短小芽胞杆菌DX01菌株Tn5转座突变株的抑菌活性筛选体系的建立

为建立短小芽胞杆菌Bacillus pumilus DX01菌株的Tn5转座突变株抑菌活性的高效筛选体系,采用发酵液平板抑菌法研究了各突变株发酵液对稻瘟病菌Magnaporthe grisea生长的影响,并测定了目标突变株的几丁质酶和蛋白酶活性及发酵液对稻瘟病菌分生孢子萌发的抑制率。从2 633个突变株中筛选出6个抑菌活性较对照菌株DX01显著变化的突变株。对照DX01发酵液对真菌孢子萌发的抑制率为36%,而突变株Tn5-901和Tn5-194则分别为96%和3%,抑菌活性与对照的差异达到极显著水平。其余4个突变株的几丁质酶、蛋白酶活性多重比较结果与发酵液平板抑菌结果并不完全一致,但发酵液平板抑菌法简单、高效,适用于短小芽胞杆菌突变株的抑菌活性初筛。     

pseP基因对蜡样芽胞杆菌0-9菌株生物薄膜形成和防治小麦纹枯病的影响

为了阐明芽胞杆菌0-9菌株的生防机制,通过敲除0-9菌株的多糖外运蛋白编码基因pseP构建基因敲除菌株0-9（ΔpseP）和基因敲除菌株的互补菌株0-9（ΔpseP∷pseP）,并比较野生型0-9菌株及其衍生菌株形成生物薄膜的能力及其对小麦纹枯病的生防效果。结果显示,pseP基因敲除后,菌株生物薄膜形成能力和对小麦纹枯病的生防作用降低,生防效果较野生型0-9菌株降低43.6%。基因互补后,互补菌株生物薄膜的形成能力和对小麦纹枯病的生防作用得到恢复,生防效果较敲除菌株提高29.2%。研究表明蜡样芽胞杆菌0-9菌株的pseP基因参与了生防菌株0-9生物薄膜的形成和对小麦纹枯病的生物防治。     

枯草芽胞杆菌NCD-2中调控因子PhoP对fengycin合成的调控作用

 PhoR/PhoP双因子调控系统是枯草芽胞杆菌中一个重要的全局性调控系统, 在低磷环境下, 感应激酶PhoR自磷酸化, 并且将获得的磷酸基团转移到调控因子PhoP上, 从而激活PhoP的调控活性。为了明确调控因子PhoP对枯草芽胞杆菌NCD-2菌株中主要抑菌活性物质-fengycin合成能力的调控作用, 本研究通过同源重组技术缺失突变NCD-2菌株中的phoP基因, 拮抗活性测定结果表明, phoP基因缺失菌株显著降低了对立枯丝核菌的抑菌活性。通过快速蛋白质液相色谱(FPLC)技术比较了NCD-2菌株野生型及其phoP基因突变子fengycin的合成能力, 结果证明, phoP基因突变菌株降低了fengycin的合成能力。对突变子进行phoP基因互补发现, 互补phoP基因可使突变子的相关性状恢复到野生型菌株水平。以上结果证明, phoP基因对枯草芽胞杆菌NCD-2菌株中fengycin的合成具有正调控功能。     

防治稻瘟病芽胞杆菌的筛选及效果评价

从232株细菌中筛选获得2株对稻瘟病菌具有明显抑菌活性的菌株S09和S170,对稻瘟病菌Magnaporthe oryzae P131菌丝的抑制率分别为90.44%和92.38%;离体叶片法测定表明,菌株S09和S170抑菌效果分别为74.62%和75.52%,与绿地康1号无显著差异。16S rDNA序列分析比对,菌株S09与短小芽胞杆菌Bacillus pumilus基因序列的同源性最高;gyrA基因测序结果表明,菌株S170与解淀粉芽胞杆菌B.amyloliquefaciens基因序列的同源性最高。结合形态观察,确定菌株S09为短小芽胞杆菌,菌株S170为解淀粉芽胞杆菌。菌株S09和S170对稻瘟病的温室防治效果分别是76.56%和80.57%。田间试验结果表明,菌株S09和S170对水稻有明显的促生和增产作用,分别提高水稻种子发芽17.37%和12.34%、促进根长18.68%和33.85%、增加株高12.44%和28.49%、降低空秕率25.17%和35.59%,还可以有效改善稻米的垩白粒率和垩白度。短小芽胞杆菌S09和解淀粉芽胞杆菌S170对叶瘟的田间防治效果分别是70.59%和73.19%,对穗颈瘟的田间防治效果分别是71.55%和74.82%,菌株S170对稻瘟病的防治效果与75%三环唑可湿性粉剂、绿地康1号处理无显著差异。     

广东省马铃薯块茎软腐病病原菌鉴定

为明确广东省惠州市马铃薯块茎软腐病的病原菌,采用常规病组织分离法获得4株菌株,通过生理生化特征和分子生物学特性对其进行鉴定,并测定了该病原菌对20个马铃薯品种的致病力。结果显示,该病原菌为革兰氏阳性菌,在LB平板上菌落有透明和不透明2种形态;除葡萄糖发酵阳性、对链霉素和青霉素具有抗性等特征不同外,BP-hd-1、BP-hd-2、BP-hd-3和BP-hd-4菌株与短小芽胞杆菌Bacillus pumilus菌株的其它生理生化特征均相同。系统进化分析结果表明,4株菌株的16S rDNA序列分别与短小芽胞杆菌AI-Khrj18(KY123871)、ML270(KC692158)、NCTC10337(LT906438)和ARD21(KX023236)的相似性为100.0%;4株菌株的gyrB基因序列均与短小芽胞杆菌AUEC29菌株(HM585095)的gyrB基因相似性最高,为99.7%～99.8%。生理生化特征及分子生物学鉴定结果表明,引起广东省马铃薯块茎软腐病的病原菌为短小芽胞杆菌。致病力测定结果显示,接种菌株BP-1后20个马铃薯品种的发病率均为100.0%,表明该病原菌对马铃薯有强致病力。     

贝莱斯芽胞杆菌Bacillus velezensis YL2021嗜铁素合成基因dhbC的功能研究

贝莱斯芽胞杆菌Bacillus velezensis YL2021是一株对多种病原细菌均有良好防治效果的生防菌,基因组中含有完整的儿茶酚型（2,3-Dihydroxybenzoate,DHB）嗜铁素合成基因簇。为了探明贝莱斯芽胞杆菌YL2021嗜铁素的功能,本研究以嗜铁素合成基因dhbC为对象,构建重组质粒pMD19-dhbc（cm）,利用同源重组技术获得突变株ΔdhbC。生防相关性状研究结果表明,与野生型菌株YL2021相比,突变株ΔdhbC泳动能力和生物膜形成能力明显下降,但是群集运动能力未发生改变。在营养丰富的LB培养基中,野生型菌株YL2021和突变株ΔdhbC均不产生嗜铁素,生长能力未发生改变,室内抑菌能力相当;但在缺铁M9培养基中,野生型菌株YL2021能产生儿茶酚型嗜铁素,生长缓慢,对供试细菌有较强的抑菌作用,突变株ΔdhbC不能产生嗜铁素,生长能力明显下降,完全丧失抑菌能力。本文结果表明,缺铁条件下,dhbC基因是贝莱斯芽胞杆菌YL2021嗜铁素生物合成的关键基因,嗜铁素是菌株YL2021发挥生防作用的重要抑菌物质。     

芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框（GenBank登录号为JQ309841）,编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。     

群体感应调控因子ComA对枯草芽胞杆菌NCD-2抑菌物质产生和生物膜形成的影响

为明确群体感应系统在枯草芽胞杆菌NCD-2菌株抑菌活性和生物膜形成中的作用,通过同源重组技术对群体感应系统中的comA基因进行定位缺失突变,分别比较了NCD-2菌株和comA基因突变子在胞外酶合成、抑菌活性、脂肽抗生素(丰产素)产生以及生物膜形成上的差异,并进一步利用实时荧光定量RT-PCR技术比较了二者生物膜基质编码基因epsA和tasA的表达情况。结果表明,通过同源重组技术获得了comA基因突变子MA-4,同菌株NCD-2相比,其在胞外蛋白酶和纤维素酶的合成能力、对番茄灰霉菌Botrytis cinerea的抑菌活性、丰产素的合成量和生物膜的形成能力上均明显下降;生物膜基质编码基因tasA的表达量下降了70%,而epsA的表达量变化不明显。表明ComA是NCD-2菌株脂肽抗生素和生物膜形成中的重要调控因子。     

转录调控因子AbrB对生防短短芽胞杆菌X23中抗生素edeines生物合成的影响

非核糖体类抗生素edeines是短短芽胞杆菌Brevibacillus brevis X23产生的主要抑菌物质，提高其产量有助于增加生防效果。通过全基因组测序及生物信息学分析挖掘短短芽胞杆菌X23的全局性调控因子，基于Red/ET同源重组技术构建温敏型敲除载体，通过双交换同源重组技术获得短短芽胞杆菌X23全局性转录负调控因子缺失的突变株，然后采用平板抑菌、液相色谱-质谱联用（HPLC-MS）和实时荧光定量PCR研究了全局性转录负调控因子敲除对短短芽胞杆菌X23不同生长时期发酵液的抑菌活性、edeines产量、ede操纵子转录水平的影响。从短短芽胞杆菌X23基因组中挖掘到了1个全局性转录负调控因子AbrB，敲除abrB基因后edeines生物合成基因簇操纵子的转录水平在生长前期显著提高，在发酵48 h后abrB突变株中edeine A和edeine B总产量比出发菌株提高了1.1倍。结果显示短短芽胞杆菌X23中AbrB是edeines生物合成过程中的负调控因子，敲除abrB提高了edeines的产量。     

杧果炭疽菌拮抗细菌的筛选鉴定及其防病效果

为寻找对杧果炭疽菌Colletotrichum gloeosporioides有效的生防菌株,从瓜叶白粉病病斑上分离得到104株细菌,通过平板对峙培养筛选出16株拮抗细菌,分别测定其在8种不同培养基中的抑菌活性,筛选出有较强抑菌作用的9A、26A和4N菌株进行室内、果园防病试验,并对具有较好防治作用的9A、26A菌株进行了鉴定。结果显示,拮抗菌9A、26A和4N分别在南瓜汁、红薯汁和马铃薯汁培养基上的抑菌活性最高,对杧果炭疽菌菌丝生长抑制率分别为88.83%、87.83%和85.87%;室内9A、26A和4N菌株菌液处理离体果实,24 h后接种炭疽菌分生孢子液,对杧果炭疽病的防治效果均达100%,与咪鲜胺对照组相同;在果园9A和26A的防病效果分别为98.4%和90.5%,显著高于对照组的防效78.8%,而4N的防效只有58.2%。根据形态、生理生化特征及16S rDNA 序列分析结果,将菌株9A和26A鉴定为枯草芽胞杆菌Bacillus subtilis。研究表明,菌株9A和26A具有较好的生防利用价值。     

Genomic sequencing globally identifies functional genes and potential virulence-related effectors of Xanthomonas axonopodis pv. malvacearum

Highly virulent strains (HVS) of Xanthomonas axonopodis pathovar (pv.) malvacearum (Xam) infect all commercial cultivars of cotton, including the long-established “immune” cultivar 101-102B. Here, we present high-quality draft sequences of a highly virulent Xam strain (GSPB2388) from Sudan, and a strain of race 18 (GSPB1386) from Nicaragua, using Illumina/Solexa paired-end sequencing. The short sequence reads were assembled into 61 scaffolds for GSPB2388 (N50 of 164 kb) and 127 scaffolds for GSPB1386 (N50 of 100 kb), with draft maps of roughly 5 Mb, which contain 4,665 and 4,520 protein-coding genes, respectively. Through gene annotation and comparisons with plant pathogen proteins, 181 and 178 potential virulence-related genes, including genes encoding a major group of type III effectors, were identified from GSPB2388 and GSPB1386, respectively. The differential effectors and sequence diversity between the HVS and race 18 may enable the identification of key factors that contribute to the high virulence of HVS. Additionally, the average nucleotide identity (ANI) between Xam and Xanthomonas axonopodis pv. citri is 98.4 %, suggesting that these strains belong to the same species.     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.     

亚洲玉米螟6-磷酸葡萄糖异构酶基因(Pgi)全序测定及分析

为进一步从核酸水平上研究亚洲玉米螟Pgi基因相关特性,采用反转录PCR及RACE等技术对该基因编码序列及DNA全序列进行了测定,与Gen Bank中其它昆虫Pgi基因相关信息进行比较分析,并构建了系统发育树。结果表明:亚洲玉米螟Pgi基因编码区序列长为1 671 bp,共编码556个氨基酸;其DNA序列全长为10 078 bp(短序列为9 311 bp),由12个外显子与11个内含子镶嵌而成;各外显子长度与鳞翅目大部分昆虫相同,介于95~188 bp之间,内含子序列总长度为8 407 bp(短序列为7 640 bp),各内含子长度介于418~1 547 bp之间,且在内含子3、4、5、11上均发现杂合现象。系统发育结果显示,大部分昆虫Pgi基因c DNA严格按物种聚类,除了双翅目的家蝇Musca domestica与黑森瘿蚊Mayetiola destructor出现一定的交叉现象外,其余没有出现交叉现象。     

棉铃虫para同源基因III-IV接头DNA片段序列分析

利用反转录 PCR技术 ,用一对特异性寡核苷酸引物 ,分离获得棉铃虫 para同源基因 III- IV接头约30 0 bp DNA片段 ,发现在 Bao D- R和 Bao D- S品系间存在 4个核苷酸差异 ,但在推导的氨基酸组成上没有差别。对比分析表明 ,分离获得的棉铃虫 III- IV接头氨基酸组成与烟芽夜蛾 hscp片段同一区域有 98.1%的氨基酸相同 ,与德国蜚蠊 CSMA的氨基酸有 93.5%相同 ,与果蝇 para基因有 88.9%的氨基酸相同     

Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.     

褐飞虱化学感受蛋白基因克隆与信号肽活性鉴定

为科学利用化学感受蛋白（chemosensory protein,CSP）绿色防控褐飞虱Nilaparvata lugens,采用同源序列比对和基因克隆对褐飞虱CSP基因进行鉴定,利用基因组分析其CSP基因簇,通过酵母信号肽分泌验证系统验证褐飞虱CSP信号肽的活性。结果显示,共克隆了15个褐飞虱CSP基因,其中有4个CSP基因为新鉴定到的;共发现了2个CSP基因簇,均位于褐飞虱2号染色体上;14个CSP的N末端序列有较强的信号肽分泌活性,其中11个活性序列与SignalP-4.1预测的序列一致,另外3个CSP的活性序列比SignalP-4.1预测的序列长。     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.