| Grape phylloxera(Daktulosphaira vitifoliae Fitch) is a devasting pest among the viticulture industry. Although there had been a large amount of researches with related to pest-caused damages on the viticulture reported, fewer were in reference to control measures of phylloxera due to the successful use of rootstocks which was considered to some extent helpful for the prevention of phylloxera. In China, the damage caused by phylloxera is still not ignorable besides that, the technique of rootstock is not commonly applied and most of the vineyards are still using self-rooted grapes, which means a control measure is necessary in the event of vineyards infected by phylloxera. The efficacy of ten plants aqueous extracts in controlling phylloxera was discussed and the probable working mechanism of tobacco or nicotine was also involved. Totally the results were showed as the following aspects:1.Bioactivities of 10 plant aqueous extracts to grape phylloxera in ChinaThe present research was conducted to evaluate the biological efficacy of plant aqueous extracts to grape phylloxera.10 different species of plants were used in this study. Toxicity and repellent activities towards the instars and life development of grape phylloxera were quantified during the vito tests. All initial tests were completed in laboratory and the results suggested that all the tested varieties revealed repellent effect against phylloxera, tobacco aqueous extracts showed an equal toxicity capacity with Movent, and the correctted mortality rate could reach 96.00%. Aqueous extracts also slowed down the developmental speed of phylloxera, while extending the developmental durations by 4~5 days to 30 d. The egg laying ability was also decreased significantly, while daily egg laying number decreased by over 21.89% on the aqueous extracts treated roots. As conclusion, on one end the study suggests that plant aqueous extracts tested in our research have biological activity to grape phylloxera and could affect normal subsist. On another end, Nicotiana tabacum has a significantly negative effect to populations of phylloxera.2. Control effect of tobacco aqueous extracts to infection and damage of grape phylloxera on grapesTwo year-old Kyoho grapes were chosen as test material in the greenhouse. The results showed that tobacco aqueous extracts had a certain alleviating effects on phylloxera infection. Both the tobacco aqueous extracts at the concentration of 20 mg?m L-1 and 50 mg?m L-1 had aninhibition to phylloxera infection. After the 21 days irrigation, nodosities numbers were respectively 42.90% and 52.80% smaller than that of infected grapes. Tobacco aqueous extracts of high concentration can inhibit the growth and soil microorganism amount. Plant growth amount increased respectively by 57.10% and 31.40%, when the amount of bacteria increased, compared with control and 50mg?m L-1 treatment. During the 7~21 days, photosynthesis ratio of tobacco treatment recovered from the infection. Tobacco aqueous extract have a control effect towards grape phylloxera in potted tests and can ease the hurts caused by phylloxera infection.3. Chemical composition evaluation of tobacco aqueous extractsDifferent chemical solutions were used to identify the probable compositions of the tobacco aqueous extracts. The results revealed alkaloid, phenols, saccharides, tannins and polypeptide components existed in the extracts, the most significant response was alkaloids.4. Toxicity equation conduction of nicotine solutions to grape phylloxera nymphsConcentration gradient of nicotine solutions were used in toxicity efficacy test of phylloxera. After the first 24 hours, mortalities were recorded and toxicity equation was conducted as y=1.0161x-0.6355, correlation ratio was r2=0.9950, and the LC50 was 0.2212mg?m L-1 under 95% confidence limit.5. Physiological and biochemical properties change of phylloxera nymphs under nicotine solution effectSublethal dose nicotine solutions(0.2 mg?mL-1) were used in the biochemical properties change evaluation. Result reveraled that phylloxera biochemical indexes changed after the nicotine treatment. Acetylcholinesterase and Cytochrome P450 were inhibited during the test time; carboxylesterase and phospho-esterase were conducted for a short period after treatment. Acid phosphatase activity didn’t change observably, while alkaline phosphatase increased during 2~4h and then decreased. Carboxylesterase had the same change trend with alkaline phosphatase. Cytochrome P450 related enzymes(including Cyt-b5 content, p-nitroanisole-Odemethylase and NADPH-Cyt-c reductase) decreased observably during the treat time.Cholinedterase reactivators(2-PAM) treatment could recover the Acetylcholinesterase activity of poisoned phylloxera. During the different treatment, phylloxera after nicotine treated 60 min and 2-PAM recover 15 min showed the highest recover rate, which could reach 52.55% wihle the rate was only 12.76% when treated by nicotine for 120 min and 2-PAM recover 15 min. RT-PCR test showed that nicotine solution could increase the expression index of nicotinic acetylcholine receptor-α-subunit gene, while 2-PAM recover treatment could decrease the expression index when ACh E were higher inhibited but also increase theexpression when ACh E were lighter inhibited. But both the biochemical and RT-PCR dates showed cholinedterase reactivator treatment didn’t have observably influence to the healthy phylloxera.6. Grape phylloxera AChR-α-subunit gene clone and analysisThe 3’end sequence of grape phylloxera ACh R-α-subunit gene were cloned and analyzed. The results reveraled this gene of grape phylloxera had a high degree Conserved Domain with some already known sequences of other insects, which had a similar ratio at 70.00~90.00%. The whole sequences we cloned totally had 982 bp, including a 550 bp open reading frame, coding a 183 aa polypeptide which has a MW at 81914.68 D. The PI was 5.12 from the divine software, and all the amino acid sequence was outside the surface of cell membrane. |
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