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توسعه و اعتبارسنجی یک روش نوین ارزیابی سریع و سهبعدی انحلال فسفات در باکتریهای گرممثبت اسپوردار | ||
| زیست شناسی خاک | ||
| مقالات آماده انتشار، پذیرفته شده، انتشار آنلاین از تاریخ 21 تیر 1405 | ||
| نوع مقاله: مقاله پژوهشی | ||
| شناسه دیجیتال (DOI): 10.22092/sbj.2026.372945.294 | ||
| نویسندگان | ||
| شکیبا رئیسی1؛ حسینعلی علیخانی1؛ علیرضا فلاح نصرت آباد* 2؛ بهمن خوشرو* 2؛ احمدعلی پوربابائی1 | ||
| 1گروه علوم و مهندسی خاک، دانشکده کشاورزی، دانشگاه تهران، کرج، ایران. | ||
| 2موسسه تحقیقات خاک و آب کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران. | ||
| چکیده | ||
| این پژوهش با هدف طراحی و ارزیابی یک محیط کشت جدید (FKR) همراه با یک بستر سهبعدی نیمهجامد برای غربالگری کمی و کیفی PSB، عمدتاً از جنس Bacillus، انجام شد. 299 جدایه گرممثبت، عمدتاً Bacillus از ریزوسفر ذرت، روی محیطهای جامد FKR و سه محیط استاندارد Pikovskaya (PVK)، NBRIP و Sperber (SP) کشت داده شدند. سپس جدایهها در لولههای حاوی FKR–BCP نیمهجامد بر اساس شاخص عمق (SD) و حجم انحلال (SV)، الگوهای رشد در گرادیان اکسیژن و ویژگیهای متابولیکی ثانویه بررسی شدند. محیط FKR بهتنهایی 55% سویهها را به عنوان PSB شناسایی کرد، در حالیکه میانگین سه محیط استاندارد تنها ۱۹٫۳٪ (۲۰، ۲۰ و ۱۸٪) بود (P < 0.05)، که بیانگر افزایش حدود ۲٫۹۵ برابری کارایی غربالگری است. سهم نتایج منفی کاذب در محیطهای استاندارد حدود ۸۰–۸۲٪ و در FKR فقط ۴۵٪ بود. در سامانه سهبعدی، طی ۲۴ ساعت نخست، بیش از ۷۵٪ سویههای فعال، ناحیه زرد مشخص در ستون نیمهجامد ایجاد کردند، در حالیکه در همان بازه، کمتر از ۵٪ روی پلیت FKR هاله قابلاعتماد داشتند (P < 0.05). سویههای برتر موفق به ایجاد عمق انحلالی بیش از ۴۰ میلیمتر شدند. بررسیها نشان داد که با تغییر رویکرد از ارزیابی دوبعدی (SI) به سهبعدی (SV)، جایگاه حدود ۱۵ درصد از سویهها تغییر میکند. در مجموع، ۶ سویه (۳٫۸٪) الگوی انحلال قلیایی، حدود ۷٪ تولید گاز و حدود ۲٪ مایعسازی محیط نیمهجامد را نشان دادند. ترکیب فرمولاسیون FKR با روش سهبعدی نیمهجامد و کمیسازی حجمی (SV)، ابزاری کارآمد برای انتخاب هدفمند سویههای مناسب توسعه کودهای زیستی فسفاته در خاکهای آهکی و قلیایی فراهم میکند. | ||
| کلیدواژهها | ||
| باسیلوس؛ غربالگری میکروبی؛ کود زیستی؛ محیط کشت نیمهجامد؛ هاله انحلال | ||
| عنوان مقاله [English] | ||
| Development and validation of a novel rapid three dimensional assay for phosphate solubilization in spore forming Gram positive bacteria | ||
| نویسندگان [English] | ||
| Shakiba Reisi1؛ Hossein Ali Alikhani1؛ Alireza Fallah Nosrat-Abad2؛ Bahman Khoshrou2؛ Ahmad Ali Pourbabaei1 | ||
| 1Department of Soil Science and Engineering, Faculty of Agriculture, University of Tehran, Karaj, Iran. | ||
| 2Soil and Water Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. | ||
| چکیده [English] | ||
| Background and Objectives: Insoluble phosphates are among the major factors limiting phosphorus nutrition in calcareous soils, and the accurate selection of efficient phosphate solubilizing bacteria (PSB) is essential for the development of P biofertilizers. This study aimed to design and evaluate a new culture medium formulation (FKR), in combination with a semi solid three dimensional platform, for quantitative and qualitative screening of PSB, mainly belonging to the genus Bacillus. Materials and Methods: A collection of 299 Gram positive bacterial isolates, predominantly Bacillus spp. obtained from the rhizosphere of maize plants under different field conditions and cultivars, was first grown on the FKR medium and on three standard media, Pikovskaya (PVK), NBRIP and Sperber (SP). The percentage of positive isolates, halo diameter and two dimensional solubilization index (SI) on plates were measured and evaluated using appropriate statistical tests (one way ANOVA and Duncan’s multiple range test at 5% probability, as well as suitable tests for comparison of proportions such as chi square or Fisher’s exact test). The performance of the same isolates was then assessed in semi solid three dimensional FKR tubes (with and without the BCP pH indicator), based on solubilization depth (SD) and solubilization volume (SV) indices, growth patterns along the oxygen gradient, and secondary metabolic traits (gas production, liquefaction, and medium alkalization). Results: The FKR medium alone identified 164 isolates as PSB (55% of the 299 isolates), whereas the mean percentage of positive isolates on the three standard media PVK, NBRIP and SP was only 19.3% (20, 20 and 18%, respectively). This difference in the proportion of positive isolates was statistically significant (P < 0.05), indicating that the FKR formulation increased screening efficiency by approximately 2.95 fold compared with the average of the three standard media. At the same time, the proportion of false negative results on the standard media was about 80–82% (239–245 isolates without halos), whereas this proportion decreased to 45% (135 isolates) on FKR, revealing part of the hidden phosphate solubilizing potential under nutrient limited standard conditions. In terms of qualitative differentiation based on SI, the FKR medium detected eight isolates in the very strong class (class A; SI ≥ 2.5), while the three standard media together detected only two isolates in this class and NBRIP did not detect any class A isolate, highlighting a systematic limitation of minimal formulations in detecting high performance strains. In the three dimensional evaluation, the semi solid FKR–BCP tube method exhibited a clear advantage over plate assays in terms of speed and clarity of response: within the first 24 h, more than 75% of the solubilizing isolates produced a distinct yellow zone along the semi solid column, whereas in the same period fewer than 5% of these isolates formed a reliable clear halo on solid FKR plates (P ≤ 0.05). Three dimensional quantification based on SD showed that top performing isolates could generate solubilization depths exceeding 40 mm along the column, and calculation of the volumetric SV index (mm³) provided a more comprehensive picture of the intensity and spatial extent of activity. Comparison of the ranking of the ten best isolates according to SI and SV revealed substantial changes in the relative position of some strains. For instance, isolate FKR S10, with the highest SI (3.50), ranked first in two dimensional assays but dropped to rank 61 in terms of SV (2,031 mm³), whereas isolate FKR S94, with a moderate SI (2.47; rank eight), showed the highest SV (10,244 mm³) and ranked first in three dimensional assays. Overall, about 15% of ranks were markedly shifted between the two approaches, indicating that two dimensional evaluation may underestimate the true potential of isolates with invasive and deep growth. From a metabolic perspective, the three dimensional system led to the discovery of phenomena that were not visible in plate assays. In total, six isolates (≈3.8% of the population evaluated in tubes) exhibited an alkalinizing solubilization pattern and produced a dark purple halo in FKR–BCP due to local pH increase, likely associated with alkaline phosphatase activity or secretion of calcium chelating and other complexing agents. Moreover, about 7% of active isolates produced gas (fine bubbles along the diffusion path) and around 2% liquefied the semi solid matrix, indicating the production of extracellular hydrolytic enzymes. Finally, three main growth patterns along the oxygen gradient of the semi solid column surface (pellicle/ring), continuous vertical, and banded (layered) were recorded, providing valuable qualitative information on the ecophysiology and respiratory preferences of the isolates. Conclusions: The results demonstrate that combining the FKR chemical formulation with a semi solid three dimensional system markedly increases the sensitivity and resolution of detection, quantification and functional differentiation of Bacillus PSB compared with classical plate based methods. By substantially reducing false negatives, enabling volumetric quantification (SV), revealing both acidic and non acidic solubilization mechanisms, and capturing secondary metabolic activities and growth patterns along oxygen gradients, this platform provides an efficient and exploratory tool for targeted selection of robust, high performance strains for the development of phosphate biofertilizers, particularly for calcareous and alkaline soils. | ||
| کلیدواژهها [English] | ||
| Bacillus, Microbial screening, Biofertilizer, Semi-solid medium, Solubilization halo | ||
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