| Plant root-knot nematode is a highly specialized type of omnivorous pathogenic nematodes. Root-knot nematodes mainly drill in the root tissues of plants to harm, forming spherical or conical white tumor. It will not only break the conducting tissue in plants, but also can spread many fungal and bacterial diseases, which eventually leads to the slow growth of plants, unusual leaf color, dysplastic root system, falling quality and production, and even causing plant death. Because root-knot nematode harm has great concealment, chemical pesticides are the main method used in the prevention and control, which not only caused the loss of the quality of agricultural products dramatically, but also polluted the environment greatly. This is a serious threat to human survival and safety.There are many different kinds of plants in China and my country is rich in natural resources. The application of botanical pesticides, as a new type of high efficiency, no residue, pollution-free green pesticides in agricultural production, has become one of the main means in the prevention and control of plant diseases and insect pests in the new century.In order to develop more sustainable and environmentally-friendly nematicides for controlling Meloidogyne incognita, nematicidal activities of 13 spore plants were studied with bioassay in vitro. On this basis, the nematicidal effects of Dryopteris crassirhizoma were systematically studied. Further the impacts were studied by the different extraction solvents and extraction methods on the nematicidal activity of Dryopteris crassirhizoma against root-knot nematode Meloidogyne incognita. The nematicidal potential of Dryopteris crassirhizoma was demonstrated in pot experiments. The total phloroglucinol content and nematicidal effects were determined in different extraction parts of Dryopteris crassirhizoma. The nematicidal mechanisms, antibacterial activity and allelopathic effects of Dryopteris crassirhizoma were also studied. The wettable powder of Dryopteris crassirhizoma was formulated.1. About 10 g of tissue from each of the 13 plant species were extracted in 100 ml of 70% ethanol for 3 days at 25℃. After extraction, the solvent was flash evaporated in a rotary vacuum evaporator. The residue was weighted and reconstituted in 5% acetone (AR) and kept as 2 mg·mL-1 stock solution for screening for nematicidal activity.Almost all the plant extracts caused significant mortality of Meloidogyne incognita larvae, as compared with the controls without plant extracts. The mortality gradually increased with the increase of exposure time. Five plant extracts (Conocephalum supradecompositum, Conocephalum conicum, Equisetum sylvaticum, Pteridium aquilinum var. latiusculum and Dryopteris crassirhizoma) gave more than 50% nematode mortality after 24 h and Conocephalum conicum exhibited the highest nematicidal activity after 24 h. Ten plant extracts gave more than 50% nematode mortality after 48 h, and Conocephalum supradecompositum and Dryopteris crassirhizoma also showed the strongest nematicidal activity after 48 h. Three plant extracts (Marchantia polymorpha, Equisetum pretense and Equisetum palustre) displayed the relatively poor activity against Meloidogyne incognita all along. When the overall activities of the tested plant extracts were compared, Dryopteris crassirhizoma showed the maximum activity after 72 h and resulted in 90.99% juvenile death.2. Four different methods and eight different solvents were used to make extracts of Dryopteris Crassirhizoma. Plant nematodes were treated by extractions at concentration of 1 mg·mL-1, and mortalities were investigated after 24h,48h and 72h treatments respectively. The optimal solvent is 70% ethanol,and the highest extraction rate is 18.70% by SuoShi method. The ideal method is to soak drug for 72 h at room temperature by cool solvent, and the adjusted mortalities of treated nematode is 86.87% after 72h.3. In pot experiments, tomato plant growth was significantly increased by using three different methods (soil drench, root dip and soil amendment at three levels of 10g、30g、50g) of application of Dryopteris crassirhizoma compared to the infested control.Moreover, the gall index values and final nematode population density were significantly reduced by all the treatments of Dryopteris crassirhizoma. Shoots and root systems of the plants treated with Dryopteris crassirhizoma developed normally and had only small galls on the lateral roots, whereas the shoots and roots from the infested control plants were severely deformed.In all treatments plant growth gradually increased with the increase of application quantum of Dryopteris crassirhizoma, whereas the gall index and final nematode population density reduced correspondingly.In contrast, it was observed that there was no significant difference in plant growth at same level among three methods of application of Dryopteris crassirhizoma. But the method of soil amendment was better for controlling the galls and final nematode population, there were significant differences at same level in comparison with soil drench and root-dip treatments.4. Determination of total phloroglucinols and nematicidal activities on different sections. The extraction of 70% ethanol was extracted successively by petroleum, chloroform, EtoAc, n-BuoH and water. The results showed chloroform section had the maximum phloroglucinol content and nematicidal activities, the phloroglucinol content amounts to 64.36% and the LC50 value is 0.39 mg·mL-1, followed by EtoAc(54.33%,0.67 mg·mL-1), n-BuoH(9.45%、0.97 mg·mL-1), petroleum(7.97%、1.73 mg·mL-1) and water extracts(0.00%、1.85 mg·mL-1). The phloroglucinol content and LC5o value of crude extract were 10.03% and 0.99 mg·mL-1.The nematicidal activities were in direct proportion to the phloroglucinol contents.5.Study on the nematicidal mechanisms of Dryopteris crassirhizoma. The nematicidal mechanisms were studied after treated by chloroform extracts of Dryopteris crassirhizoma at the concentration of 1.0 mg·mL-1 for 24 h.The results showed that the ultrastructures of Meloidogyne incognita were damaged significantly.SEM observation showed that the cuticular tissue was sloughed off from the body surface of nematodes,the typical annuli disappeared and the internal tissue was exposed.TEM observation showed that the integrity of muscular cells was compromised, and the structure of the protoplasts severely damaged. The nerve fibers were fragmented into many pieces and lacked integrity.The nutritional contents (protein and total glucoside) were significantly lower than control, the activities of metabolic enzymes for detoxication (CAT and GST) were significantly lower than control, the inhibitory rate of acetylcholinesterase activities amounted to 32.07%, the acetylcholine content was significantly higher than control, the Na+、K+-ATPase activities was significantly lower than control.After Meloidogyne incognita was treated by Dryopteris crassirhizoma, the defensive structures and ultramicroscopic structures were changed, the metabolisms were impeded and metabolic enzymes expressed imbalancedly. All the factors led to Meloidogyne incognita die.6. Formulation on the wettable powder of Dryopteris crassirhizoma.The optimum formula was 3% calcium lignin sulfonate,2% sodium lignin sulfonate,4% sodium dodecyl sulphate,50% Dryopteris crassirhizoma and 41% silica white. The results showed that the wettable powder of Dryopteris crassirhizoma had the good nematicidal activity in pot experiments. The desease index was 24.04% and the control effect amounted to 68.11% when the application amount was at a level of 50g-pot-1.7.Research on the antibacterial activity of Dryopteris crassirhizoma. The crude extract of Dryopteris crassirhizoma was extracted successively by petroleum, chloroform, EtoAc, n-BuoH and water, and divided into five pats. The antibacterial activities of every part were determined against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus flavus and Rhizopus nigricans.The testing results showed that five parts all had certain antibacterial effects on Aspergillus flavus and Rhizopus nigricans, the chloroform and EtoAc parts had the stonger inhibitory effects, and the water part had the minimum inhibitory effect. The four parts of petroleum, chloroform, EtoAc, n-BuoH all had some antibacterial effect on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Saccharomyces cerevisiae, while the water part had no effect. Under the condition of the same dose,the n-BuoH part had the minimum bacteriostatic action, the petroleum part had the maximum bacteriostatic action on Saccharomyces cerevisiae. The chloroform and EtoAc parts showed no bacteriostasis on Saccharomyces cerevisiae, and the n-BuoH part showed no bacteriostasis on Staphylococcus aureus and Saccharomyces cerevisiae under the condition of low dose.8. Research on the allelopathic effects of Dryopteris crassirhizoma. The crude extract of Dryopteris crassirhizoma was extracted successively by petroleum, chloroform, EtoAc, n-BuoH and water, and divided into five pats. The allelopathic effects of five parts were measured on the seeds of Semen Raphani belonging to Cruciferae, Cucumis sativus belonging to Cucurbitaceae and Zeal mays belonging to Gramineae. Compared with the controls, the petroleum and n-BuoH parts showed the inhibiting effects on the germination rate and germination potential of Semen Raphani seeds, and the petroleum part showed the significant inhibition effect; the chloroform, EtoAc and water had the promoting effects on Semen Raphani seeds. Under the condition of high concentration (1.00 mg·mL-1), the chloroform part showed the significant promoting effect, while the EtoAc and water parts showed the significant inhibition effects.Compared with the controls, the petroleum and n-BuoH parts showed the promoting effects on the germination rate and germination potential of Cucumis sativus seeds, but there was no significant influences; The chloroform and EtoAc parts had the inhibition effects on Cucumis sativus seeds belonging to Cucurbitaceae, and the EtoAc part showed the significant inhibition effect at the concentration of 1.00 mg·mL-1; The water part showed the promoting effect under the condition of low concentration and the inhibiting effect under the condition of high concentration, but all there were no significant influences.Compared with the controls, the petroleum and water parts produced the inhibition effects on the germination rate and germination potential of Zeal mays belonging to Gramineae, and that the petroleum part showed the significant inhibition effect; the chloroform and n-BuoH parts produced the promoting effects on Zeal mays, while There were no significant differences between promoting effects; the EtoAc part showed the promoting effect under the condition of low concentration and the inhibiting effect under the condition of high concentration, but all there were no significant differences.The five parts of Dryopteris crassirhizoma all had no significant differences on the dry weights of Semen Raphani belonging to Cruciferae, Cucumis sativus belonging to Cucurbitaceae and Zeal mays belonging to Gramineae after germination and growth.In a word, through this study it can be determined that Dryopteris crassirhizoma had good nematicidal activity, and had inhibitory effect on various fungi and bacteria.what’s more, it is safe for the germinations and growths of plant seeds.The developed wettable powder of Dryopteris crassirhizoma had a certain practical value and application prospect, and it can provide theoretical basis for the development and utilization of the botanical pesticide from Dryopteris crassirhizoma. |
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