稻瘟病菌MoRho2基因功能初步分析

稻瘟病菌(Magnaporthe oryzae)是研究病原真菌生长发育及致病机理的重要模式生物。Rho家族蛋白是一类具有GTP酶活性的GTP结合蛋白,在多种细胞信号转导通路中起着分子开关的作用。通过生物信息学方法分析Mo Rho2的理化性质、蛋白结构域等信息,并推测其潜在功能。为进一步明确Mo Rho2蛋白的功能,利用基因敲除技术获得Mo Rho2基因的敲除突变体。表型分析结果表明,与野生型菌株相比,Mo Rho2敲除突变体的菌丝生长速度、分生孢子形态、附着胞形态、产孢量、对植物的毒力等方面均无明显的差异,但分生孢子萌发和附着胞形成略滞后于野生型菌株。突变体能够产生正常的侵入栓并侵入洋葱表皮。综上所述,Mo Rho2基因可能参与稻瘟病菌分生孢子的萌发和附着胞的形成,结果为进一步揭示基因Mo Rho2的生物学功能奠定基础。     

香蕉枯萎病菌假定分泌蛋白SP10的功能分析

为了研究尖孢镰刀菌古巴专化型假定分泌蛋白基因SP10的在致病过程的作用,构建SP10基因敲除菌株,分析SP10敲除菌株的表型特征。结果表明:SP10基因敲除菌株在菌丝生长、产孢和菌落形态等方面无明显变化,但SP10基因敲除菌株对巴西蕉的致病性减弱;在接种30 d后,采用共聚焦显微镜显微观察香蕉根系,结果显示,SP10基因敲除菌株可在根系维管束中定殖。上述结果表明,假定的分泌蛋白SP10参与调控尖孢镰刀菌对香蕉的致病性,但不参与该病原菌生长和发育的调控。此结果为进一步研究尖孢镰刀菌分泌蛋白的功能奠定了基础。     

绿僵菌对荔枝椿象的室内致病力测定

在室内用喷雾法测定了4株绿僵菌对荔枝椿象3～4龄若虫的致病力.结果表明,绿僵菌致病力强弱与菌株种类和接种剂量有密切关系.菌株对荔枝椿象若虫致病力的强弱顺序为J813MA4J86CMA3.在接种菌株J813孢子浓度为1×108个/mL的情况下,对荔枝椿象若虫的LT50为7.63 d.J813菌株对荔枝椿象若虫具有很强的二次侵染能力.     

绿僵菌对甘蔗螟虫的室内致病力测定

在室内用喷雾法测定了4株绿僵菌对甘蔗螟虫3～4龄若虫的致病力。结果表明,绿僵菌致病力强弱与菌株种类和接种剂量有密切关系。先接虫后喷施菌剂时,菌株对甘蔗螟虫若虫致病力的强弱顺序为JF813HS1MA4JF883;先喷施菌剂后接虫时,菌株对甘蔗螟虫若虫致病力的强弱顺序为MA4HS1JF813JF883。在接种各菌株孢子浓度为1×108个/mL时,菌剂对甘蔗螟虫若虫的致死率高。其中JF813菌株对甘蔗螟虫若虫具有很强的二次侵染能力,HS1菌株在两种处理方式中致病力稳定。     

稻瘟病菌MGG14093基因功能的初步研究

根据之前的基因芯片分析,发现在稻瘟病菌侵染水稻的过程中,许多基因被诱导表达。MGG14093为其中一个推测为编码分泌蛋白的未知功能基因,在病菌侵染过程中该基因表达量上调2.906。利用生物信息学在线软件对该基因编码蛋白的特性进行初步分析,并通过RT-PCR扩增进一步分析该基因在稻瘟病菌侵染水稻过程中的表达动态,利用基因敲除和过量表达技术对该基因的功能进行初步研究。结果表明:该基因编码蛋白为定位于胞外的分泌蛋白,没有已知的蛋白结构域,但可能存在几处氨基酸活性位点。MGG14093基因敲除和过量表达后,对稻瘟病菌的营养生长、产孢及附着胞分化没有显著的影响,但该基因过量表达后导致病菌的致病力下降,表明该基因可能参与病菌的致病过程。本研究结果为进一步揭示MGG14093基因的生物学功能奠定了基础。     

绿僵菌对东亚飞蝗的室内致病力测定

在室内用喷雾法测定了6株绿僵菌对东亚飞蝗的致病力,结果表明,其致病力强弱与菌株种类和接种剂量有密切关系,菌株对东亚飞蝗的致病力由强到弱顺序为MA3>MA4>MB6>MA6>MA5>MA11。接种MA3菌株的孢子浓度在1.0×108个/mL情况下,对4龄蝗蝻的LD50为4.46d。用不同剂量的MA4菌株的孢子油悬浮剂涂抹法接种4龄蝗蝻,发现其LD50仅为15.39个/头。另外,观察发现,MA3菌株具有很强的2次侵染能力。     

玉蜀黍尾孢菌CzVelB基因功能分析

以获得的野生型和CzVelB敲除突变体菌株为供试菌株,分别对其进行生物学及致病力测定,在此基础上分析毒素和细胞壁降解酶的活性。结果表明,与野生型相比,敲除CzVelB后菌丝生长速率及颜色无明显差异,但产孢量下降56.6%,且孢子萌发率下降,萌发过程滞后1 h。致病性分析发现,敲除CzVelB后菌株致病性明显下降,病情指数、病斑大小,突变体均低于野生型。分析毒素对叶片的致病性发现,突变体所产生的毒素低于野生型产生的毒素。敲除突变体菌株ΔCzVelB及野生型菌株在活体内外均能产生5种细胞壁降解酶,其中敲除CzVelB后,5种离体病原菌细胞壁降解酶活性明显下降。分析侵染过程中5种细胞壁降解酶发现,CzVelB主要通过影响CX和PMG在接种前期12 h内起重要作用;在接种后期(72～96 h),CzVelB主要通过影响PGTE的活性调控玉蜀黍尾孢菌致病力。     

尖孢镰刀菌古巴专化型4号生理小种fpd1基因敲除与表型分析

为了研究尖孢镰刀菌古巴专化型4号生理小种fpd1基因的功能,构建了该基因敲除突变体并对其表型特征进行了分析。结果表明,与野生型菌株相比,fpd1基因敲除突变体生长减慢,产孢量显著降低,对巴西蕉的致病性明显减弱;fpd1基因可能在尖孢镰刀菌古巴专化型的产孢、生长发育和致病性等方面具有重要作用。本研究结果为进一步研究fpd1基因的功能和尖孢镰刀菌的致病机理奠定了基础。     

香蕉枯萎病菌4号生理小种cat1基因敲除与表型分析

前期蛋白质组学研究发现尖孢镰刀菌古巴专化型4号生理小种(Foc4)中的过氧化氢酶1(cat1)基因受到巴西蕉诱导上调表达,为研究cat1基因在Foc4侵染香蕉过程中的作用,利用同源重组方法敲除Foc4的cat1基因,并对获得的敲除突变体开展表型分析和致病性测定。结果表明:cat1基因缺失对Foc4的菌丝、孢子形态、菌落生长、抗渗透压胁迫和细胞壁选择性压力等没有影响;但引起菌株抵抗外源氧胁迫、细胞壁穿透能力、纤维素利用能力减弱和对巴西蕉的致病性减弱。显示cat1基因的产物通过清除香蕉分泌的活性氧在Foc4对香蕉的致病过程中起作用,并参与病原菌对寄主纤维素成分的代谢。此结果为进一步深入研究香蕉枯萎病菌的致病机理奠定基础。     

香蕉枯萎病菌4号生理小种smy1基因的功能分析

Smy1是一种参与调控真菌生长的驱动蛋白。本研究克隆了香蕉枯萎病菌4号生理小种（Foc 4）驱动蛋白基因smy1,并采用同源重组技术获得smy1敲除的野生型Foc 4的smy1敲除突变体。通过测定突变体菌丝形态、生长速度、产生孢子量、对香蕉苗的致病力及对氰烯菌酯的敏感性,用于研究smy1在香蕉枯萎病菌的生长发育及对氰烯菌酯抗药性中的作用。结果表明,与野生型Foc 4相比,smy1敲除突变体生长缓慢、菌丝畸形、产孢量增加,对巴西蕉苗的致病力减弱,但对氰烯菌酯的抗性水平变化不大。由此推断smy1基因可能在Foc 4的生长发育、产孢以及致病力等方面具有重要作用。     

不同剂型草甘膦对金龟子绿僵菌MA4菌株生长发育的影响

测试3种不同的草甘膦剂型在不同浓度下对MA4菌株孢子萌发、菌落生长、产孢量的影响。结果表明,飞达红88.8％草甘膦铵盐可溶性粒剂对孢子萌发与菌落生长抑制作用最强,10％草甘膦水剂对产孢的抑制作用最大,95％的氨盐影响均相对较小。同时研究发现,草甘膦各剂型中的不同助剂对金龟子绿僵菌菌株的生长与产孢的影响较大。     

橡胶树胶孢炭疽病菌致病相关基因CgATG8的功能分析

ATG8是病原真菌中细胞自噬过程中的重要基因,并参与致病过程。基于橡胶树胶孢炭疽菌HBCg01全基因组数据库,采用PCR和RT-PCR扩增得到橡胶树胶孢炭疽病菌的CgATG8基因并进行序列分析。采用Split-Marker Recombination法获得该基因的敲除转化子。表型分析结果表明,缺失该基因后,橡胶树胶孢炭疽病菌的致病力丧失,产孢能力、孢子萌发率、附着胞形成率和膨压均降低,而且孢子萌发和附着胞的形成延迟。     

Direct seeding as an alternative to transplanting for guayule in southeast Queensland

Guayule (Parthenium argentatum Gray) is a source of high quality rubber and low-allergenic latex. Commercial potential of guayule to produce high value latex products has increased due to the increased incidence of deadly diseases in humans. The objective of this study was to investigate the potential of direct seeding in southeast Queensland as an alternative to establishment by the current high cost transplanting method. Experiments were conducted for 2 years at Gatton, Queensland using physical and chemical seed treatments. NaOCl plus GA₃, osmo-priming with polyethylene glycol, seed pelleting and seed tape planting were tested. Planting depth had a significant influence on seedling emergence. Emergence at 18 mm depth (1.5 seedlings/m) was significantly reduced compared with 10 mm depth (2.9 seedlings/m). Osmo-priming was effective in increasing germination from 36 to 47%. It also improved seedling emergence and vigour. Osmo-priming significantly increased establishment (7.5 seedlings/m) compared with untreated seed (3.3 seedlings/m). NaOCl plus GA₃ did not have a significant influence on germination (38%) or seedling emergence (5.0 seedlings/m) but increased survival at 42 days after planting. Establishment as a percentage of emerged seedlings was high for both osmo-primed and NaOCl plus GA₃ treatments with 89 and 88% respectively, whereas untreated seed had only 70% survival. Osmo-priming also increased vigour as indicated by increased root length (101 mm), shoot height (123 mm), and seedling dry matter (379 mg/seedling) compared with the control (83, 107 mm and 206 mg/seedling, respectively).     

不同处理方法对菜用黄麻种子萌发的影响

为了提高菜用黄麻种子的发芽率,保证出苗的整齐度,采用沙子研磨、热水浸种、98%浓硫酸浸种和40%氢氧化钠浸种共4种方法对菜用黄麻桂麻菜1号和桂麻菜2号种子进行处理。结果表明:沙子研磨、热水浸种、98%浓硫酸浸种处理对种子的发芽势和发芽率均有显著地影响。桂麻菜1号和桂麻菜2号在种子与沙子体积比1:4、研磨时间为8 min时,种子发芽率最高分别为78.0%、86.3%;热水80℃浸种8 min时,种子发芽率最高分别为86.3%、83.0%;98%浓硫酸浸种处理30 min时,桂麻菜1号发芽率最高达78.3%,桂麻菜2号在浸种10 min时,发芽率最高达87.3%。40%氢氧化钠浸种处理对菜用黄麻种子发芽率的影响不明显。在这4种菜用黄麻种子处理方法中,热水浸种方法易于操作,处理时间短,成本低廉,适合生产上推广运用。     

细胞膜质子泵基因超表达促进水稻种子萌发

 构建了质子泵基因OsA8（AJ440219）超表达的水稻株系（OE1）,以其T2代种子为材料,研究细胞膜质子泵基因超表达对种子萌发的影响。结果表明,质子泵超表达的种子不仅发芽速度快,而且初生根生长也比野生型快。鉴定了OsA8及其他质子泵基因在转录水平上的表达差异,并通过两相分离法纯化了初生根系质膜的H+ ATPase,检测其活性。通过蛋白质免疫杂交技术分析比较超表达的水稻株系与野生型水稻根系H+ ATPase酶蛋白浓度的差异。结果显示,超表达材料根系OsA8基因的表达量高于野生型,其单位膜蛋白中质膜H+ ATPase蛋白的浓度也显著高于野生型,因而其根系质膜H+ ATPase活性高于野生型。超表达材料OE1的种子萌发速率和初生根的生长速率均快于野生型的主要原因很可能就是质子泵基因OsA8在OE1水稻种子中超量表达所引起的下游生理效应。     

UvSMEK1, a Suppressor of MEK Null,Regulates Pathogenicity,Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Rice false smut, which is caused by Ustilaginoidea virens, is an emerging disease of rice spikelets in rice-growing areas worldwide. However, the infection mechanism of U. virens on rice spikelets is still unclear. Here, we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase(MEK) null(UvSMEK1) in U. virens that is conserved among filamentous fungi. Compared with wild type U. virens strain P-1, Uv SMEK1 deletion mutants were defective in pathogenicity and conidial germination. In addition, conidiation of UvSMEK1 deletion mutants was significantly reduced on yeast extract tryptone(YT) plates, but increased in YT broth compared with the wild type. Compared with Uv SMEK1 expression level during the vegetative mycelia and conidiation stages, UvSMEK1 dramatically increased during infection of rice florets. Surprisingly, the Uv SMEK1 deletion mutants exhibited higher tolerance to H2 O2 and NaCl. In summary, presented evidence suggested that UvSMEK1 positively regulated pathogenicity, conidial germination and conidiation in YT broth, and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses. The results enhance our understanding of the regulatory mechanism of pathogenicity of U. virens, and present a potential molecular target for blocking rice infection by U. virens.     

Soak conditions and temperature following sowing influence the response of maize (Zea mays L.) seeds to on-farm priming (pre-sowing seed soak)

On-farm seed priming of maize (i.e. soaking seed in water overnight, 17 h) in the semi-arid tropics has been shown to improve crop establishment and yield but the benefits can be variable. In previous work, this variation was thought to result from the interaction between primed seeds and conditions in the seedbed following sowing. To investigate this interaction, experiments were carried out under controlled temperature conditions that represented those of seedbeds in the semi-arid tropics. The conditions during seed priming and during subsequent germination and emergence were investigated as potential causes of variable seedling emergence relative to that of untreated seeds. Priming advanced emergence from moist sand cores at 30 °C/20 °C (day/night), reduced emergence at 35 °C/28 °C and delayed and reduced emergence at 40 °C/28 °C. In drier cores (drained 8 days) at 35 °C/28 °C, priming advanced germination time and did not decrease final percentage emergence. These results confirmed earlier field observations that the effect of priming in maize can vary and lead to positive, neutral or negative effects. Priming decreased the optimum and ceiling temperatures for germination, and unlike untreated seeds, the relationship between germination rate and sub-optimal temperature was not linear in all cases. Pre-humidifying seeds did not enhance the effect of priming, but aerating the soak water did. These results indicate that variable results from priming recorded in the field were not due to imbibitional damage, but may result from the negative effects of hypoxia during soaking exacerbated by moist conditions and high temperatures at sowing. These effects differed between seedlots of different cultivars, indicating the importance of selecting more tolerant maize hybrids to reduce variation in response to priming following sowing in the field. The implications of these findings for the use of primed seeds in the field are discussed.     

Seed treatment of sorghum varieties with brine (NaCl) solution for control of Striga hermonthica in Sorghum

Field trials were conducted in 1997 and 1998 wet seasons to determine the effect of seed treatment of different sorghum varieties with brine (NaCl) on the control of Striga hermonthica in sorghum. The varieties of sorghum used were ICSV 1002, ICSV 1007 (improved resistant varieties from ICRISAT, Mali), BES (a local improved variety), and War-warabashi (a local susceptible variety). Different concentrations of brine at 0.5, 1.0, 1.5, and 2.0 M were used. The results show that the varieties, ICSV 1002 and ICSV 1007 had significantly higher establishment count than the BES and War-warabashi varieties, but had shorter plants. Similarly, the ICSV 1002 and ICSV 1007 and War-warabashi varieties had significantly larger LAI values than the BES variety, although the BES significantly out yielded them. The susceptible War-warabashi variety supported the highest number of Striga emergence than the improved varieties. The effect of brine treatments on plant establishment, growth and yield of sorghum under Striga infestation shows that there was a decrease in crop stands with increase in brine concentration, with the least value at 2.0 M brine treatment, which was damaging at this rate. Plant height and, leaf area index (LAI) of sorghum increased with increasing concentration of brine to a maximum at 1.5 M and declined at 2.0 M brine treatment. The 1.5 M brine treatment produced the highest grain yield. Striga shoot emergence significantly declined with increasing brine concentration reaching the lowest values at 1.5 and 2.0 M brine treatments. The interaction between sorghum varieties and brine concentrations shows that War-warabashi with 1.5 M brine treatment had the tallest plants and largest LAI, while BES and 1.5 M brine produced the highest grain yield. In general, the interaction between the varieties and all the brine concentrations significantly reduced Striga emergence. Irrespective of the sorghum varieties the use of brine at 1.5 M was found optimal for controlling Striga emergence resulting in increased crop growth and grain yield. This implies that which ever variety farmers are using either resistant, tolerant or susceptible, they could easily use brine at 1.5 M to effectively control Striga and increase in grain yield. The mechanism by which brine successfully controlled Striga, is, however, not clear and needs to be investigated.     

Germination responses of the halophyte Chloris virgata to temperature and reduced water potential caused by salinity,alkalinity and drought stress

Chloris virgata is considered a useful grass species for grassland restoration in northern China. However, little information exists concerning the germination responses of this species to temperature and water potential caused by stress conditions. Experiments were conducted in growth chambers to assess the effect of temperature, salinity, alkalinity, drought and the interactions of temperature and stress on seed germination. Seeds were germinated at three diurnal temperature regimes, with four water potentials in NaCl, NaHCO₃ and PEG solutions. Results showed that optimal germination under stress occurred at 15–25 °C, and germination percentages and rates were inhibited by either an increase or decrease in temperature from the optimal temperature. The inhibitory effects of the low water potential caused by salinity and drought on germination were greater at 25–35 °C, but seeds were subjected to more stress despite the relatively higher water potential because of the alkalinity at this temperature. The recovery percentage under salinity was highest at ?1·2 MPa at 15–25 °C, and more than 80% of seeds also germinated at this water potential after they were transferred from drought stress. However, seeds lost their viability in higher alkalinities under all temperatures, and at 25–35 °C, there was lower recovery percentage under stress. Results suggest that salinity, alkalinity and drought stress have different impacts on seed germination, and the tolerance to stress of C. virgata seeds is affected by the interactions of temperature and water potential caused by salinity, alkalinity and drought. Chloris virgata shows potential utility as a promising grass species in salinity–alkalinity and drought‐stressed environments.