رديابي همزمان باكتري هاي مهم بذرزاد لوبيا با استفاده از روش Multiplex-PCR

Abstract Bean (Phaseolus vulgaris L.), as a source of plant protein, plays an important role in human diets, and therefore, its cultivation is common in many countries, including Iran. Among the common diseases of bean plants, three bacterial diseases common bacterial blight (Xanthomonas axonopodis pv. phaseoli, Xap), bacterial wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens, Cff), and halo blight (Pseudomonas savastanoi pv. phaseolicola, Psp) are considered the most significant seed-borne diseases. These diseases have caused substantial damage to bean fields in Iran in recent years. Due to the uniform production and distribution process of edible and seed beans in Iran, these bacterial pathogens can potentially spread from contaminated areas to other bean-growing regions. Therefore, ensuring that bean seeds intended for cultivation are free of these pathogenic bacteria is crucial for managing the three diseases: common bacterial blight, bacterial wilt, and halo blight. In this regard, developing a rapid, cost-effective, and easy-to-use method for detecting Xap, Cff, and Psp in bean seeds was the primary objective of this study. For this purpose, after confirming the morphological, pathogenicity, physiological, and biochemical characteristics, three selected isolates—Xap58, Cff19, and Psp26—belonging to the Xap, Cff, and Psp pathovars were chosen for molecular detection tests via PCR. Initially, amplification of the expected DNA fragments of these bacteria was confirmed using specific primer pairs: X4c/X4e (730 bp), Cff-for2/Cff-rev4 (306 bp), and P5.1/P3.1 (502 bp). Subsequently, through serial dilution of bacterial DNA and the use of specific primers combined with gradient-PCR, the optimal conditions for a 20-µl reaction were determined to include 20 ng/µl DNA concentration, 5 pmol primer concentration, and an annealing temperature of 57°C for the amplification of the target DNA fragments in PCR, duplex PCR, and triplex PCR assays. By serially diluting bacterial suspensions (10⁷ cfu/ml) of Xap58, Cff19, and Psp26, extracting their DNA via the boiling method, and conducting PCR assays, it was found that all three isolates could be detected up to a dilution of 10⁻³ in individual PCR and duplex PCR assays and up to 10⁻¹ in triplex PCR assays. To eva‎luate the sensitivity of the three PCR methods for pathogen detection, white bean seeds were artificially inoculated using immersion in individual, dual, and triple bacterial suspensions of Xap58, Cff19, and Psp26. After air-drying the seeds, at intervals of 0, 2, 4, 8, 16, 32, 64, and 128 days, two seeds from each treatment were immersed in nutrient broth, incubated on a shaker at 150 rpm for 18 hours, and then subjected to DNA extraction. The results of PCR reactions using these samples demonstrated that all three pathogens (Xap58, Cff19, and Psp26) could be detected on bean seeds for up to 128 days using individual PCR, duplex PCR, and triplex PCR methods. Further optimization of this triplex PCR method can facilitate its use for detecting bean seed contamination. Keywords: Common bacterial blight, bacterial wilt of beans, halo blight of beans, duplex PCR, triplex PCR

مسعود بهار

بهرام شريف نبي

صادق زارعي , حميدرضا عشقي زاده