Molecular and Biochemical Studies on Some Aflatoxin-Producing Fungi in Egypt
Heba Yousef Rizk Mohamed;
Abstract
Aflatoxin producing fungi are serious plant pathogens attacking several economical host plants results in remarkable defect in economic quality.
Aspergillus flavus is imperfect filamentous fungus that is opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals and insects. It has a worldwide distribution in the environment. Although it is universally found in air, soil, dust and water. So, it cause many destructive diseases of many agricultural crops such as maize (corn), cotton, groundnuts (peanuts) and stored grains, which lead to quantitative and qualitative losses of yield and produce the most toxic and potent carcinogenic metabolites such as aflatoxins and other mycotoxins.
This study was aimed to determine the biochemical and molecular variability within various isolates of Aspergillus flavus from peanut and corn considering some factors such as geographical distribution and the difference of plant hosts. Make early detection and quantification of Aspergillus flavus and aflatoxins producing ability in two important agricultural crops (corn and peanuts) and soil and to evaluate various approaches for management of Aspergillus flavus using hydrogen peroxide and gallic acid, which may lead to improvement in human health, food safety and agricultural economy. SUMMARY
131
To achieve this aim, the following approaches were fulfilled:
1. Agricultural crops samples were collected from various geographical areas.
2. Fungal isolation, culturing and identification of A. flavus by morphological aspects.
3. Screening of A. flavus isolates for aflatoxins production.
4. Quantitative estimation of aflatoxins concentration.
5. Molecular Characterization of A. flavus Isolates including:
Random Amplified Polymorphic DNA Method (RAPD)
Internal Transcribed Spacer (ITS) Analysis
Conventional Specific PCR
6. Quantitative estimation of A.flavus by Real Time PCR (qPCR) reaction using the Light Cycler instrument.
7. Effect of hydrogen peroxide and gallic acid on growth rate, catalase activity and aflatoxins production of A. flavus.
The results obtained can be summarized as follows:
1) Pathogenic isolates of Aspergillus flavus from corn and peanut were isolated, purified and identified.
2) Aspergillus flavus isolates were screened for the ability of aflatoxins production and data showed that all tested isolates were aflatoxin-producing isolates.
3) Quantification of aflatoxins levels in A. flavus isolates using HPLC showed that aflatoxigenic fungi isolates have different ability to aflatoxin production. In general isolates of A. flavus
SUMMARY
132
have isolated from peanut have ability to aflatoxin production more than those isolated from corn. Aflatoxin B1 was produced in all isolates, whereas production of aflatoxin G2 was more restricted. Gharbia’s corn isolate recorded the highest rate of aflatoxin content while in Beni Suef’s peanut isolates (Isolate No. 23) recorded the highest content of aflatoxin. On the other hand, Gharbia’s isolate recorded the highest amount of total aflatoxin and aflatoxin B1 followed by Beni Suef isolate (Isolate No. 23).
4) Analysis of DNA fingerprinting indicated that there was a considerable genetic variation within the A. flavus isolates isolated from corn and peanut. Since, in A. flavus the results showed that primers 1, 3 and 5 were able to distinguish between the isolates from Upper and Lower Egypt governorates. Since the Upper Egypt, isolates fell in one similarity group while isolates of the Lower Egypt governorates fell in another similarity group. When eight primers were used to detect the genetic variability among Aspergillus flavus isolates from peanut, considerable degrees of genetic variability were observed. Some of the used primers placed a single isolate in a separate clustering group giving an indication of the high genetic variability between this isolate and the others as shown in primers (1, 5, 6, and7) with similarity percentage 71.73%, 42.29%, 79.62% and 64.55%. Regarding geographic isolation, cluster analysis using the UPGMA method for thirty-five isolates of Aspergillus flavus isolated from corn and peanut using primer (3 and 5) clearly separated the isolates into two main clusters. The first main cluster include A. flavus isolates
SUMMARY
133
isolated from corn and second main cluster include A. flavus isolates isolated from peanut with similarity percentage 63.76 % and 5.10 % respectively.
Aspergillus flavus is imperfect filamentous fungus that is opportunistic pathogen causing invasive and non-invasive aspergillosis in humans, animals and insects. It has a worldwide distribution in the environment. Although it is universally found in air, soil, dust and water. So, it cause many destructive diseases of many agricultural crops such as maize (corn), cotton, groundnuts (peanuts) and stored grains, which lead to quantitative and qualitative losses of yield and produce the most toxic and potent carcinogenic metabolites such as aflatoxins and other mycotoxins.
This study was aimed to determine the biochemical and molecular variability within various isolates of Aspergillus flavus from peanut and corn considering some factors such as geographical distribution and the difference of plant hosts. Make early detection and quantification of Aspergillus flavus and aflatoxins producing ability in two important agricultural crops (corn and peanuts) and soil and to evaluate various approaches for management of Aspergillus flavus using hydrogen peroxide and gallic acid, which may lead to improvement in human health, food safety and agricultural economy. SUMMARY
131
To achieve this aim, the following approaches were fulfilled:
1. Agricultural crops samples were collected from various geographical areas.
2. Fungal isolation, culturing and identification of A. flavus by morphological aspects.
3. Screening of A. flavus isolates for aflatoxins production.
4. Quantitative estimation of aflatoxins concentration.
5. Molecular Characterization of A. flavus Isolates including:
Random Amplified Polymorphic DNA Method (RAPD)
Internal Transcribed Spacer (ITS) Analysis
Conventional Specific PCR
6. Quantitative estimation of A.flavus by Real Time PCR (qPCR) reaction using the Light Cycler instrument.
7. Effect of hydrogen peroxide and gallic acid on growth rate, catalase activity and aflatoxins production of A. flavus.
The results obtained can be summarized as follows:
1) Pathogenic isolates of Aspergillus flavus from corn and peanut were isolated, purified and identified.
2) Aspergillus flavus isolates were screened for the ability of aflatoxins production and data showed that all tested isolates were aflatoxin-producing isolates.
3) Quantification of aflatoxins levels in A. flavus isolates using HPLC showed that aflatoxigenic fungi isolates have different ability to aflatoxin production. In general isolates of A. flavus
SUMMARY
132
have isolated from peanut have ability to aflatoxin production more than those isolated from corn. Aflatoxin B1 was produced in all isolates, whereas production of aflatoxin G2 was more restricted. Gharbia’s corn isolate recorded the highest rate of aflatoxin content while in Beni Suef’s peanut isolates (Isolate No. 23) recorded the highest content of aflatoxin. On the other hand, Gharbia’s isolate recorded the highest amount of total aflatoxin and aflatoxin B1 followed by Beni Suef isolate (Isolate No. 23).
4) Analysis of DNA fingerprinting indicated that there was a considerable genetic variation within the A. flavus isolates isolated from corn and peanut. Since, in A. flavus the results showed that primers 1, 3 and 5 were able to distinguish between the isolates from Upper and Lower Egypt governorates. Since the Upper Egypt, isolates fell in one similarity group while isolates of the Lower Egypt governorates fell in another similarity group. When eight primers were used to detect the genetic variability among Aspergillus flavus isolates from peanut, considerable degrees of genetic variability were observed. Some of the used primers placed a single isolate in a separate clustering group giving an indication of the high genetic variability between this isolate and the others as shown in primers (1, 5, 6, and7) with similarity percentage 71.73%, 42.29%, 79.62% and 64.55%. Regarding geographic isolation, cluster analysis using the UPGMA method for thirty-five isolates of Aspergillus flavus isolated from corn and peanut using primer (3 and 5) clearly separated the isolates into two main clusters. The first main cluster include A. flavus isolates
SUMMARY
133
isolated from corn and second main cluster include A. flavus isolates isolated from peanut with similarity percentage 63.76 % and 5.10 % respectively.
Other data
| Title | Molecular and Biochemical Studies on Some Aflatoxin-Producing Fungi in Egypt | Other Titles | دراسات جزيئية وكيميائية حيوية على بعض الفطريات المفرزة للافلاتوكسين فى مصر | Authors | Heba Yousef Rizk Mohamed | Issue Date | 2014 |
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