A total of seventeen isolates of Aspergillus flavus from maize were collected from different agro-ecological zones of Tamil Nadu, India. The isolates were tested for their ability to produce aflatoxin B 1 (AFB 1 ) in vitro by indirect competitive enzyme-linked immunosorbent assay (ELISA). The amount of AFB 1 produced by the isolates of A. flavus ranged from 1.9 to 206.6 ng/ml. Among the various isolates of A. flavus, the isolate AFM46 produced the highest amount of AFB 1 . DNA was extracted from A. flavus isolates and their molecular variability was investigated by using restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) regions of ribosomal DNA. PCR amplification with ITS1 and ITS4 primers resulted in the amplification of a product of approximately 600 bp. Digestion of the PCR products with the restriction enzymes EcoRI, HaeIII and TaqI produced fragments of different sizes. Analysis of the genetic coefficient matrix derived from the scores of RFLP profiles showed that minimum and maximum per cent similarities among the tested A. flavus strains ranged from 0 to 88%. Cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA) clearly separated the isolates into five groups (group I-V) confirming the genetic diversity among the A. flavus isolates from maize.
The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.
The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.
Integrated management of aflatoxin B 1 contamination of groundnut (Arachis hypogaea L.)with Burkholderia sp. and zimmu (Allium sativum L. Allium cepa L.) intercroppingThe efficacy of intercropping of the medicinal plant zimmu (Allium sativum L. )Allium cepa L.) and an antagonistic bacterium Burkholderia sp. strain TNAU-1 was evaluated separately and in combination for control of Aspergillus flavus infection and aflatoxin B 1 contamination in groundnut (Arachis hypogaea L.) under field conditions. The Burkholderia sp. strain TNAU-1 inhibited the growth of all the toxigenic and non-toxigenic isolates of A. flavus in vitro and the inhibition zone ranged from 3.1Á11.3 mm. A talc-based powder formulation of the Burkholderia sp was developed for field application. When groundnut seeds were treated with the formulation of Burkholderia sp., significant increases in root length, shoot length and seedling vigor were observed. Zimmu intercropping (once in every three rows of groundnut) alone significantly reduced the A. flavus infection and aflatoxin contamination in groundnut. Seed treatment at 10 g/kg or soil application at 2.5 kg/ha on 30, 45, 60 days after sowing with the formulation of Burkholderia sp. also resulted in significant reduction in the infection by A. flavus and aflatoxin B 1 contamination in kernels. The maximum percentage reduction in aflatoxin B 1 content was achieved by the combination of zimmu intercropping, seed treatment and soil application of Burkholderia sp. Seed treatment followed by soil application with the formulation of Burkholderia sp. recorded the highest pod yield in both the field trials. Zimmu intercropping or seed treatment or soil application with formulation of Burkholderia sp. significantly reduced the population of A. flavus in the soil. Application of Burkholderia sp. through seed or soil resulted in high colonization of Burkholderia sp. in rhizosphere soil, and the rhizosphere population increased with increase in the age of the crop. A colony PCR assay was developed using primers made to Burkholderia sp. specific sequence of the ribosomal DNA for accurate detection and confirmation of Burkholderia sp.
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