السيطرة الاحيائية واللااحيائية على نمو الفطر Aspergillus flavus والتعبير الجيني للجينين aflDو aflR وانتاج الافلاتوكسين B1 == Biotic and Abiotic Control on Aspergillus flavus Growth, aflD and aflR Expression and Aflatoxin B1 Production
Author name:
لبيد عبد الله نجم السعد
Supervisor name:
عدنان عيسى البدران | سامي عبد الرضا الجميلي
General topic:
Biology
Specific topic:
Biotechnologies
Degree:
Doctorate
University:
University Of Basrah - College Of Education For Pure Sciences
Language:
English
University location:
Basrah
First pages:
24T3569 - p.pdf
Abstract:
Fifteen isolates of Aspergillus flavus (AFL1 - AFL15) were isolated from multiple sources included soil, compost, seeds, fruits, feed and air. The isolates were identified morphologically then confirmed by molecular techniques. Only thirteen isolates were confirmed positively (AFL1 - AFL5 and AFL8 - AFL15) while two of them were not. A phylogenetic analysis was made which revealed that the isolates grouped according to their source of isolation. The aflR profile and phenotypic AFB1 production confirmed that all thirteen isolates posses the ability to produce AFB1 with significant differences (P ≤ 0.05) among them. Growth rate profile was performed under 30ºC which showed significant differences (P ≤ 0.05) among isolates. According to the above tests, A.flavus AFL14 was selected to be the experimental isolate for the rest of experiments in this study. Comparing to A.flavus NRRL3357 type strain, the impact of temperature, water activity aw and isolate factors was examined to determine the limits and optimum ecological conditions of growth of A.flavus AFL14. The results displayed that the best growth rate was (7.217 mm/day) at 0.98 aw where the lowest was (4.069 mm/day) at 0.9 aw with no growth at 0.85 aw while the impact of temperature demonstrated by outweigh of growth rate at 35ºC (6.201 mm/day) followed by 30ºC (5.272 mm/day) which exceeded 25ºC and 40ºC (4.604 and 4.051 mm/day), respectively.Bacillus subtilis (isolates : BSS1, BSS2, BSS3, BSS4 and BSW) and Pseudomonas fluorescens (isolates : PFMst and PFDL) were elected to be used as biocontrol agents. All the isolates were passed the biochemical identification tests moreover, BSS4 and BSW isolates of B.subtilis and PFMst and PFDL isolates of P.fluorescens were confirmed molecularly which used later in all biocontrol experiments in this study. The ability ofbiocontrol agents to inhibit fungal growth was investigated and the resultsshowed significant inhibition impact represented by significant outweighof B. subtilis BSS4 (99%) on the rest of B. subtilis isolates followed by P.fluorescense PFDL and PFMst (92.29 and 86.19) %, respectively, with nosignificant differences (P ≤ 0.05) among them. The extra - cellular abilityof biocontrol agents to degrade AFB1 showed a high degradation abilityIpreformed by B. subtilis BSS4, BSW isolates and P. fluorescence PFMst,PFDL isolates (100, 100, 97.805 and 97.396%), respectively. Thedegradation residues administrated to rats to determine their effect onbiosystems, the blood parameters showed a significant reduction ofWBC, HB, RBC, and P.C.V while there was a significant increase inUrea, Glutamic pyruvate transaminase (GPT) Glutamic oxaloacetictransaminase (GOT) and MCV in AF+DMSO treatment comparing tothe negative and positive control. Water activity and biocontrol factorswere examined to control aflD and aflR expression and phenotypic AFB1production. When the experiment held using Nutrient Agar medium, asignificant inhibition of aflD and aflR expression (P=0.000 and 0.003),respectively performed by BSW at 0.94 aw comparing to 0.98 aw whileno significant inhibition was observed in the rest of the treatments. Allbiocontrol agents revealed high significant reduction of aflD and aflRexpression (P≤0.001) in each water activity level separately whichconfirmed by HPLC results. The phenotypic results showed that 0.94 awexceeded 0.98 aw in AFB1 reduction (2011 and 4280 ng/gm),respectively which agreed with gene expression results. When MaizeMeal Agar used as a medium, aflD expression presented a highsignificant increase at 0.98 aw relating to the NM level of each individualtreatment (P= 0.000 - 0.03) except PFDL+AFL14 which showed nosignificant aflD expression between both levels. aflR expression revealeda high significant reduction (P= 0. 000) caused by 0.98 PFDL+AFL14and 0.98 BSW+AFL14, respectively, while no significant differenceswere observed in the rest of the treatments or control. The HPLC resultsdisplayed a high significant reduction of AFB1 at 0.98 aw (8447 ng/gm)compared to (219000 ng/gm) at NM aw. The biocontrol agents caused ahigh significant reduction for both aflD and aflR expression (P=0. 000 - 0.043) under each individual water activity level while, HPLC resultsshowed non significant low reduction to the AFB1 performed by BSS4and BSW (25160 and 44790 ng/gm), respectively, followed by asignificant increase in AFB1 amount caused by PFMst and PFDL(267600 and 184100 ng/gm), respectively
