اثر باکتری‌های حل‌کننده فسفات بر میزان آلکالوئیدهای مورفین، پاپاورین، و نوسکاپین خشخاش (papaver somniferum l.)

تاکنون بیش از 40 نوع آلکالوئید مختلف در گیاه دارویی و ارزشمند خشخاش (papaver somniferum l.)، شناخته شده که مهم‌ترین آن‌ها مورفین، کدءین، تباءین، نوسکاپین و پاپاورین است. بیوسنتز این آلکالوئیدها ممکن است به‌شدت تحت تاثیر انواعی از الیسیتورهای زنده و غیرزنده قرار گیرد. پژوهش حاضر با هدف بررسی اثرات سویه‌های باکتریایی با قابلیت حل‌کنندگی فسفات معدنی به‌عنوان الیسیتورهای زنده، بر میزان تولید آلکالوئیدهای مورفین، پاپاورین، و نوسکاپین در گیاه خشخاش انجام شده است. در این پژوهش، قابلیت حل‌کنندگی فسفات معدنی توسط چهار سویه باکتریایی enterobacter xiangfangensis s2،pantoea dispersa s7 ، pantoea stewartii s25 و pseudomonas canadensis s36 به‌روش کمّی و به کمک محیط کشت مایع sperber ارزیابی شد. در شرایط گلخانه، اثر محلول‌پاشی بوته‌های خشخاش با سوسپانسیون سویه‌های باکتریایی (cfu.ml^-1 10^8) بر میزان آلکالوئیدهای مورفین، پاپاورین، و نوسکاپین موجود در کپسول، ساقه، و برگ خشخاش بررسی شد. آلکالوئیدها به‌روش الکلی استخراج و توسط دستگاه hplc ردیابی شدند. تمامی آزمون‌ها در قالب طرح آماری کاملاً تصادفی و با سه تکرار به‌ازای هر تیمار انجام شدند (p<0.05). نتایج نشان داد که بیشترین (458/67 میکروگرم بر میلی‌لیتر) و کمترین (130/47 میکروگرم بر میلی‌لیتر) میزان حل‌کنندگی فسفات به‌ترتیب مربوط به سویه‌های s2 و s36 بود. در تیمار با سویه‌های باکتریایی مشخص شد که سطح مورفین موجود در ساقه و برگ و نیز کپسول در اغلب موارد افزایش معنی‌داری در قیاس با شاهد را نشان داد. میزان پاپاورین موجود در ساقه و برگ کاهش معنی‌داری داشت، ولی در کپسول تغییرات معنی‌داری نداشت. نوسکاپین موجود در ساقه و برگ نیز افزایش معنی‌داری داشت و از 0/8 میلی‌گرم بر گرم وزن خشک در شاهد به 8/12 در تیمار s2 رسید. درصورتی‌که میزان نوسکاپین کپسول فقط در تیمار با s2 و s36 افزایش معنی‌داری داشت و سایر سویه‌ها اختلاف معنی‌داری با شاهد نداشتند. به این ترتیب با انتخاب سویه‌های باکتریایی سازگار و کارآمد از گروه باکتری‌های حل‌کننده فسفات، می‌توان سطح آلکالوئیدهای مورفین، پاپاورین، و نوسکاپین در اندام‌های هوایی گیاه دارویی و ارزشمند خشخاش را به‌طور چشمگیری افزایش داد.

the effect of phosphate solubilizing bacteria on the amount of morphine, papaverine, and noscapine alkaloids of papaver somniferum l.

introduction so far, more than 40 different types of alkaloids have been known in poppy (papaver somniferum l.) as a valuable medicinal plant, the most important of which are morphine, codeine, thebaine, noscapine, and papaverine. the biosynthesis of these alkaloids may be strongly influenced by a variety of biotic and abiotic elicitors. in fact, microbes as biotic elicitors can affect the production of poppy alkaloids. among them, plant growth promoting rhizobacteria (pgpr) can be noticed, which stimulate and improve plant growth through various mechanisms such as mineral phosphate solubilization, plant hormone production, siderophores secretion, nitrogen fixation, etc. the use of pgpr agents can not only lead to an increase in plant biomass, but simultaneously, due to their role as biotic elicitors, they cause to an increase in the biosynthesis of secondary metabolites in plants. these biotic elicitors target plants’ defense mechanisms and result in triggering a series of metabolic changes throughout the plant. the use of pgpr agents to stimulate the plant to produce secondary metabolites has several advantages: first, in some plants, defensive metabolites are active biological compounds that lead to the induction of food production with high added-value in the plants. secondly, physiologically, with the increase in the synthesis of secondary metabolites, the resistance of the plant against pathogens also increases. accordingly, the present study was performed with the aim of investigating the effects of bacterial strains with the ability to solubilize inorganic phosphate as biotic elicitors on the amount of morphine, papaverine, and noscapine alkaloids in p. somniferum. materials and methods in this research, the solubility of inorganic phosphate by four bacterial strains including enterobacter xiangfangensis s2, pantoea dispersa s7, pantoea stewartii s25, and pseudomonas canadensis s36 was evaluated quantitatively using sperber broth medium. under greenhouse conditions, the effect of foliar spraying of p. somniferum plants with a suspension of the bacterial strains (108 cfu.ml-1) on the amount of morphine, papaverine, and noscapine in the plants’ capsules, stems, and leaves was investigated. about three weeks after the appearance of capsules in poppy plants, the aerial parts of the plants (stems, leaves, and capsules) were sprayed with the bacterial suspensions. one week after foliar spraying, poppy plants were harvested in order to determine the amount of the desired alkaloids. three pots were considered for each treatment and there were three poppy plants in each pot. alkaloids were extracted based on an alcoholic method and detected using hplc. morphine and noscapine standards were prepared at a concentration of 1000 μg.ml-1 and papaverine standard at a concentration of 250 μg/ml. then the mixture was prepared in proportions of 1, 1:50, 1:10, 1:50 and 1:100 and injected into the hplc set to draw the calibration curve. all the experiments were conducted in a form of completely randomized design with three replications for each treatment (p<0.05).results and discussionthe results showed that the highest (458.67 µg.ml-1) and the lowest (130.47 µg.ml-1) phosphate solubility were related to s2 and s36 strains, respectively. s7 and s25 strains were not statistically significantly different from each other and after s2 strain, they were placed in the second statistical position. in the bacterial strains’ treatments, the level of morphine in the stems and leaves as well as the capsules increased significantly in most cases compared to the control. the amount of papaverine in the stems and leaves decreased significantly, but it had no significant changes in the capsule. also, noscapine showed a significant increase in the stems and leaves and reached from 0.8 mg.g-1 dw in the control to 8.12 in the s2 treatment. while, the amount of noscapine increased significantly in the capsules, only in the s2 and s36 treatments. other strains did not show significant differences with the control for noscapine content in the capsules. the results showed that the interaction effects of the type of the alkaloids and the use of phosphate solubilizing bacterial strains on the concentration of the studied alkaloids in poppy stems, leaves and capsules are significant (p<0.01). conclusionit can be concluded that there is no need to apply genetic engineering to increase the production of valuable secondary metabolites by medicinal plants. rather, this goal can be achieved much cheaper by using bacterial elicitors. accordingly, by selecting compatible and efficient bacterial strains with phosphate solubilizing activity, the amounts of morphine, papaverine, and noscapine alkaloids in the aerial parts of p. somniferum as a valuable medicinal plant can be noticeably increased.