تأثير ريزوبيوم بر توليد گلومالين توسط Rhizophagus irregularis در همزيستي با گياه شبدر تحت سطوح نيتروژن

The Effect of Rhizobium on Glomalin Production by Rhizophagus irregularis in Symbiosis with Clover Plant under Different Levels of Nitrogen

گلومالین یك تركیب گلیكوپروتئینی ویژه است كه توسط قارچ¬های راسته گلومرال از رده گلومرومایكوتا تولید می¬شود و نقش كلیدی در ذخیره كربن آلی و نیتروژن خاك دارد. همچنین در تشكیل خاكدانه¬های پایدار و استقرار جوامع غنی میكروبی در خاك نقش بسزایی دارد. آزمایشی در قالب طرح فاكتوریل در پایه بلوك‌های كامل تصادفی با سه تكرار طراحی شد و گیاه شبدر (Trifolium repense L.)با قارچ Rhizophagus irregularis و باكتری Rhizobium leguminosarum bv. Trifolii تلقیح شد. چهار سطح نیتروژن (0، 2, 6 و 10 میلی‌مولار به فرم نیترات) بوسیله محلول غذایی نیومن و رومهلد ایجاد شد. گلدان‌ها با محلول غذایی آبیاری شدند. گیاهان شبدر پس از 12 هفته برداشت شد. گلومالین در بستر شن (SG) و گلومالین ریشه‌ای (RG) پس از استخراج از خاك، به روش بردفورد اندازه¬گیری شد. با افزایش سطوح نیتروژن میزان SG به طور معنی‌داری كاهش یافت (01/0 p <)؛ به طوری‌كه در سطح دو میلی‌مولار 05/63 درصد كاهش در مقدار SG نسبت به تیمار شاهد بدون نیتروژن مشاهده گردید. در حضور باكتری ریزوبیوم تولید گلومالین SG توسط قارچ‌های آربوسكولار به طور معنی‌داری افزایش یافت (01/0p <). بیشترین مقدار گلومالین در بستر شنی در حضور باكتری ریزوبیوم و قارچ AM و در سطح بدون نیتروژن بود. مقدار RG با افزایش غلظت نیتروژن افزایش پیدا كرد. در سطح 10 میلی‌مولار نیتروژن، RG نسبت به سطح صفر، 2 و 6 میلی‌مولار سرب به ترتیب افزایش 92/12، 91/11 و 44/1 درصدی داشت.

Introduction: Glomalin is a specific glycoprotein produced by the fungi belonging to phylum Glomeromycota and plays a key role in soil carbon and nitrogen storage. This also has a significant role in the stable aggregates formation and establishment of microbial communities in soil. Assimilated plant C which is allocated to the mycorrhizal fungus, appears as a recalcitrant glycoprotein (glomalin) in cell walls of hyphae and spores. Considering global warming due to increasing greenhouse gases, this phenomenon cab be important in carbon sequestration and reducing CO2 in atmosphere. Chemical fertilizers can affect symbiotic relations of these fungi, which in turn affect glomalin production. Materials and Methods: In a factorial completely randomized design with three replication, clover plants (Trifolium repense L.) were included with Rhizophagus irregularis and/or Rhizobium leguminosarum bv. Trifolii. Four levels of nitrogen (0, 2, 6 and 10 mM as nitrate) in Newman & Romheld nutrient solution were applied to the pots containing 1.5 kg sterile sand. The pots were daily irrigated with nutrient solution containing the above-mentioned levels of nitrogen. Clover plants were excised after 12 weeks of growth. Fine roots were cleaned with %10 KOH and then stained using lactoglycerol trypan blue. Root colonization percentage was determined by grid line intersections method (GLM) described by Norrif et al (1992). For glomalin extraction, hyphal or root samples were autoclaved at 121 ⁰C with 50 mM sodium citrate buffer for 60 min in three cycles. Sand glomalin (SG) and root glomalin (RG) were measured by Bradford method after extraction. Nitrogen concentration in shoot and root was measured according to the standard method. Results and Discussion: By increasing nitrogen level, the SG significantly decreased (p < 0.01), and at 2 mM, a 63.5 % decrease in SG was observed with relative to the nitrogen-free control. In the rhizobial treated pots, SG production increased by fungal inoculation (p < 0.01). The interaction between bacteria and AM was also significant in production of SG. At the presence of rhizobium bacteria, glomalin production by AM fungi increased significantly. The changes of glomalin content were not impacted by the presence of bacteria in the uninoculated pots with fungi. The highest amount of SG was recorded in the co-inoculated plants with nitrogen-free level. The amount of RG enhanced by increasing nitrogen concentration in nutrient solution. At 10 mM, RG increased by 12.90 %, 11.91 % and 1.44 % compared to the levels of 0, 2 and 6 mM, respectively. As the nitrogen level increased, the percentage of root colonization increased with respect to the control. Nitrogen concentration in shoot and root was enhanced by N increment to 10mM. Conclusion: Carbon sequestration via glomali synthase by AM fungi is an important pathway for capturing CO2 from atmosphere. Field management measures help AM development of glomalin production. Based on our results, co-inoculated plants with AM and rhizobuim seem to positively affect the production of this glycoprotein. On the other hand, SG decreased significantly by increasing nitrogen concentrations in the nutrient solution. RG, however, increased significantly as a result of increased nitrogen in both fungal inoculations. The highest amount of RG was recorded in the co-inoculated plants with 10mM level. Glomalin synthesis by the fungi is positively affected by the soil nitrogen availability. Nitrogen is the main constituent of this glycoprotein. Plant photosynthates are translocated to the fungal organs via roots and mainly utilized for glomalin synthesis in hyphal and spore cell walls. During this process, nitrogen plays an important role as a constituent of the glycoprotein. The Bradford method was used for glomalin determination in this study. The method is not specific for glomalin and can also measure other glomalin related proteins and glycoproteins. Other proteins increased by N fertilization can hence be measured based on Bradford method. Once plant assimilates are translocated to the fungi, they may be transformed to the nitrogenous compounds if sufficient nitrogen sources are available. Accordingly, a considerable amount of fixed carbon is assimilated in fungal organs and soil particles. It can be concluded that carbon sequestration by arbuscular mycorrhizal symbiosis in terrestrial ecosystems can be improved by N fertilization at optimum level. In addition, the presence of rhizobium bacteria can meet the nitrogen requirement of plants through biological stabilization of nitrogen.