جداسازي و شناسايي باكتري‌هاي حل‌كننده پتاسيم از سنگ شيل‌گلاكونيتدار استان گلستان و تعيين برخي خصوصيات محرك رشدي آنها

Isolation and identification of potassium solubilizing bacteria of the shale stones containing glauconite in Golestan Province and determination of growth promoting properties

هواديدگي زيستي كاني‌هاي سيليكاتي منبع عمده پتاسيم براي گياهان در شرايط طبيعي مي‌باشد. هدف از اين مطالعه، جداسازي و شناسايي باكتري‌هاي حل‌كننده پتاسيم از سنگ شيل­گلاكونيت­دار در استان گلستان و تعيين برخي خصوصيات محرك رشدي آنها بود. تعداد پنج نمونه يك كيلوگرمي از خاك ريزوسفر گندم از عمق صفر تا 30 سانتي‌متري و پنج نمونه يك كيلوگرمي از سنگ شيل گلاكونيت‌دار جمع‌آوري و به آزمايشگاه انتقال داده شد. پس از جداسازي و خالص­سازي جدايه­ها، سنجش ميزان آزادسازي پتاسيم در محيط كشت الكساندروف حاوي مسكويت و گلاكونيت به روش نورسنجي شعله­اي انجام شد. شناسايي جدايه­ها براساس ويژگي­هاي بيوشيميايي آنها انجام گرديد. آزمون‌هاي محرك رشدي در جدايه­هايي كه از لحاظ ظاهري متفاوت بوده و ازتوانمندي بالايي در آزادسازي پتاسيم برخوردار بودند صورت پذيرفت. در نهايت بهترين جدايه با استفاده از توالي نوكلئوتيدي ژن 16S rRNA شناسايي شد. از تعداد 40 جدايه اوليه، 20 جدايه از خاك ريزوسفري و 20 جدايه از خاك حاصل از پودر سنگ شيل گلاكونيت‌دار جداسازي شدند. آزادسازي پتاسيم توسط 10 جدايه برتر نشان داد كه بيشترين مقدار پتاسيم آزاد شده مربوط به جدايه‌ 39 به ميزان 2/34 ميلي‌گرم بر ليتراز مسكويت و 8/31 ميلي‌گرم بر ليتر از گلاكونيت بود. بيشترين مقدار سيدروفور، اكسين و انحلال فسفر نامحلول توسط جدايه شماره 39 مشاهده گرديد. نتايج توالي نوكلئوتيدي ژن16S rRNA نشان داد كه جدايه 39 متعلق به گونه آرتروباكتر فنانترنيوورانس بود. اين مطالعه نشان داد باكتري­هاي بومي از توانايي خوبي در آزاد كردن پتاسيم از منبع شيل گلاكونيتي برخوردارند.

Introduction Potassium is one of the essential and macro elements in the growth of plant cells. This element plays an important role in improving the quality of agricultural products. The amounts of available potassium levels in most soils decrease more quickly and potassium balance is disturbed in many fields. Cultivation and lack of the application of potassium fertilizers in agricultural soils of Iran have caused the depletion of potassium and the amount of available potassium in most soils has reached below the critical level. The compensation of depleted potassium in the soil through indigenous resources and use of potassium bio-fertilizers is therefore very important. Weathering of silicate minerals by bacteria is considered as one of the essential K source for plant growth and development. The objective of this study was to isolate and identify potassium solubilizing bacteria from the shale containing glauconite mineral in Golestan Province and determine some traits related to plant growth promotion and selecting a superior strain in order to incubate in wheat lands. Materials and Methods Accordingly, a total of 5 samples 1 kg of rhizosphere of wheat from a depth of 0 to 30 cm and 5 samples 1 kg from shale stone (containing glauconite) were collected from Aitamir formation in Golestan Province in May 2015 and were transferred to the laboratory of Gorgan University of Agricultural Science and Natural Resources. Isolates were transferred to Aleksandrov media containing glauconite and muscovite and incubated for 10 days and the isolated strains were stored in the refrigerator at 4 oC. The amount of potassium release in solutions after 10 days was measured. Some biochemical and morphological properties of isolates were determined based on standard methods. PGPR tests were done in the isolates which were morphologically different and had high potential in releasing K. Finally, a strain whith high ability in releasing potassium and growth promoting properties was identified using nucleotide sequence of 16S rRNA gene. Results and Discussion Results showed that 40 strains from the first stage, 20 strains from rhizospherial soil and 20 strains from the soil resulting from glauconite mineral powders were isolated. Biochemical and potassium release tests showed that the highest released potassium was related to isolate No. 39 with an amount of 34.2 mg l-1 in muscovite, and 31.8 mg. l-1 in glauconite. The amount of siderophore produced in the superior strains showed that the lowest and the highest ratios of the diameter of the colony, were 1.12 and 3.1 related to isolate No. 19 and No. 39, respectively. The highest and the lowest auxins produced were also related to the isolate No. 39 and No. 27 with the amount of 52.25 and 5.15 mg per liter, respectively, measured at 72 and 96 hours. The results showed that the soluble phosphorus between different isolates was significantly different (P <0.05), its greatest concentration at 72 hours was related to isolates No. 39 with an amount of 295 mg per liter and the lowest at 24 hours was related to isolate No. 31 with an amount of 80 mg per liter. Also, the production of hydrogen cyanide test showed that none of the isolates was capable of producing siderophore. The obtained results from nucleotide sequence of 16S rRNA gene showed that the selected strain belonged to Arthrobacter phenanthrenivoran species. Conclusion It can be concluded that silicate bacteria contribute to the dynamics and mineralizing of elements in the soil and eventually K release from glauconite containing shale minerals mainly by reducing rhizosphere pH, the secretion of organic anions and complex formation with the surface cations of mineral and secreted extracellular Polysaccharides and soluble compounds and decomposition of soil organic matter. Among 40 isolated strains and the various tests and the results of released potassium in both minerals, the results showed that the potential of potassium releasing was different between the tested strains. Moreover, this study showed that in addition to the effect of these strains on potassium releasing, siderophore production, auxin and inorganic phosphate solubility, they can be effective in plant growth and in land inoculation. This study revealed the potential of indigenous bacteria species in the release of K from shale containing glauconite. It is anticipated that shale containing glauconite can provide a part of the need of the crops for potassium.