柑橘木虱两地理种群的内共生菌群落组成及传菌能力

柑橘黄龙病(Huanglongbing,HLB)是经柑橘木虱Diaphorina citri传播的最主要柑橘病害之一,危害严重时能对柑橘产业造成毁灭性的破坏。为了鉴定福建和海南2个地理种群柑橘木虱的内共生菌群落组成,本研究对16SrRNA部分保守序列进行PCR扩增,并利用特异性引物对不同内共生菌进行了感染率检测;另外,还通过人工接虫的方法,探索柑橘木虱成虫在带黄龙病菌蕉柑Citrus reticulata cv.Tankan上的获菌能力,以及带菌柑橘木虱成虫对黄岩蜜橘C.reticulata cv.Subcompress的传菌能力。研究发现,这2个地理种群的柑橘木虱含有相同的内共生菌组成,包括α-Proteobacteria,Wolbachia spp.,γ-Proteobacteria,mycetocyte symbionts,β-Proteobacteria,Oxalobacter和β-Proteobacteria,Herbaspirillum,而且这2个地理种群柑橘木虱的4种内共生菌的携带率均在95%以上。柑橘木虱成虫在带菌蕉柑上饲菌28d后,带菌率可达到82%,而带菌柑橘木虱成虫在黄岩蜜橘上传菌75d后,可导致橘树整体带菌。本研究为柑橘木虱的进一步研究和防虫治病途径提供了一些理论依据。

西瓜砧木种传镰刀菌的种类鉴定及其致病性研究

本研究针对神力砧木种胚和福田将军砧木种壳分离得到的2个镰刀菌分离物,结合形态特征及分子生物学的方法进行了鉴定,并研究了其对西瓜砧木种子发芽的影响和幼苗的致病性。结果表明:2个分离物均为尖孢镰刀菌(Fusariumoxysporum)。2个尖孢镰刀菌分离物对西瓜砧木种子发芽有显著的影响,发芽势、发芽率、发芽指数、活力指数等指标均显著降低,且能导致西瓜砧木幼苗出现典型的枯萎症状,经柯赫氏法则检验证明其具有致病性。西瓜砧木种子传带尖孢镰刀菌并具有致病性为国内首次报道。

甘蓝种传镰刀菌的鉴定及其致病性研究

针对春甘45和红亩紫甘蓝种子上分离得到的两株镰刀菌(Fusarium-CG和Fusarium-HM),结合形态学特征及rDNA ITS序列分析进行了鉴定,并研究了其对甘蓝种子发芽的影响和幼苗的致病性。结果表明:2株镰刀菌均为尖孢镰刀菌(Fusarium oxysporum);对甘蓝种子发芽有一定的影响,发芽势、发芽率、发芽指数、活力指数等指标均降低,其中106个/mL孢子悬浮液处理后,活力指数显著降低;能导致甘蓝幼苗出现典型的枯萎症状,柯赫氏法则检验证明其具有致病性。甘蓝种子传带尖孢镰刀菌并具有致病性为国内首次报道。

不同种类寄主对柑橘黄龙病菌侵染能力及虫媒适应性的影响

柑橘木虱Diaphorina citri是柑橘黄龙病菌“Candidatus Liberibacter asiaticus”的主要媒介昆虫,探明不同植物种类寄主对柑橘黄龙病菌侵染能力及柑橘木虱适应性的影响,在设计黄龙病防控策略及寄主抗性研究中具有参考价值。本文选取蕉柑Citrus reticulata、蜜柚Citrus grandis、黄皮Clausena lansium和九里香Murraya exotica为研究材料,对比其田间植株黄龙病发病率和柑橘木虱数量及带菌情况,并在实验室条件下比较柑橘木虱对不同寄主的适应性(包括发育历期、存活率、携菌能力和传菌能力)。结果发现:(1)田间蕉柑和蜜柚的感染率最高,分别为47%和37%;而黄皮和九里香的带菌率较低,分别为18%和4%,且菌浓度均较轻微;(2)田间九里香上柑橘木虱卵、若虫及成虫数量均最多,蕉柑和黄皮其次,蜜柚最少,但蕉柑和蜜柚上柑橘木虱的携菌率和携菌量最高;(3)4种寄主上柑橘木虱卵和1~3龄若虫的发育历期没有显著性差异,柑橘木虱4龄、5龄若虫和总若虫历期在九里香上最短,而在蜜柚上最长;4种寄主上4龄、5龄若虫存活率没有显著性差异,但蜜柚上木虱卵的孵化率、1~3龄及总若虫存活率最低。(4)蕉柑和蜜柚上带菌木虱的携菌能力及传菌能力最高,其次为黄皮,九里香最低。该研究证明了除了寄主植物本身抗病性差异外,虫媒对寄主植物的适应性差异也是造成田间黄龙病发病差异的重要因素。

细菌群体感应信号分子——酰基高丝氨酸内酯的检测

革兰氏阴性菌根据信号分子N-酰基高丝氨酸内酯(AHLs)的浓度可以监测周围环境中自身或其他细菌的数量变化,当信号分子达到一定浓度阈值时,能启动相关基因的表达来适应环境的变化,这一调控系统被称为细菌的群体感应(quorumsensing,QS)系统。快速简便而有效地检测细菌是否以及产生何种信号分子成为深入研究和了解细菌群体感应的重要手段。现对信号分子AHLs敏感的用于检测不同的信号分子AHLs的微生物传感菌进行综述,并对其检测能力进行了讨论。

兼具苯酚降解和禾谷镰刀菌生防功能菌的活性验证

为同时解决农业用地存在的苯酚污染和土传禾谷镰刀菌病害问题,通过微生物学方法筛选高效降解苯酚的微生物,进一步从中复筛出对禾谷镰刀菌病害具有抑制作用的"多功能菌",并通过温室试验验证该种多功能菌在降解土壤苯酚污染和防治土传镰刀菌病害的效果。本研究共分离得到的苯酚降解菌中有3株对禾谷镰刀菌具有良好的抑制效果,其中菌株PBC01在70 h内对500 mg·L^-1的苯酚降解率为99.57%,该菌株对玉米禾谷镰刀菌的抑制率为79.38%,经16s rRNA基因测序鉴定该种微生物为Rhodococcus sp.。温室培养试验结果显示该菌株能显著降低土壤中的苯酚含量,40 d内将土壤中苯酚降低84.20%。同时,菌株PBC01也可减缓禾谷镰刀菌对玉米株高、叶绿素含量、生物量的抑制作用。

黄瓜种传镰刀菌粗毒素检测及其致害作用

为检测黄瓜种传镰刀菌产生的粗毒素中镰刀菌酸含量及其对种子和幼苗的致害作用,采用乙酸乙酯和乙醚萃取法提取其中的镰刀菌酸并利用HPLC法进行定量分析,通过粗毒素处理黄瓜种子及幼苗根部测定其对种子发芽的影响和幼苗的致枯萎作用。结果表明:供试7株种传镰刀菌SC-CQ-P、HLJ-XC-S、LN-ZN203-S、GD-HY-S、HN-XH-S、LN-ZN6-S和HB-TS-R粗毒素中都检测到镰刀菌酸,质量浓度分别为3 574.71、866.43、291.10、123.32、856.13、358.27和327.71μg/mL。种传镰刀菌粗毒素对黄瓜种子发芽具有一定的抑制作用;粗毒素浸渍黄瓜幼苗根部导致幼苗枯萎,并且其对幼苗的致枯萎作用随着镰刀菌酸浓度的升高和处理时间的延长而加重。

黄瓜种传镰刀菌种类的鉴定及其致病性研究

为明确我国主栽黄瓜品种的种子中携带镰刀菌的种类及其危害,对来自我国黄瓜主产区21个黄瓜品种的种子进行种传镰刀菌检测,从种胚和种子外部分离得到镰刀菌分离物9个,采用形态学及分子生物学的方法进行鉴定,并研究其对黄瓜种子发芽和幼苗致病性的影响。结果表明:9个分离物中,4个分离物为尖孢镰刀菌(Fusarium oxysporum),4个分离物为串珠镰刀菌(F.moniliforme),1个分离物为再育镰刀菌(F.proliferatum)。4个尖孢镰刀菌分离物对黄瓜种子发芽均有显著影响,发芽指数、活力指数、根长和鲜重等指标均显著降低,且能导致黄瓜幼苗出现典型的枯萎症状,经柯赫氏法则检验证明其具有致病性。其他分离物对黄瓜种子发芽也有一定的抑制作用,但没有致病性。

Genetic Study on JS399-19 Resistance in Hyphal Fusion of Fusarium graminearum by Using Nitrate Nonutilizing Mutants as Genetic Markers

Twenty-two nitrate nonutilizing （nit） mutants were recovered from five wild-type isolates of Fusarium graminearum and fifty nit mutants were recovered from three JS399-19-resistant mutants of F. graminearum cultured on MMC medium. Some biological properties were compared between nit mutants and their parental isolates. The results showed that there were no significant differences in growth rate, cultural characters or pathogenicity between JS399-19-resistant nit mutants and their parental isolates. But the conidial production and the sexual reproduction ability changed to some extent. There was no cross resistance toward chlorate and JS399-19 in F. graminearum and the resistance could be stable through 20-time subcultures. Therefore, the nit could be used as a genetic marker for studying the genetics of JS399-19 resistance in E graminearum, which was used to study JS399-19 resistance transferability in hyphal fusion. Resistance in JS399-19 could not be transferred by hyphal fusion or could be transferred with low chance between two compatible isolates, which would delay the development of JS399-19 resistance in the field.

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens

Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe （Aloe barbadensis Miller） by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride （25 rain） for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride （10 min）. Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium （30 rain） in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24℃. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHAI05 and C58CI was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHAI05 than to C58C1. Infected tissues were selected using G418 （10.0-25.0 mg/L） to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.

The Factors of Genetic Transformation for Indica Rice Kasalath

Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.

Establishment of Green Cotton Regeneration System

[Objective]The aimed to optimize transformation system for Agrobacterium-mediated green cotton shoot tip and provide reference for genetic engineering-assisted breeding of color cotton. [Method]With shoot tip of natural green cotton as receptor and by using Agrobacterium-mediated transformation method,the optimum conditions of regeneration system were obtained. [Result]Combining with vacuum infiltration,3-old-day shoot tips were infected for 10 minutes in Abarobacterium suspension with OD600 value about 0.5 followed by co-culture for 24 h on the medium containing MS＋l mg/L KT,pH value of 5.8. The regeneration culture was conducted in MS medium containing l mg/L KT and increasingly 30,50 and 70 mg/L Kan. The highest transformation frequency was 34.6%,and the positive resistant plantlets of green cotton 9804 were obtained. [Conclusion]Green cotton is more beneficial than white cotton to conduct genetic transformation.

应用F(ab')_2^-酶联吸附分析法检测大麦黄花叶病毒

F(ab′)_2酶联免疫吸附分析法(F(ab′)_2-ELISA)成功地用于大麦黄花叶病毒(BaYMV)的常规检测和诊断.其步骤是先用稀释1000—4000倍的抗血清F(ab′)_2包被反应板,加待测样品和稀释1000倍的同种抗血清或IgG,然后再加A蛋白碱性磷酸酯酶和底物,测定OD值。比较试验表明,ELISA稀释缓冲液加入1％小牛血清或1％全脂奶粉,BaYMV的测检灵敏度可提高达2.5—5.0ng/ml,病叶汁液检测终浓度为稀释1600—3200倍。我国BaYMV分离物与英国分离物的血清学性质完全一致。BaYMV在大麦病株中以叶部含量较高,茎中含量次之,根部测不出病毒。检测和诊断田间样品,即使有的样品已不新鲜,也均能得到满意的结果。此方法也成功地用于大麦温和花叶病毒(BaMMV)、小麦黄花叶病毒(WYMV)、燕麦花叶病毒(OMV)和燕麦金色条纹病毒(OGSV)等禾谷多粘菌传麦毒的检测,这S种病毒的血清学关系研究表明,除BaYMV和WYMV之间具有血清学关系以外,其余彼此均不反应。

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

Study on Population Genetic Structure and Pathogenicity of Ustilaginoidea virens from Anhui Province

In order to determine population genetic structure and pathogenicity of Ustilaginoidea virens in the major rice-growing areas of Anhui Province, total 92 U. virens strains were collected from 28 rice-planting counties （cities） of Anhui Province. Their genetic diversity was analyzed by using REP-PCR （repetitive extragenic palindromic sequence PCR）, and pathogenicity was determined with artificial inoculation method. The results showed that U. virens in rice-growing regions of Anhui Province had a rich genetic diversity. At the similarity level of 0.76, the 92 U. virens strains could be classified into 7 groups. Significant differences were found in pathogenicity among the 24 U. virens strains belonging to different groups, which showed no association with territorial source of U. virens strain or cluster method adopted by this study. Strain pathogenicity and rice varieties showed significant specificity.

细菌菌群传感效应信号分子高丝氨酸内酯平板分析法的优化

目的利用指示菌紫色杆菌CV026菌株,建立和优化稳定的细菌菌群传感效应信号分子高丝氨酸内酯平板分析方法。方法优化受测铜绿假单胞菌高丝氨酸内酯的提取方法,分析铜绿假单胞菌培养时间对信号分子检测的影响。另一方面,优化指示菌株CV026的培养基成分,并优化紫色菌素的提取方法,以完善信号分子的检测反应。结果恒温水浴挥发法可有效提取高丝氨酸内酯,并且发现当铜绿假单胞菌的培养时间为3d时,高丝氨酸内酯最易被检出。进一步,试验结果显示添加L-Try可提高指示菌株紫色菌素的产量,并利于高丝氨酸内酯的检测。此外,比较发现采用乙醇溶解紫色菌素的方法可更高效的测定紫色菌素的量。结论本课题建立的检测方法将更有效的检测细菌菌群传感系统信号分子高丝氨酸内酯,将有利于细菌菌群传感机制研究和抑制细菌菌群传感药物的开发工作。

Population Genetic Structure of Magnaporthe oryzae in Rice Blast Epidemic Areas in Hubei Province

Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.

Investigation of fecal coliform and typical enteric virus in representative beaches of China

Through investigating ten recreational marine beaches in China, we aimed to detect the occurrence of human enteric viruses in coastal bathing beaches and find a correlationship, if any, between the presence of enteric viruses in surface seawater and the concentrations of fecal coliforms, the conventional indicator of fecal pollution. In this study, twenty seawater samples were assayed for fecal coliforms and human pathogenic enteric viruses （hepatitis A viruses, rotaviruses, polioviruses） analysis. Enteric viruses were detected by RT-PCR, in 20 sample sites, 5%, 40%, 40% were positive for the presence of human hepatitis A viruses, rotaviruses, polioviruses, respectively. Seven of 20 sites are suffering from severe fecal contamination, based on traditional plate counts of fecal coliform outnumbering the established thresholds for recreation. Additionally, statistical analysis presented that no correlation was found between bacterial indicators and viruses in surface seawaters. The data confirmed that indicator bacteria in water are not reflective of the presence of enteric viruses in marine waters. Thus, current recreational water quality standards of both bacterial and viral indices should be reevaluated.

Optimization of Agrobacterium Mediated Genetic Transformation System of Tomato Meifen No.1

[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS ＋ 2.0 mg/L 6-BA＋ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105（OD = 0.4）for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1.