Inhibitive Mechanisms Of Chrysophanol Against Sphaerotheca Fuliginea

Powdery mildew, caused by Sphaerotheca fuliginea Poll., is one of the most important diseases on cucumbers. Fungicides and resistant cultivars are the major means to control the disease on cucumber. However, with intensive and long-term use of fungicides, pathogens have developed resistance, and the control effect was significantly decreased. Therefore, it is a main issue to develop new fungicides with high efficiency, low toxicity and low-residue to control powdery mildew of cucurbit crops.In this study, chrysophanol a free anthraquinone compound was tested for its control effect against S. fuliginea under lab and field conditions. We established a bioassay method for screening fungicides to control cucumber powdery mildew, and did a research on its protective and curative effect on cucumber powdery mildew. An appropriate formulation for chrysophanol was made and tested in field experiments. The mode of action of chrysophanol on cucumber powdery mildew was investigated by means of microscope and electronic microscope. At the same time, respiratory inhibition effect on spores was tested, and the resistance-related enzymatic gene expression profiling of chrysophanol–treated leaves were detected.The follows are the major results.1. A bioassay method for screening fungicides against S. fuliginea, a pathogen causing cucumber powdery mildew was established. The results showed that a highly susceptible cultivar"Xinchangchunmici"was a suitable cultivar for bioassay test, with the appropriate seedling age of 5~10 d, the spore culturing-time of 10~15 d and the spore concentration of 30~40 spores per field of vision (10×10 times under microscope) in the suspension. Cotyledon-spraying method or leaf-disc method was suitable for bioassay, and Trypan Blue staining method was used for detecting the development of S. fuliginea on leaf surface.2. The protective and curative effect of chrysophanol on the development of S. fuliginea was determined by cotyledon-spraying method. The results showed that chrysophanol effectively reduced the disease index, the protective and curative EC50 values were 33.36μg/mL and 90.10μg/mL respectively, while the protective EC50 for Sulfur DF was 59.75μg/mL and the curative EC50 for Difenoconazole WG was 13.55μg/mL. 3. The action mode of chrysophanol on the development of S. fuliginea and the uptake transmission of chrysophanol on cucumber cotyledons and leaves were determined. Results showed that control effect could reach 84.83% 20 days after foliages spraying with chrysophanol at 100μg/mL concentration, the same as 500μg/mL 50% Sulfur SC. At cotyledon and leaf tests, chrysophanol showed certain cross-layer and little transverse effect, while almost no acropetal and basipetal uptake transmission effect in cucumber.4. Formulation process was test using 47% chrysophanol, and 5% chrysopanol suspension concentrate (5% chrysopanol SC) was achieved. Field tests showed that 5% chrysopanol SC had a control effect as high as 88.89% at 69 g (a.i)/hm2 concentration.5. Inhibitive effect of chrysophanol on spore germination and mycelia growth of S. fuliginea was observed from histopathology. Rusult showed that, when 2.4μg/mL chrysophanol was used as preventive treatment, the results showed that chrysophanol depressed the growth of mycelia and reproduction of new conidia. The average germination rate was about 66.6% 24 h after inoculation in untreated control, while there was no germination in the treatment with chrysophanol. The average germ tuber length was about 414.7μm 48 h after inoculation in untreated control, while only 40.1μm in the treatment with chrysophanol. After inoculation for 144 h the maximum number of conidia linked in a chain were 8 with most of them between 6 to 7 in the control, while that was only 2 in chrysophanol treatment and most of them was 1. The disease index in cotyledon of cucumber was 72.2 in the control 7 days after inoculation, while only 2.1 in the treatment with chrysophanol. The protective control effect was 97.1% much higher than 65.4% of 50% Sulfur SC. 6. In this paper the ultrastructure of powdery mildew on cucumber leaves was studied using electronic microscope after the leaves were treated with chrysophanol. Results showed that when chrysophanol was used as preventive treatments, most of spores couldn't infect host for spores cell wall ruptured, and fungal development was affected, including conidia shrank, the few germinating tube malformed and shrank, hyphae expanded slowly. Infection peg immediately surrounded by dense host cell wall papilla; a few of infection pegs could formed abnormal haustorium, cytoplasm in these haustorium aggregated and very dense, and organelles disintegrated. In the curative treatment, chrysophanol affected fungal survival, resulting in broken cell wall of germ tubes, swelling and collapse of hyphal tips, hyphal malformation, delayed and reduced sporulation. The morphological changes induced by chrysophanol at the ultrastructural level were reflected by more dense host cell wall papilla, host cell walls infected or adjacent to haustoria were thickened, haustorium deformation, vacuolization, abortion and necrosis, empty cavity appeared and vacuole enlarged; space along the extrahaustorial membrane was filled with black and amorphous electron-dense substance, organelles in haustorium disintegrated, expressed disintegration tendency. When chrysophanol was used as eradicate treatments, it affected fungal survival, resulting in distorted, malformation and collapse of hyphal and spores.7. Results revealed that chrysophanol had significant inhibition effect on metabolic pathways, and the most important pathway of chrysophanol inhibiting powdery mildew was HMP. The superpose rateof chrysophanoland Na3PO4 was 10%.8. Several resistance-related enzymatic activities of the leaves of cucumber were detected after treated by chrysopanol. Results showed that phenylalanine ammonia-lyase (PAL), chlorophyll, lignin and total phcnolic content was increased, while the activity change of peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) was not obvious. The activities of PAL reached its maximum at two and seven days respectively after inoculation, and the activities were 4.9 times of the acetone-treated, while the activities of uninoculation chrysopanol-treatment was lower. The content of chlorophyll was increased whether inoculation after chrysopanol treated, the content was 1.8 times of the acetonetreated at three days after inoculation.The content of lignin reached its maximum at two days after inoculation, the lignin content was 4.9 and 6.8 times respectively compared to the start time of inoculation and uninoculation chrysopanol-treatment, while the lignin content was stable of acetone-treated. The content of total phcnolic was increased after chrysopanol treated, while the content reached its maximum at one and five days respectively after inoculation, which were higher 36% and 23% than acetone-treated at the same time.9. Results showed that the expression of PAL and Chitinase gene in cucumber leaves could be induced by chrysopanol detected by Quantitative Real Time PCR. The expression of PAL were 9.5 times of that at starting time at two days after inoculation and reached its maximum at two days after inoculation. The expression of chitinase reached its maximum at one day after inoculation, and expression of chitinase were 3.8 times of that at starting time.