydrogenase gene (g3pdh), heat shock protein 60 gene (hsp60), and DNA-dependent RNA polymerase subunit II gene (rpb2) showed that the four isolates were clustered in different subgroups of B. cinerea. [Conclusion] The pathogens causing leaf blotch and head rot on confectionery sunflower were identified as B. cinerea. This is the first report of leaf blotch and head rot caused by B. cinerea on confectionery sunflower in the Northwest China. For the first time, we discover that B. cinerea can produce two types of microsclerotia. The heat stress at 30℃ induced the B. cinerea isolates uncapable of forming sclerotia to form sclerotia, and the sclerotium-forming ability of the mutants could be inherited.