江西稻瘟病菌稻巨座壳致病性分化年度动态变化

为明确江西水稻种植区稻巨座壳菌（稻瘟病病菌）的年际变化规律,本研究采用7个中国鉴别寄主与30个单基因鉴别品系两套鉴别寄主分别鉴定分析了2006-2018年间从江西37个水稻主要种植县市分离的1 161个稻瘟病单孢菌的生理小种、致病力、致病类型与无毒基因型等。研究结果表明,江西稻瘟病菌可以分成7群49个生理小种,其中ZA、ZB、ZC群为优势种群,ZB13为优势小种,出现频率为18.00%,以毒性较强的强致病力菌株为主;江西稻瘟病菌在生理小种构成、优势小种、致病力年际变化方面均具有3-5年的周期性;江西历年稻瘟病菌的致病类型较为丰富且存在年度差异,菌株致病类型占各年度总菌株数的82.79%-98.21%,优势致病型菌株占当年总菌株的3.57%-5.77%;历年稻瘟病菌的无毒基因个数为24-29个,其中Avr-Pizt、Avr-Piz5、Avr-Pik、Avr-Pik(C)在历年供试菌株中出现频率较高,说明与之相对应的抗瘟基因在江西抗病育种与抗性品种布局中具有较好的应用价值。     

江苏省稻瘟病菌有性态的研究

用标准菌株对1997－1999年在江苏吴江市、宜兴市、通州市、高邮市和赣榆县采集的325个稻瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为22．77％,可育率仅为7．08％。不同年份、不同地区采集的稻瘟病菌菌株的性亲和力和交配型有较大的差异,三年的交配率分别为26．61％、8．26％和33．64％;通州地区和赣榆地区菌株的交本相对较高,分别为26．15％和25．42％,宜兴地区菌株的交配率较低,只有15．38％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,1997年29个可交配菌株中有21个菌株表现为MAT1－2交配型,而1999年36个可交配菌株均为MAT1－1交配型。用江苏省稻瘟病菌的可育菌株进行互交,25个组合中只有6个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

云南疣粒野生稻稻瘟病抗性

野生稻(Oryza rufipogo)保存有许多栽培稻(O. sativa)不具备或已经消失的优异基因资源, 是扩大栽培稻遗传背景、改良产量与品质、提高抗病虫害及抗逆境能力的重要基因库。疣粒野生稻(O. meyeriana)是中国3种野生稻资源之一, 主要分布在云南。为进一步了解其稻瘟病抗性, 首先利用来自不同稻作区的稻瘟病菌株, 通过注射接种法对疣粒野生稻进行系统的稻瘟病抗性鉴定, 发现疣粒野生稻对接种的所有稻瘟病菌株都感病。进一步采用3'/5' RACE方法, 从疣粒野生稻中克隆了水稻同源基因Pid2和Pid3, 并构建过表达转基因株系对基因功能进行了研究。结果表明, Pid2和Pid3与疣粒野生稻中同源基因间在DNA和氨基酸水平上有较大的序列差异, 过表达转基因的日本晴植株对稻瘟病菌的敏感性与对照相似。推测疣粒野生稻在自然接种条件下, 表现出的抗稻瘟病表型很可能是其旱生叶片结构特征形成了对稻瘟病菌侵染的天然屏障。对控制疣粒野生稻这一类性状基因资源的挖掘和利用, 有利于优良抗性水稻品种的培育。研究结果为疣粒野生稻的研究利用提供了新信息和新思路。     

转基因改良水稻抗稻瘟病的策略及其进展

稻瘟病是由子囊菌引起的广泛发生在世界各水稻产区的主要真菌病害。由于病原菌致病性的高度分化,使得对稻瘟病很难控制和防治。长期实践证明,培育抗病品种是稻瘟病抗病育种的主要目标。随着基因工程的发展,利用转基因技术导入外源基因改良稻瘟病抗性已成为一条新途径。现有研究表明,通过某些抗病基因、抗真菌蛋白基因、杀菌肽基因的克隆和转育,可以培育出获得对稻瘟病广谱抗性的水稻品种(系)。     

进境乌克兰玉米中夹杂向日葵间座壳菌的鉴定

自进境乌克兰玉米中夹杂的向日葵籽上,采用常规平板分离法获得1株疑似向日葵间座壳菌Diaporthe helianthi的菌株76782-5。形态特征观察结果表明,该菌株在PDA上产生分生孢子器和大量β型分生孢子,但未见有性阶段。经rDNA ITS和β-tubulin基因序列比对分析,发现该菌株与GenBank中多个向日葵间座壳菌菌株的基因序列同源性达99%以上,位于同一个发育分支。致病性测定结果表明,该菌株可侵染向日葵根茎部,引起典型茎溃疡病症状。研究结果表明,进境乌克兰玉米中夹杂的向日葵籽上携带有向日葵间座壳菌D. helianthi,这是我国首次截获该种检疫性真菌。     

江苏省稻瘟病菌有性态的研究*

用标准菌株对１９９７～１９９９年在江苏省吴江市,宜兴市、通州市、高邮市和赣榆县采集的３２５个猪瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为２２．７７％,可育率仅为７．０８％。不同年份、不同地区采集的猪瘟病菌茵株的性亲和力和交配型有较大的差异,三年的交配率分别为２６．６１％、８．２６％和３３．６４％;温州地区和赣榆地区菌株的交配率相对较高,分别为２６．１５％和２５．４２％,宜兴地区菌株的交配率较低,只有１５．３８％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,１９９７年２９个可交配菌株中有２１个菌株表现为ＭＡＴ１－２ 交配型,而１９９９年３６个可交配茵株均为ＭＡＴ１－１交配型。用江苏省稻瘟病菌的可育菌株进行互交,２５个组合中只有６个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

稻瘟病菌稻巨座壳存活因子基因MoSVF1的功能分析

稻巨座壳引起的稻瘟病是一种重要的农作物真菌病害,该病害的发生和流行限制了水稻的产量,威胁全球粮食安全。细胞凋亡和自噬在稻巨座壳的致病性中起重要作用,抗细胞凋亡的存活因子Svf1在禾谷镰刀菌和核盘菌中被报道与致病力相关,但是其在稻巨座壳中的详细功能却并未见报道。本研究对MoSvf1进行序列分析,发现Svf1在真菌中高度保守。对其进行基因敲除并观察表型,发现Mosvf1突变体菌落生长减慢,菌丝形态异常,但其产孢量相比野生型有显著性增多。说明MoSvf1正调控稻巨座壳的营养生长,负调控孢子的形成。Mosvf1突变体的附着胞形成和致病力无可见缺陷。为进一步完善MoSvf1的互作调控网络,我们通过IP-MS的方法鉴定MoSvf1的互作蛋白,发现其中包含多个与胞内信号传导及细胞凋亡有关的蛋白。对MoSvf1互作蛋白的分析有助于进一步揭示MoSvf1的调控模式和作用途径,为阐明稻巨座壳的生长和凋亡打下重要基础。     

江苏省稻瘟病菌的毒性多样性及水稻品种的抗病性

在13个已知日本抗病基因品种上检测1997－1999年采集自江苏省吴江,赣榆,通州,高邮和宜兴等5个代表地区的324个稻瘟病菌株的毒性,结果可将上述菌株划分为90种毒性类型,表明江苏省稻瘟病菌存在着丰富的毒性多样性,毒性类型组成在地区间存在较大的差异,并且随着时间的推移,稻瘟病菌毒性类型组成有差异加大的趋势,在已知抗病基因品种上测定江苏省稻瘟病菌的毒力,结果显示：Pi-k^3,Pi-ta,Pi-ta^2和Pi-sh等抗病基因对江苏省的稻瘟病菌的抗谱很窄,而Pi-i,Pi-z,Pi-z^t和Pi-b等抗病基因的抗谱比较宽,可作为抗源加以利用,用6个代表性毒性类型菌株接种江苏省80筱水稻主载品种和新育成品种,品种抗性分析表明,上述水稻品种中的籼稻和杂交稻对江苏省稻瘟病菌具有较高的抗性,而粳稻品种的抗性较差,上述研究结果为利用水稻品种抗性多样性控制稻瘟病提供了依据。     

贵州梨芽枯间座壳菌属Diaporthe的种类

为明确引起贵州梨芽枯病的间座壳菌种类,本研究进行了病害调查、病芽采集和病菌分离及纯化,根据其菌落特征与ITS序列鉴定,共获得94个间座壳属Diaporthe菌株,从中选取40个代表菌株进行多基因(ITS、TEF、CAL、HIS和TUB)系统发育分析和形态学观察,结果显示它们分属于间座壳属的8个种,包括核桃间座壳菌D. biguttulata、榆树间座壳菌D. eres、椭圆间座壳菌D. ellipsoidea、梭状间座壳菌D. fusicola、香港间座壳菌D. hongkongensis、塞纳间座壳菌D. sennae、大豆间座壳菌D. sojae和蜜柑间座壳菌D. unshiuensis,其中D. ellipsoidea、D. biguttulata和D. sennae为侵染梨的首次报道。玉露香梨离体枝条接种结果显示,8种间座壳的代表菌株均可产生与田间相同的症状,分离比最高的D. eres致病力较强,表明它们均为梨芽枯病的病原菌,其中D. eres为病原优势种。本研究首次报道了引起梨芽枯的间座壳菌种类组成,为该病的发生规律及防控技术研究提供了理论依据。     

单孢分离菌株的异核不对称特性揭示梯棱羊肚菌为假同宗结合真菌

本文以基因组数据显示只具有MAT1-1 idiomorph的单孢菌株YPL6-3和只具有MAT1-2 idiomorph的单孢菌株YPL6-1为材料,研究它们子代的子囊果、单孢菌株群体和同一子囊中8个单孢菌株的交配型分布情况。YPL6-3和YPL6-1菌株分别隔离栽培和互补混栽均能形成正常的子囊果,其子囊果菌柄交配型分布与亲本菌株有关。PCR扩增检测235株子代单孢菌株的交配基因出现有趣现象：一些菌株的MAT1-1-1基因电泳条带强,而MAT1-2-1基因的条带弱;另一些菌株则MAT1-1-1基因条带弱,而MAT1-2-1基因条带强。同时也有两基因条带都强或者一基因条带强,另一基因无条带的菌株。从3个子囊果中共挑取了10个子囊,并对每个子囊中的单孢进行独立分离,PCR扩增并电泳检测交配基因时也出现了上述相同的情况。若MAT1-1-1强,则MAT1-2-1弱或无,这样的菌株在同一子囊中不会超出4个,反之亦然。利用长片段PCR扩增YPL6-1和YPL6-3菌株的全长MAT idiomorph,利用Nanopore测序技术对扩增子进行单分子实时测序,2次重复实验的序列比对发现：菌株YPL6-1中存在99.63%和99.81%的MAT1-2 idiomorph分子及0.37%和0.19%的MAT1-1 idiomorph分子;菌株YPL6-3中存在99.45%和99.74%的MAT1-1 idiomorph 分子及0.55%和0.26%的MAT1-2 idiomorph分子。从而证实,这2个基因组测序和PCR扩增都只能检测到一种交配型的菌株,其实是异核菌株,只是2种交配型核的数量占比存在较大偏离。根据上述现象推断,梯棱羊肚菌的子囊孢子都是异核的,萌发后形成的单孢菌株具有异核不对称特点,从而推测梯棱羊肚菌是一种特殊的假同宗子囊菌,同时也揭示了梯棱羊肚菌单孢出菇的真相。     

Engineering broad-spectrum disease-resistant rice by editing multiple susceptibility genes

Rice blast and bacterial blight are important diseases of rice (Oryza sativa) caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively. Breeding rice varieties for broad-spectrum resistance is considered the most effective and sustainable approach to controlling both diseases. Although dominant resistance genes have been extensively used in rice breeding and production, generating disease-resistant varieties by altering susceptibility (S) genes that facilitate pathogen compatibility remains unexplored. Here, using CRISPR/Cas9 technology, we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M. oryzae. We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo. Remarkably, a triple mutant of all three S genes had significantly enhanced resistance to both M. oryzae and Xoo. Moreover, the triple mutant was comparable to the wild type in regard to key agronomic traits, including plant height, effective panicle number per plant, grain number per panicle, seed setting rate, and thousand-grain weight. These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broad-spectrum resistance.     

稻镰状瓶霉对水稻白叶枯病的生物防治

本文研究了野生稻暗色有隔内生真菌稻镰状瓶霉与水稻的共生关系,通过根部接种DsRED荧光标记菌株,观察稻镰状瓶霉在水稻根系内的定殖,并对其抗白叶枯病特性进行调查,结果发现：经叶部接种白叶枯病菌后,对照组发病严重,病级集中在7级和9级,分别占比43.33%和34.67%,病情指数为79.26;相比之下,接种了稻镰状瓶霉的水稻植株发病较轻,叶片病斑面积小,并伴随零星黑色过敏性坏死斑出现,未发病植株占8%,72%的植株病级为1级,病情指数仅为15.26。稻镰状瓶霉对水稻白叶枯病的防治效果达到80.75%。稻镰状瓶霉的定殖能够引起叶片中超氧化物歧化酶、过氧化氢酶和过氧化物酶活性极显著提高,分别是对照组的5.26、12.08和10.53倍;诱导PR1a和PR1b显著上调表达8.71和3.37倍,AOS、OsSAUR2和EL5基因显著下调表达0.28、0.57和0.65倍。利用野生稻内生真菌防控水稻白叶枯病,在国内外尚属首次,可为水稻白叶枯病的生物防治提供新途径。     

致死性中线肉芽肿分离的不规则毛霉与米根霉致小鼠非典型增生研究

致死性中线肉芽肿（LMG）与鼻眶脑真菌病（ROCM）均为发生于面中线部位的损毁性溃疡,有时LMG可合并真菌感染,后者常被认为是继发。我们从LMG组织中分别分离出不规则毛霉（PUTH 10050641）和米根霉（PUTH 20111112）,病人均经抗真菌治疗获得痊愈,我们因此推测PUTH 10050641和PUTH 20111112可能参与诱导非典型增生及CD-56和Ki-67抗原表达。为论证上述推测,我们用ICR小鼠设计了一项观察研究。方法为小鼠背部皮下接种PUTH 10050641和PUTH 20111112,分别于接种后1周、2周、3周、4周和5周进行皮肤标本培养和组织学分析,包括直接免疫荧光和免疫组化特征。结果成功诱发感染并形成溃疡,病理表现为多形细胞浸润性炎症、血管侵入。此外,一些区域出现非典型增生、CD-56和Ki-67抗原表达,其中Ki-67高表达围绕真菌菌丝周围。上述研究表明不规则毛霉PUTH 10050641和米根霉PUTH 20111112参与了诱导非典型增生及NK细胞聚集。     

水稻主栽品种空育131抗稻瘟病位点的扫描及其基因组重构建

空育131粳稻(Oryza sativa ssp.japonica)品种因具有早熟质优、丰产稳产及耐低温冷害等优点成为黑龙江省的第一大主栽品种。为了挽救其近年来由于感染稻瘟病而从生产上退出的局面,通过对主栽品种空育131基因组的重测序和扫描,明确其遗缺多个优良抗稻瘟病Pi基因(Pi2、Pi9、Pi36、Pi5-1、Pb1、Pid3、Pi25、Pikh、Pi1、Pik-m、Pik-p和Pi56t等),并通过回交育种的方法,将MP水稻材料中的Pb1广谱抗瘟基因片段导入空育131染色体组中。该基因组的再构建过程尽可能不改变原品种的其它优良性状,并利用控制目标导入片段长短的策略来缩短Pb1位点附近的连锁累赘。在目前得到的导入系中,目标导入片段长约700 kb,背景回复率为99.38%。表型鉴定结果显示,该导入系可能和亲本MP水稻材料发挥同等的抗瘟能力。     

Mating-type Distribution and Fertility Status in Magnaporthe grisea Populations from Argentina

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.     

Significant difference in pathogenicity between MAT1-1 and MAT1-2 isolates in the wheat pathogen Mycosphaerella graminicola

Five Mycosphaerella graminicola populations from four geographic regions (Australia, Israel, Switzerland, and the USA) were assayed for neutral RFLP markers and mating type idiomorphs. On average, 25-30 genetically distinct isolates were selected from each population and their pathogenicity was measured on two wheat cultivars in a common garden experiment conducted in a greenhouse. A significant difference in pathogenicity was found between MAT1-1 and MAT1-2 isolates. On average, MAT1-1 isolates had 14-22% greater pathogenicity than MAT1-2 isolates. The pattern of higher pathogenicity in MAT1-1 isolates was consistent across four geographical populations and on two wheat cultivars. A uniform and continuous variation in pathogenicity was found among isolates within each mating type, but no genetic differentiation in selectively neutral RFLP loci was found between mating types, consistent with the hypothesis that differences in pathogenicity were not due to the effects of specific pathogenicity genes or non-random genetic backgrounds.     

Determining the Fertility Status of Setaria Infecting <Emphasis Type="Italic">Magnaporthe grisea</Emphasis> Isolates with Standard Testers and Identification of Tolerant Cultivar of <Emphasis Type="Italic">Setaria italica</Emphasis>

A total of 128 isolates of Setaria-infecting Magnaporthe grisea strains were obtained from different states of South India which includes sampling sites from Tamil Nadu, two from Karnataka, one from Andhra Pradesh and Kerala. Out of the selected 128 isolates 30 strains were tested with MAT1-1 and MAT1-2 fertile standard testers to determine their mating type. None of the 30 Setaria isolates produced perithecia with fertile testers. However, when monoconidial isolates were mated among themselves, isolates from the same field produced only barren perithecia and the tester isolates were able to mate readily with finger millet isolates. This is the first report of the mating-type studies on Setaria infecting Magnaporthe grisea with standard testers. This result indicates that the Setaria infecting population is infertile. In pathogenicity assay, it was found that 9 out of the 22 Setaria accessions were highly susceptible to Setaria strains of the blast fungus and seven cultivars/accessions were resistant to blast pathogen. Various virulence reactions were scored according to Standard Evaluation System.