Detection and Analysis of Magnaporthe oryzae Avirulence Genes AVR-Pib,AVR-Pik and AvrPiz-t in Heilongjiang Province

【Objective】The objective of this study is to investigate the distribution and variation mechanism of avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t in Magnaporthe oryzae strains from different regions and years in Heilongjiang Province, to understand the pathogenic phenotypes of different avirulence gene alleles, and to provide a reference for utilization and distribution of resistance cultivars in Heilongjiang Province.【Method】Based on the avirulence gene sequences published in NCBI, specific primers were designed to amplify full length and the coding sequence (CDS) regions of three genes, respectively. From 2016 to 2017, 335 M. oryzae strains in different regions of Heilongjiang Province were collected and isolated, and their DNA was PCR-amplified using avirulence genes primers and analyzed by agarose gel electrophoresis. The PCR products with different band patterns and from representative strains of different regions were selected for sequencing. The sequencing results were compared with the corresponding avirulence gene sequences for base and amino acid. The pathogenic phenotype of M. oryzae strains with different variant types was determined based on the rice resistance to single-gene lines.【Result】The specific bands of AVR-Pib, AVR-Pik and AvrPiz-t were detected in PCR detection and appeared in different distribution frequencies and mutation types, indicating that these 3 avirulence genes were all distributed in Heilongjiang Province. The average amplification frequency of the 3 avirulence genes was 75.52%, 87.16% and 85.67%, respectively. Among them, 4 types of band (bandless, high band, mid to high band and low band) of AVR-Pib were detected by electrophoresis analysis and 5 variant types AVR-Pib (1-1, 1-2, 2, 3, 3-1) were detected by PCR product sequencing. The genotypes AVR-Pib-1-1, AVR-Pib-1-2, AVR-Pib-2 and AVR-Pib-3-1 are newly discovered variant types, of which genotypes AVR-Pib-1-1 and AVR-Pib-1-2 are insertions of transposon Pot2 but with different insertion sites. The genotype AVR-Pib-2 has a small fragment insertion in the upstream of CDS region. The genotype AVR-Pib-3-1 base sequence has 4 differences from the original sequence, namely 32 (C/G) 35 (T/A) 36 (T/A) 38 (T/A), and the amino acid translation was terminated prematurely. Pathogenic analysis showed that except for the normal genotype AVR-Pib-3, the other alleles lost their avirulence functions. Seven AVR-Pik alleles (D, A, B, C, E, F, F2) were detected after PCR product sequencing, and the alterations in the nucleotide sequences of these alleles all resulted in amino acid missense mutations. The 7 AVR-Pik alleles have been reported previously. The avirulence gene AvrPiz-t was analyzed by electrophoresis and sequencing of PCR products, and 2 types of band (high band and normal band type) and 4 genotypes of AvrPiz-t (A, B, C, D) were revealed. Among them, AvrPiz-t-A is the original genotype, while AvrPiz-t-B has a base A insertion at position 191, causing premature termination of amino acid translation. Genotype AvrPiz-t-C is a newly discovered allelic type, characterized by the presence of a nucleotide variation at position 17 (T/C) and the insertion of base C at position 19 compared with type A, leading to the frameshift mutation and premature translation termination. The high band type avirulent genotype AvrPiz-t-D was sequenced and verified as having an insertion of the Pot3 transposon. Rice single-gene lines infection showed that the strains with AvrPiz-t-A were avirulent to Piz-t line due to Piz-t recognition, whereas the strains with AvrPiz-t (B, C, D) were virulent to Piz-t line due to lost the ability recognized by Piz-t.【Conclusion】The avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t of M. oryzae in Heilongjiang Province are widely distributed and the types of variation are abundant. The results of this study can provide a reference for breeding and popularizing rice cultivars with corresponding disease-resistance genes.     

4种花椒提取物对玉米象成虫熏蒸活性的研究

在30℃下采用广口瓶密闭熏蒸法熏蒸48h和72h,测定青椒精油、红椒精油、一级麻素及二级麻素时玉米象Sitophilus zeamais Motschulsky成虫的熏蒸致死作用及LC50值。结果表明LC50值大小顺序为一级麻素〉二级麻素〉红椒精油〉青椒精油．进一步研究熏蒸时间和温度时青椒精油及红椒精油熏蒸活性的影响,结果表明熏蒸时间越长,LC50值越小;温度越高,校正死亡率越大,表明2种花椒精油的持效期较长,在高温下能更好地发挥药效。     

福建省水稻霜霉病诊断和病原鉴定

水稻霜霉病诊断特征为植株矮缩,叶片黄化、产生黄褐色条斑.利用显微镜检查染色病组织内的病原菌藏卵器和卵孢子,通过观察病原菌的培养性状、形态特征和致病性测定,该病原菌被鉴定为大孢指疫霉水稻变种.     

晚稻细菌性条斑病流行趋势预测模型的研究

本文在分析万安县１９８９～１９９４年晚稻细菌性条斑病发生的历史资料和相关气象资料的基础上,应用灰色关联度选择预报因子,根据模糊综合评判原理组建模型,预报晚稻细菌性条斑病的流行趋势,经回测符合率达１００％,并在１９９５年、１９９６年和１９９７年应用该模型指导万安县晚稻细菌性条斑病的防治,取得了显著的经济效益。     

米根霉HZS6生产高光学纯度L-乳酸发酵条件的研究

通过研究和控制初始发酵条件,比较产物L-乳酸、D-乳酸组成变化,得出提高L-乳酸光学纯度的米根霉发酵生产条件：在pH6．0、温度34℃、初始葡萄糖浓度100g／L、NH。NO3 2g／L、添加1．5g／L L-乳酸的适合条件下,米根霉HZS6能够发酵生产光学纯度99％以上的L-乳酸,其转化率达到80％,终浓度为81．3g／L。     

根癌农杆菌介导的稻瘟病菌近等菌系的构建与初步应用*

 利用根癌农杆菌C58C1介导转化1株致病力极弱的野生型稻瘟病菌菌株CY2,成功地获得3000多个转化子,混合接种于携带25个不同抗病单基因的水稻近等基因系上,获得4株单胞致病突变体;继代培养和PCR检测表明,这些突变体均为T-DNA插入CY2的后代,而非自发突变或外源菌株污染。将4株突变体与实验室已保存的20株致病突变体分别回接水稻近等基因系,推断其携带的致病基因类型,结果表明突变体分别针对13个抗性单基因的18个单基因系发生了突变,其中针对Pi-a,Pik-s,Pi-sh单基因系表现出较高的侵染频率。由于这些突变体均来自同一个菌株,仅致病性存在差异,属于近等菌系,利用其分析出102个水稻品种中的34个品种的抗病基因组成,其中,抗病基因Pik-s,Pi-a,Pi-sh,Pi-19(t)分布频率较高,分别达70.59%,61.76%,52.94%和44.12%,且带有这类基因的糯稻品种多于杂交水稻。     

曲霉型豆豉用高产蛋白酶菌种的选育

从曲霉型阳江豆豉曲中筛选出5株菌落形态不同的曲霉(Aspergillus spp.),通过纯种发酵制备豆豉曲,测定了豆豉曲中碱性蛋白酶和中性蛋白酶的活性,从中筛选出1株蛋白酶活性较高的菌种,并对该曲霉进行ITS r DNA序列分析,发现该曲霉与Aspergillus oryzae strain P6B2(JX429926)的同源性最高,故鉴定为米曲霉(Aspergillus oryzae).以该米曲霉为出发菌株,通过紫外线诱变和硫酸二乙酯化学复合诱变处理,选育得到优良高产蛋白酶活性的突变株,其中性蛋白酶酶     

部分云南粳稻品种的抗瘟性分类

 根据云南部分粳稻品种对7个菌系的反应型，将596个粳稻品种划分为35群。借助单基因鉴别品种，推断了50个品种的基因型。应用致病谱广的北方小种，筛选出一批新抗源，满足抗病育种对抗源的需求，也为系统的抗病基因分析奠定物质基础。     

石菖蒲提取物对玉米象的驱避和触杀作用

测定了石菖蒲4种溶剂提取物对玉米象成虫的驱避和触杀作用。结果表明:石菖蒲的4种溶剂提取物对玉米象成虫均有很好的驱避效果,60 h驱避等级均达到Ⅲ和Ⅳ级。4种溶剂的提取物均对玉米象成虫有明显的触杀作用,毒力回归方程及LC50分别为:无水乙醇提取物:y=4.37 2.13x,1.97 mg/cm2;丙酮提取物:y=4.53 1.42x,2.14 mg/cm2;乙酸乙脂提取物:y=4.52 2.10x,1.69 mg/cm2;石油醚提取物:y=4.23 2.32x,2.15 mg/cm2。