一种用玉米拟轮枝镰孢穗腐病病粒制备接种体的简易方法

制备接种体是玉米穗腐病抗性鉴定中的一个重要环节, 本研究提出了一种用玉米拟轮枝镰孢穗腐病病粒制备接种体的简易方法, 即将上年的拟轮枝镰孢穗腐病带菌病粒按一定重量加入无菌水洗脱制成接种体。田间接种试验结果表明, 采用本方法简易制备的接种体接种后可获得与常规制备接种体相当的接种效果, 且在-18℃冻存15 d仍可达到新鲜的常规制备接种体接种后的水平。使用简易制备接种体对33个玉米品种进行了田间穗腐病抗性鉴定, 21个品种表现为感病或高感。与实验室常规培养制备接种体的方法相比, 本制备方法简便易操作, 用时短, 成本低, 无需专业仪器设备, 能够一次制备大量接种体, 可广泛用于玉米种质材料和新品种的拟轮枝镰孢穗腐病田间抗性鉴定及抗性材料筛选等。     

引起玉米穗腐病的两种镰孢菌双重PCR快速检测体系的建立

禾谷镰孢(Fusarium graminearum)和拟轮枝镰孢(Fusarium verticillioides)是引起玉米穗腐病的两种主要病原菌。为了建立这两种病原菌的快速和定量检测体系,本研究分别设计了针对F.graminearum和F.verticillioides的基因特异性引物,建立了双重PCR反应体系,并从DNA浓度、引物浓度和退火温度3个方面对反应体系进行了优化。结果表明,退火温度为54℃和58℃时扩增效果较好,DNA模版的检测灵敏度可以达到6.25 ng·μL~(-1);F.graminearum和F.verticillioides引物最佳终浓度配比为0.2μmol·L~(-1)/0.4μmol·L~(-1)。本研究建立的双重PCR对同时鉴别引起玉米穗腐病的两种主要镰孢菌提供了准确、快速、灵敏的检验检疫技术。     

亚洲玉米螟为害对玉米镰孢穗腐病发生程度的影响

为了明确亚洲玉米螟[Ostrinia furnacalis(Guenée)]在玉米雌穗不同发育期为害对玉米镰孢穗腐病发生程度的影响,分别在春播和夏播玉米吐丝、灌浆和乳熟期,以亚洲玉米螟为试虫,以轮枝镰孢菌为供试菌,单独或复合接种侵染。结果表明,无论春播还是夏播玉米,镰孢穗腐病病情指数均以复合处理最高,为31.57～88.80,单独接虫处理为16.27～55.00,单独接菌处理为19.02～59.47;玉米镰孢穗腐病的发生程度受玉米雌穗发育期和亚洲玉米螟为害程度的影响,其玉米螟为害级别为吐丝期>灌浆期>乳熟期;受螟害的介导作用,春播玉米雌穗被害率、螟害级别和镰孢穗腐病的发病率均显著重于夏播玉米,但夏播玉米镰孢穗腐病的病情指数高于春播玉米。螟害的介入明显加重了玉米镰孢穗腐病的发生,严重程度与雌穗发育时期密切相关。     

玉米穗腐病和茎基腐病镰孢菌间相关性的RAPD分析

 利用随机扩增多态性DNA (RAPD)技术对玉米穗腐病和茎基腐病镰孢菌之间的相互关系进行了研究,证实了引起2种病害的串珠镰孢菌同源性很高,在遗传上具有较高的相似性,病菌不易受地域或环境和寄生部位选择作用的影响,同一类型的串珠镰孢菌可以是穗腐病和茎腐病的共同病原菌。禾谷镰孢菌间的遗传变异性很强,易受地域、环境和寄生部位选择作用的影响,出现明显的分化现象,穗腐病和茎基腐病可以由同一禾谷镰孢菌分化类型侵染所致,也可由不同分化类型侵染所致。     

海南玉米穗腐病病原菌分离鉴定及优势种的遗传多样性分析

为了阐明海南省玉米穗腐病病原菌的种类,结合形态学和分子生物学方法对83份病样进行了病原菌的分离和鉴定,最终获得12种病原菌,包括拟轮枝镰孢(Fusarium verticillioides)、层出镰孢(F.proliferatum)、亚粘团镰孢(F.subglutinans)、木贼镰孢(F.equiseti)、新知镰孢(F.andiyazi)、棘孢木霉(Trichoderma asperellum)、拟康宁木霉(T.koningiopsis)、卵孢木霉(T.ovalisporum)、喙刚毛球腔菌(Exserohilum rostratum)、玉蜀黍平脐蠕孢(Bipolaris maydis)、草酸青霉(Penicillium oxalicum)和可可毛色二孢(Lasiodiplodia theobromae)。其中,拟轮枝镰孢分离频率占65.06%,为优势种。以拟轮枝镰孢的EF-1α、histone 3和β-tubulin基因序列为基础进行多基因家系分析,以明确海南省与内陆部分省份不同菌株之间的遗传关系。结果表明,海南省内菌株之间具有丰富的遗传多样性,而且海南省和内陆省份菌株之间存在高频率的基因交流。     

安徽省玉米穗腐病主要致病镰孢菌的分离与鉴定

为明确安徽省玉米穗腐病主要致病镰孢菌的种类,采用单孢分离、形态学鉴定和分子生物学鉴定的方法,对2017年采集自安徽省6个玉米主产区——合肥市、蚌埠市、淮北市、阜阳市、亳州市和宿州市的玉米穗腐病病样进行了分离鉴定。在获得的455株镰孢菌中,拟轮枝镰孢菌、禾谷镰孢复合种、层出镰孢菌、尖孢镰孢菌和黄色镰孢菌的分离频率分别为59.13%、21.28%、13.68%、5.12%和0.79%。其中拟轮枝镰孢菌在各地的分布最广,属于安徽省的优势致病镰孢菌。     

Effect of environmental conditions on spore production by Fusarium verticillioides, the causal agent of maize ear rot

Silk infection by Fusarium verticillioides is caused by conidia produced on maize crop residues and results in kernel infection and consequent accumulation of fumonisins. Studies were carried out in both controlled and field conditions to understand the dynamics of sporulation on maize residues. The effect of temperature (5°C to 45°C) and incubation time (3 to 41 days) on spore production on maize meal agar was described by a logistic model that accounted for 85% of variability. The rate parameter depended on the length of incubation and the asymptote on temperature. Maximum sporulation occurred at 27°C, with a progressive increase between 5°C and 27°C and then a rapid decline, with no sporulation at 45°C. Fusarium verticillioides strains from different geographic origins showed different sporulation capabilities, with similar optimum temperatures. Pieces of stalk residues inoculated with F. verticillioides and placed above the soil between rows of maize crops, in 2003 to 2005, produced conidia continuously and abundantly for some weeks, particularly during the period after silk emergence, with an average of 1.59 × 10⁷ conidia g⁻¹ of stalk, over a wide range of environmental conditions. Sixty-seven percent of variability of the spore numbers found on stalks was accounted for by a multiple regression model. Precipitation (rain or overhead irrigation) in the 14 days before stalk sampling decreased the number of spores, whilst the number of days with conducive conditions of moisture (i.e. days with rainfall, average relative humidity >85% or vapour pressure deficit <4 hPa) and greater degree-days (base 0°C) in the 14 and 3 days before sampling, respectively, increased sporulation.     

The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides

The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi.     

玉米穗、茎腐病镰孢菌相互关系的血清学研究

运用血清学方法研究了沈阳、河北、北京、吉林4个地区玉米穗、茎腐病镰孢菌在病原学上的相互关系。结果发现:各地区玉米穗、茎腐病串珠镰孢菌(Fusarium moniliforme)具有高度的同源性,亲缘关系近。各地区玉米穗、茎腐病禾谷镰孢菌(F.graminearum)的亲缘关系因地域不同而有差异。沈阳穗、茎腐病禾谷镰孢菌之间差异明显,存在生理分化现象;吉林玉米穗、茎腐病病原物存在较高的相似性,但也有一定的差异;而河北玉米穗、茎腐病禾谷镰孢菌具有高度的同源性。     

玉米禾谷镰孢茎腐病和穗腐病抗性鉴定接种方法的比较

田间采用不同人工接种鉴定方法对92份玉米种质同步进行玉米禾谷镰孢茎腐病和穗腐病抗性评价与筛选, 明确了东北春玉米产区玉米禾谷镰孢茎腐病?穗腐病抗性鉴定的最佳接种方法:茎节注射法在玉米禾谷镰孢茎腐病抗性精准鉴定中可更好地评价玉米种质的抗病性, 综合考虑接种方法的实用性和可操作性, 菌土覆盖法对大体量的规模化种质进行田间抗病性鉴定更具有优势; 双牙签法对玉米穗腐病抗性鉴定中在种质接种后发病的严重度?田间进行接种的可操作性和实用性等方面均优于花丝通道法?同时筛选出对玉米禾谷镰孢茎腐病抗性稳定的种质52份, 对穗腐病抗性稳定的种质66份, 对2种病害均表现稳定抗性的兼抗种质33份?     

产抗菌素2,4-二乙酰基藤黄酚假单胞杆菌的分离及其防治沙打旺根腐病的室内测定

从山东、内蒙古、北京等地土壤中分离筛选出荧光菌 50 0 0余株 ,其中 1 2 0 0余株为抑制性荧光菌。经PCR检测 ,获得 73株 2 ,4 二乙酰基藤黄酚 (2 ,4 DAPG)产生菌。平板筛选结果表明 ,2 ,4 DAPG产生菌CPF 1 0和 2P8对沙打旺根腐病菌Sad1和Sad2均有较好的抑制效果 ,其中CPF 1 0的抑菌带宽分别为 5.0和 1 2 .0mm ;2P8抑菌带宽分别为 3 .5和 7.0mm。温室试验两次调查表明 ,CPF 1 0对沙打旺根腐病防治效果最好 ,达 63 .5%和 67.8% ;2P8防效也在 40 %左右 ,且均达到极显著水平。 2 ,4 DAPG产生菌可以显著促进沙打旺植株根系发育 ,CPF 1 0处理后地上部株高差异不显著 ,但鲜重和干重与对照相比有极显著的增加 ,说明两菌株菌剂处理可以促进植株生长。根部定殖结果表明 ,两菌株在沙打旺根部都有一定的定殖能力 ,在根表种群数量比较稳定 ,根内细菌数量在调查时间内呈逐渐上升的趋势     

Relationships of immature and adult thrips with silk‐cut,fusarium ear rot and fumonisin B1 contamination of maize in California and Hawaii

Field experiments were conducted in California and Hawaii in order to investigate the relationships between thrips feeding in maize ears and fusarium ear rot and silk‐cut symptoms. Half the plots in each experiment were treated with insecticides following pollination. Thrips populations within ears were enumerated at six stages of ear development. Grain was examined microscopically and the percentages of kernels with silk‐cut and ear rot symptoms were quantified by weight. Fumonisin B₁ contamination in grain was measured by ELISA. Immature stages of thrips predominated, and maximum thrips populations occurred 21 days after pollination. Insecticides reduced thrips numbers, as well as silk‐cut, ear rot symptoms and fumonisin B₁ contamination. Immature thrips populations were more strongly correlated with silk‐cut/ear rot symptoms (R = 0·75) and fumonisin B₁ accumulation (R = 0·53), than were adult thrips (R = 0·48 and 0·36, respectively). Silk‐cut kernels all had ear rot symptoms and the percentage of kernels with symptoms was highly correlated with fumonisin B₁ contamination (R = 0·84). Results suggest that thrips are not occasional feeders, but can complete a substantial portion of their life cycle on maize ears. The results also indicate that thrips activity may be a cause of silk‐cut symptoms, and this may be the mechanism that connects thrips activity with fusarium ear rot and fumonisin contamination of grain.     

荧光假单胞菌PEF-5#18防控番茄枯萎病的定殖机理

通过电转法成功将EGFP表达质粒p EGFP20转入对番茄枯萎病具有优良防病效果的荧光假单胞菌PEF-5#18体内,并研究了该生防菌在番茄根际土壤与植株根、茎内部组织的定殖情况。结果表明,处理25 d后,标记型与野生型生防菌处理均能有效控制番茄枯萎病害并增加植株鲜、干重量。与病原菌阳性对照相比,野生型菌株的防治效果为76.92%,鲜重增加194.10%,干重增加564.71%;接种后的PEF-5#18能良好定殖于番茄根际土壤并扩展进入植物根茎内部生长,其分布数量呈现出根际土壤(1.05×106 CFU/g)根(6.75×104 CFU/g)茎(1.25×102 CFU/g);与病原菌阳性对照相比,接种PEF-5#18对番茄根际土壤的可培养细菌总数影响差异不显著,但可显著提高根(增幅为8.42%)、茎(增幅为14.78%)内的可培养细菌总数以及根际土壤pH(增幅为1.61%);接种PEF-5#18能显著降低根际土壤与根、茎内病原菌侵染数量;激光共聚焦镜检发现,接种25 d后,PEF-5#18能大量分布于番茄根系表面、根内木质部、根内皮层细胞、根内细胞间隙、茎内维管及其周围细胞。     

利用伏马毒素合成基因对玉米种子寄藏镰刀菌的分子检测

我国玉米种子普遍受到拟轮生镰刀菌(Fusarium verticillioides)的侵染,该菌产生的伏马毒素对国家种质库中玉米种质的安全保存有着潜在的影响。fum1基因是F.verticillioides等镰刀菌中伏马毒素生物合成的必需基因。选用3对已知引物Fum5F/Fum5R、P1/P2和P3/P4分别对从玉米种子中分离出的9株镰刀菌和1株阴性对照菌链格孢菌进行PCR扩增检测,在F001和F003菌株中扩增出相应的特异性表达片段。测序表明,这些片段的长度分别为846bp、888bp和703bp,与已知fum1基因(AF155773)的同源性达99%以上。3对引物的检测灵敏度达到88pg/mL。结果表明,这3对引物都能够有效地检测产生伏马毒素的镰刀菌菌株,但引物Fum5F/Fum5R所扩增片段完全来自fum1基因内部,具有更强的特异性。在此定性检测基础上,需要进一步研究定量检测方法并进一步研究伏马毒素对农作物种质保存的影响。     

密度、施肥、种植方式及杀虫剂处理对玉米穗腐病及伏马毒素污染的影响

为明确不同种植密度、施肥水平、耕作方式以及杀虫剂处理对玉米穗腐病发生及籽粒中伏马毒素污染水平的影响,于2014—2015年以浚单20、先玉335为供试玉米品种进行田间试验,通过改变种植密度、施肥水平、耕作方式及使用杀虫剂氯虫苯甲酰胺处理,在收获期调查玉米穗腐病的病情指数,采用高效液相色谱柱后衍生法检测玉米籽粒中伏马毒素含量。结果表明,先玉335和浚单20在田间发生穗腐病的病情指数及其籽粒中伏马毒素含量均随种植密度的增加而显著增大,在密度为7.5万株/hm~2时,其病情指数在2014年分别为16.00和10.33,在2015年分别为18.15和16.38,伏马毒素含量在2014年分别为2.38、1.53 mg/kg,在2015年分别为3.23、2.20 mg/kg,均显著高于密度为6.0万株/hm~2和4.5万株/hm~2时;穗腐病的病情指数及伏马毒素含量随着施肥量的增加而增大,在施肥量为675 kg/hm~2时,病情指数在2014年分别为12.35和8.37,在2015年分别为15.11和13.30,伏马毒素含量在2014年分别为1.35、0.85 mg/kg,在2015年分别为2.39、2.19 mg/kg,均显著高于施肥量为450 kg/hm~2及225 kg/hm~2时;耕作方式对穗腐病病情指数及伏马毒素含量无显著影响,旋耕后播种方式下穗腐病病情指数及伏马毒素含量略小于贴茬直播方式;使用杀虫剂氯虫苯甲酰胺处理后,能显著降低穗腐病发生的严重程度及伏马毒素的污染水平。     

玉米穗腐病样品中层出镰刀菌的分离与鉴定

为了解豫西南玉米穗腐病的病原菌,于2009年9月在河南省南阳市6个县区采集玉米穗腐病样品,进行病原菌的分离与单孢纯化.依据致病性、形态及分子检测结果进行病原菌鉴定.研究结果表明,分离得到的105株镰刀菌中,层出镰刀菌Fusarium proliferatum为70株,占分离镰刀菌的66.7％,属优势种群.在基于核糖体基因内转录间隔区(rDNA-ITS)与翻译延长因子1α基因(tef)序列构建的系统发育树中,3株代表菌株Fp1、Fp2和Fp3分别与GenBank登记的层出镰刀菌在自举值89％和91％水平相聚同一群.层出镰刀菌的生长温度为13～34℃,最适温度为28℃;在pH5.0和6.0各有1个生长高峰;碳源对该菌营养生长的影响较为稳定,而氮源对其营养生长影响的变幅较大.     

甘肃玉米穗腐病样品中轮枝镰孢菌的分离鉴定及生物学特性

 为了明确甘肃玉米穗腐病的病原种类, 于2009年9月在甘肃省四大生态区采集玉米穗腐病样品, 以组织分离法进行病原物的分离培养, 对分离得到的镰孢菌菌落进行纯化和单孢分离后, 以形态学为基础, 参照Leisle分类系统进行鉴定。结果表明:共分离到271株镰孢菌菌株, 其中以轮枝镰孢菌(Fusarium verticillioides)和黄色镰孢菌(F. culmorum)出现频率高, 属优势种。按照柯赫氏法则对沈单16和金穗96832进行致病性测定, 证实了轮枝镰孢菌对玉米果穗的致病性。选取2株轮枝镰孢菌菌株进行EF-1α(tef) 基因序列分析, 将PCR产物回收测序后在GenBank上比对, 菌株GSLT4-3-2与GenBank中登记的轮枝镰孢菌FN179339、FN179345和FN179338;GSTS24-2-1与轮枝镰孢菌FN179343、FN179346、FN179340、FN179344和 FN252384亲缘关系最近, 序列同源性达100%。轮枝镰孢菌GSYC17-2-1的生长温度范围为15～35℃, 最适温度为28℃, pH 值为8;菌落在pH值为4～10的培养基上能够迅速扩展, pH值为7时产孢量最大;碳源对轮枝镰孢菌菌丝生长影响相对稳定, 而氮源对其营养生长影响的变幅较大;完全黑暗条件下, 菌丝扩展最快, 12 h光暗交替对产孢量有明显的促进作用;病原菌菌丝致死温度为70℃, 10 min。     

河北省玉米穗腐病病原菌鉴定及潜在产伏马毒素镰孢菌系统发育分析

 为明确河北省夏玉米区玉米穗腐病的发生情况、病原菌组成及地区对病原菌种类的影响,本团队于2016年和2017年在玉米收获前期对玉米穗腐病的发生情况进行调查,并通过生物学方法对随机采回的样本进行分离鉴定。田间调查结果表明,与2016年相比,2017年河北省玉米种植面积有所减少,玉米穗腐病的发病率有所下降。分离鉴定结果表明,引起河北省夏玉米区穗腐病的优势病原菌为拟轮枝镰孢菌(Fusarium verticillioides),分离频率为63.49%,其他病原菌如层出镰孢菌(F. proliferatum)、禾谷镰孢菌(F. graminearum)、变红镰孢菌(F. incarnatum)、黑曲霉(Aspergillus niger)、黄曲霉(A. flavus)、草酸青霉(Penicillium oxalicum)和哈茨木霉(Trichoderma harzianum)的分离频率分别为19.05%、6.35%、1.59%、14.29%、3.17%、9.52%和1.59%。为明确伏马毒素基因在潜在产伏马毒素镰孢菌鉴定中的作用,拟轮枝镰孢菌、层出镰孢菌和藤仓镰孢菌(F. fujikuroi)分别以EF-1α和FUM1基因序列为基础构建系统发育树。结果表明,两个系统发育树拓扑结构相似,伏马毒素基因可用于潜在产伏马毒素镰孢菌种的鉴定,基于FUM1基因的种间遗传距离大于基于EF-1α基因的种间遗传距离,而种内遗传距离的结论则与之相反。     

The influence of flavonoids in maize pericarp on fusarium ear rot symptoms and fumonisin accumulation under field conditions

Breeding efforts have been undertaken to increase resistance of maize to fusarium ear rot (FER) and to fumonisin accumulation. Flavonoids in the pericarp of the kernels are considered particularly able to reduce the fumonisin accumulation. The aim of this 2‐year field study was to assess the effect of flavonoids on FER symptoms and fumonisin contamination in maize kernels using two isogenic hybrids, one providing pigmentation in the pericarp (P1‐rr) and the other without it (P1‐wr). FER incidence (FERi), FER severity (FERs), the incidence of infections caused by Fusarium spp. in symptomless kernels (FF) and fumonisin contamination (FUM) were assessed in both hybrids. Significant differences between the two hybrids were detected mainly in 2012 trials where P1‐rr showed lower FERi (P < 0·01), FF (P < 0·05) and FUM (P < 0·1) than P1‐wr. Site, characterized by local temperature and precipitation, played a relevant role in modelling all the measured variables, as its effect was highly significant in both years, whether they were considered individually or altogether. The interaction of hybrid with location was a significant (P < 0·001) source of variation only for FF. FF, together with FERi, was also significantly (P < 0·001) influenced by the interaction of hybrid with year. In general, FUM was more influenced by year and location parameters, such as temperatures during late ripening, than by flavonoid presence in kernel pericarp. The results indicate that flavonoid pigments alone may not be an important component in the resistance of maize to fumonisin accumulation.