| The interaction between Ustilago esculenta and Zizania latifolia produces swollen structures on the upper part of plants,which we called as the jiaobai.Jiaobai is an important aquatic vegetable.Fungus survives within the host in the form of mycelium for a long-term period,and transferred to new plants with the vegetative propagule.Sometimes,a large number of teliospores produce in swollen structures of plants,which are called as the huijiao.Although teliospores germination can produce basidiospores,a large number of observations suggest that basidiospores with different mating type genes can not fuse into dikaryon mycelium,failing to infect the host.A few studies showed occasionally,basidiospores with different mating type genes can fuse into mycelium,but no sufficient evidence was confirmed.Therefore,in this study,different mating type genes in a large of basidiospores were determined and artificially synthesized mating type gene-coding proteins were evaluated.1.Mating gene type expression analysis of U.esculentaAnalyze of the expression of MFA genes and PRA genes of U.esculenta by local blast shows that all genes related to mating type were expressed.There are only a2 and a3 in U.esculenta from jiaobai plants,and only a1 and a3 in U.esculenta from huijiao plants in cv Zhejiao 2.The type of PRA is the identification basis of mating type.2.Fungal isolation and mating type genes determinationSix isolates were isolated from huijiao tissue.Fifty single-spore isolates were isolated from basidiospores produced by teliospores.Similarly,four isolates were isolated from jiaobai tissue.Forty-two single-spore isolates were isolated from basidiospores produced by teliospores from plant tissue treated with low temperature.Mating type genes were determined by PCR amplification.Analysis of mating type genes demonstrate that five isolates were a1 locus(MFA1.2-PRA1-MFA1.3)and 45 isolates were a3 locus(MFA3.2-PRA3-MFA3.1)from huijiao samples.The a1/a3 rate was 1:9.Six isolates with dikaryon contained a1 locus(MFA1.2-PRA1-MFA1.3)and a3 locus(MFA3.2-PRA3-MFA3.1).In jiaobai samples,analysis of mating type genes displayed that five isolates are a2(MFA2.3-PRA2-MFA2.1)locus and 37 isolates are a3(MFA3.2-PRA3-MFA3.1)locus.The rate of a2/a3 is 1:7.2.Four isolates with dikaryon contained a1(MFA1.2-PRA1-MFA1.3)and a3(MFA3.2-PRA3-MFA3.1).Based on the results of mating type analysis,eight isolates with a1 and a3 loci were selected from huijiao samples and they were grouped into 16 combinations for demonstration of pheromone activity with cross streaking method.The test results showed that no mycelia were observed in all combinations.Similarly,ten isolates with a1 and a3 loci were selected from jiaobai or huijiao samples and they were grouped into 25 combination but pheromone activity was demonstrated by mixed basidiospores with different pheromone loci.The test results displayed that no mycelia were observed in all combination.3.Activity testing of synthetic pheromone amino acid sequencesThe mating type genes of U.esculenta has highest homology with S.reilianum by gene analysis.Based on the pheromone gene,amino acid sequences(17 amino acids)and protein sequence(38 amino acids and 40 amino acids)of the modified pheromone sequences were synthesized artificially.Four single-spore isolates were selected,they were grouped into four combination including H9(a1)and H10(a3),H9(a1)and J4(a3),H10(a3)and J24(a2)as well as J4(a3)and J24(a2)and then added two kind of synthetic pheromone(17 amino acids)solution for determination of pheromone activity.Similarly,four combinations were added synthetic protein pheromone solution for each treatment,respectively for pheromone activity.However,the results showed that synthetic pheromones could promote the mycelium-like or fuzzy filaments in several combination.However,microscopic observation displayed that these fuzzy filaments were made up of basidiospores.DAPI nuclear staining showed that these fuzzy filaments were single nucleus. |
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