| Helminthosporioid fungi caused many important diseaseson wheat,corn and other gramineae.These diseases can cause great losses to the yield of wheat and maize and seriously threaten China's food security.The mainly diseases were caused by Helminthosporioid fungi on wheat and corn are corn northern leaf bligt,southern leaf blight of corn,northern corn leaf spot,Curvularia leaf spot of maize and wheat root rot disease.These diseases are not easy to identify in the early stage of disease,and their symptoms are similar.The conventional pathogen identification and detection techniques need to spend a lot of time and energies.So,fast,accurate and early detection of five pathogens will play a great role in the prevention and control of the five diseases.In this study,we designed two pairs of the specific primes and utilized three pairs of the specific primes which they had been reported to establish the molecular detection system of Helminthosporioid fungi disease in wheat and maize.The main results are as follows.?1?Molecular detection of corn southern leaf blight and northern corn leaf spot pathogensThe species-specific PCR primes X-EF-F/X-EF-R and Y-EF-F/Y-EF-R for B.maydis and B.zeicola were designed respectively after multiple sequence alignment of part genome sequence in elongation factor 1? of B.maydis and B.zeicola with the other allied species of Bipolaris.A 205 bp single DNA fragment from the isolates of B.maydis was amplified and a 137 bp single DNA fragment from the isolates of B.zeicola was also amplified,no product was amplified with DNA from other fungi.1pg·?l-1B.zeicola genomic DNA and 10 pg·?l-1 B.maydis genomic DNA were detected by species-specific primers.0.1 pg·?l-1B.maydis genomic DNA were detected by nest PCR.Total DNA was extracted from the diseased tissue which come from maize leaves inoculated with B.zeicola and B.maydis,the identical 137 bp and 205 bp bands were amplified.The maize leaves were inoculated in the field with spore suspension,the pathogen can be detected from symptom-free leaves in third days.Infected corn samples were collected randomly from Zhenzhou,Anyang,Xiping,Huaiyang and Tanghe in Henan Province.Total DNA from maize leaves were detected by PCR using two pairs of specific primers.The results showed that B.maydis was detected in Zhengzhou and B.zeicola was not detected,and the two pathogens were not detected in other regions.?2?Multiple detection of some important diseases in maize leaves.A pair of primers were designed according to the elongation factor 1? gene of B.maydis that is X-EF-F/X-EF-R.At the same time,take Exserohilum turcicum specific primer JB586/JB589 and Curvalaria lunata specific primer ClgD2/ClgD3 to established a multiple PCR detection for 3 pathogens.The experimental results showed that three purpose pathogens could be detected from the mixed DNA by using the three pairs of specific primers of X-EF-F/X-EF-R,JB586/JB589 and ClgD2/ClgD3.A pair of primers were designed according to the elongation factor 1? gene of B.zeicola that is Y-EF-F/Y-EF-R.At the same time,take E.turcicum specific primer JB586/JB589 and C.lunata specific primer ClgD2/ClgD3 to establish a multiple PCR detection for 3 pathogens.The experimental results showed that three purpose pathogens could be detected from the mixed DNA by using the three pairs of specific primers of Y-EF-F/Y-EF-R,JB586/JB589 and ClgD2/ClgD3.?3?Preliminary study on Tup1 gene function of B.sorokiniana.Wheat root rot caused by B.sorokiniana can cause significant losses in wheat yield.In recent years,with the wheat straw returning in the field,the pathogens have been annual accumulated.Wheat root rot has expanded.In view of the fact that wheat root rot are difficult to control and could cause great losses in the wheat yield,it is important to study the pathogenic mechanism of this fungus.In this study,we taken the core gene Tup1 of the repressor as a breakthrough point,and the Tup1 gene was knocked out by the homologous recombination method.The function of the B.sorokiniana Tup1 gene was preliminarily explored by the reverse genetics method.The deletion mutant was obtained by the gene knockout strategy combined with the reconstructive gene and the homologous recombination,and was further verified by Southern hybridization.By comparing with the wild type,the growth rate of the knockout mutant was obviously slowed down,the knockout mutant could not produce spore and mycelium deformity,and it was very sensitive to the pressure screening of hydrogen peroxide and KCl.The knockout mutants was inoculated on wheat and barley leaves and the base of the stem,it could not infect the wheat and barley.The pathogenicity of the mutant on wheat and barley was significantly decreased by inoculation.The above results showed that the Tup1 gene of B.sorokiniana was involved in controlling the growth and development of the B.sorokiniana,and influencing its pathogenicity to wheat and barley. |
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