禾谷镰孢荧光定量检测体系的建立及其在玉米苗枯病上的应用

为实现对田间土壤中禾谷镰孢Fusarium graminearum的定量检测,本研究构建了土壤含菌量与玉米苗枯病病情指数的回归模型。基于禾谷镰孢甾醇14α-去甲基化酶基因CYP51C序列,设计特异性引物HQ1-F/HQ1-R,利用引物建立实时荧光定量PCR(RT-q PCR)体系,选取4个玉米自交系品种进行室内苗枯病接种试验,调查其病情指数,利用RT-qPCR体系检测土壤禾谷镰孢含菌量,并对病情指数和土壤禾谷镰孢含菌量进行回归。结果表明,仅禾谷镰孢扩增出目的条带并且可从多种病原菌土壤中检测出。RT-qPCR的熔解曲线具有单一吸收峰,扩增曲线的循环阈值与模板浓度呈良好的线性关系,扩增效率为104.7%,标准曲线为y=-3.2137x+34.9560(R~2=0.9968),最低可检测到1 pg/μL的DNA。随着土壤禾谷镰孢接菌量的增加,单位土壤禾谷镰孢含菌量呈线性增加,即y=13.603x-85.370(R~2=0.9998)。4个玉米品种的病情指数与土壤禾谷镰孢含菌量的回归曲线分别为y=0.0789x+22.0590(R~2=0.7949)、y=0.0304x+7.8686(R~2=0.9579)、y=0.0458x+23.7540(R~2=0.5420)、y=0.0471x+32.0760(R~2=0.6753)。     

玉米内生菌L10的分离、鉴定及拮抗活性

为获得对玉米茎腐病主要病原菌禾谷镰孢Fusarium graminearum有明显拮抗作用的玉米内生菌,采用平板对峙法从成熟健康玉米茎秆中筛选禾谷镰孢拮抗菌株,并分析其抗菌谱;通过形态特征、生理生化特性及16S rDNA序列分析进行菌种鉴定;利用盆栽生防试验检测其对玉米茎腐病的防治效果。结果表明,共分离获得了164株玉米内生细菌菌株,其中L10菌株对禾谷镰孢具有较好的抑制效果,抑菌圈半径达1.68 cm;该菌对玉米大斑病菌Setosphaeria turcica、层出镰孢F. proliferatum、禾谷镰孢F. graminearum、拟轮枝镰孢F. verticilliodes、玉米弯孢叶斑病菌Curvularia lunata、玉米小斑病菌Bipolaris maydis、立枯丝核菌Rhizoctonia solani、茄链格孢Alternaria solani共8种植物病原菌均有拮抗作用,尤其对禾谷镰孢抑制效果最佳;结合形态特征、生理生化性质及16S rDNA序列分析,将L10菌株鉴定为多粘类芽胞杆菌Paenibacillus polymyxa。L10菌株脂肽类物质对禾谷镰孢菌具有较好的抑制活性,且盆栽生防试验结果显示该菌株对玉米茎腐病具有一定的防治效果。表明菌株L10对玉米镰孢茎腐病的防治具有一定潜力。     

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?     

20%烯·戊·恶种衣剂研制及对大豆镰孢根腐病的防效

为研制一种防治大豆镰孢根腐病的高效、低毒种衣剂,采用菌丝生长速率法测定了96%烯效唑(uniconazole)原药、97%戊唑醇(tebuconazole)原药、98%恶霉灵(hymexazol)原药对引起大豆根腐病的禾谷镰孢、尖镰孢和腐皮镰孢的毒力,筛选最佳复配配比。利用湿研磨法制备种衣剂,并进行室内盆栽苗验证。结果表明:烯效唑、戊唑醇和恶霉灵的配比为3∶1∶16时,对供试3种镰孢菌均表现为增效作用。室内盆栽苗验证试验结果表明,播种前,采用20%烯·戊·恶悬浮种衣剂1.0～2.0 g/kg处理大豆种子对大豆发芽和出苗没有显著影响,并可降低株高,且各浓度处理对大豆尖镰孢和腐皮镰孢根腐病的相对防效均达83.3%以上,对尖镰孢根腐的防效高于5%戊唑醇悬浮种衣剂,对腐皮镰孢和禾谷镰孢根腐病与5%戊唑醇悬浮种衣剂防效相当。本研究开发的20%烯·戊·恶悬浮种衣剂为进一步开展大豆根腐病田间化学防治及推广应用奠定了基础。     

玉米穗腐病菌的拮抗菌筛选、鉴定及降解DON毒素能力测定

穗腐病是玉米生产上的重要病害，在造成产量损失的同时，产生的毒素严重危害人畜健康。为获得兼具降解毒素功能的玉米穗腐病菌的生防菌株，以脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）毒素为靶标，从玉米病穗上分离筛选到一株能够降解该毒素的细菌菌株TP，经菌落形态特征、结合16S rDNA和gyrB基因序列分析，初步鉴定为贝莱斯芽胞杆菌Bacillus velezensis。对峙试验和复筛共培养结果显示，菌株TP对禾谷镰孢F18的抑制率分别达到70.63%和99.5%。经HPLC检测，TP在含DON的无机盐溶液中培养10 d后，其对DON的降解率为98.97%。以上研究结果表明，贝莱斯芽胞杆菌TP能拮抗禾谷镰孢菌，且能高效降解DON毒素，该菌株在防治禾谷镰孢菌引起的玉米穗腐病以及降低毒素危害中具有广阔的应用前景。     

杀菌剂对玉米穗腐病菌的毒力及毒素产生的影响

为筛选防治玉米穗腐病同时减少病原菌毒素积累的化学药剂,采用菌丝生长速率法、涂布平板法分别测定了4种药剂多菌灵、百菌清、吡唑醚菌酯和氰烯菌酯对玉米穗腐病的3种主要致病镰孢菌拟轮生镰孢菌、层出镰孢菌和禾谷镰孢菌菌丝生长和孢子萌发的影响,并采用高效液相色谱法测定了4种药剂对其毒素积累的影响。结果表明:层出镰孢菌菌株127-1和禾谷镰孢菌菌株125-3的最佳产毒时间为第10天。供试4种药剂对3种镰孢菌菌丝生长的EC50值范围为0.18~5.71μg/m L,对孢子萌发的EC50值范围为0.02~8.14μg/m L,均表现出一定的抑制作用。但对3种镰孢菌产伏马毒素、玉米赤霉烯酮和脱氧雪腐镰孢菌烯醇的影响不尽相同,吡唑醚菌酯浓度在1~500μg/m L时能够抑制拟轮生镰孢菌和层出镰孢菌产生伏马毒素,氰烯菌酯能够在0.05~0.8μg/m L浓度范围内显著抑制禾谷镰孢菌产生玉米赤霉烯酮,当多菌灵浓度为0.05~0.5μg/m L时,几乎可以完全抑制禾谷镰孢菌产生脱氧雪腐镰孢菌烯醇。     

氰烯菌酯对禾谷镰孢菌的生物活性及其内吸输导性研究

氰烯菌酯(2-氰基-3-氨基-3-苯基丙烯酸乙酯,JS399-19) 是一种对禾谷镰孢菌具有专化活性的新型杀菌剂。研究表明,该药在离体条件下对禾谷镰孢菌Fusarium graminearum抗多菌灵菌株及野生敏感菌株的菌丝生长均有很高的抑制活性,平均EC50值分别为0.117±0.036和0.107±0.020 μg/mL 。氰烯菌酯不能抑制禾谷镰孢菌的分生孢子萌发,但能引起抗多菌灵菌株及敏感菌株的分生孢子萌发后的芽管畸形。活体条件下,氰烯菌酯灌根处理对小麦赤霉病有一定防效,在叶片间的输导性较差,不能被小麦穗颈吸收;该药在同一张叶片上仅表现为向上输导,对处理部位的下部几乎没有防效。用400 μg/mL氰烯菌酯和400 μg/mL多菌灵穗部喷雾处理前1 、2 、3 d接种禾谷镰孢菌分生孢子,氰烯菌酯对小麦赤霉病的防效分别为95%、75%和62%;处理后1 、2 、3 d 接种,防效分别为88%、78%和73%,而对照药剂多菌灵的防效较差。说明氰烯菌酯对小麦赤霉病有优异的保护和治疗作用,在禾谷镰孢菌对多菌灵已产生抗性的地区,氰烯菌酯可以作为一种很好的替代药剂用于防治小麦赤霉病。     

禾谷镰孢FgCYP51B蛋白F511L突变对分生孢子产量及其对烯唑醇敏感性的影响

Phe511是禾谷镰孢甾醇-14α-脱甲基酶FgCYP51B活性口袋中的一个重要氨基酸。本研究中,我们探究了FgCYP51B蛋白中该位点突变后对禾谷镰孢主要生物学表型的影响,并通过分子对接探讨了可能的原因。结果表明禾谷镰孢FgCYP51B-F511L突变体在菌落形态、生长速率等表型上与野生型菌株PH-1没有明显差异。但是F511L突变导致分生孢子的产量严重降低,对烯唑醇的敏感性增强。分子对接发现突变后亮氨酸的长侧链使得烯唑醇的甲基发生扭转,侧链朝向发生改变,更有利于与蛋白受体形成较强的疏水作用,这可能是导致FgCYP51B-F511L突变体对烯唑醇敏感性增强的原因。     

黄芪根腐病发生危害与防治

2001～2004年对河西走廊绿洲灌区黄芪根腐病发病危害情况、病原和不同栽培条件下发病因子进行了调查。结果表明,在河西走廊黄芪根腐病发病率为21.4%,致病菌主要是尖镰孢菌(Fusarium oxysporum Schlecht.)和腐皮镰孢菌[Fusarium solaniun(Mart.)Sacc]。灌水不当或在黄芪结实期遇连续阴雨是引起黄芪根腐病大面积发生危害的直接因子,提出了相应的防治方法。     

MB和CMC液体培养基对禾谷镰孢产孢水平的影响

禾谷镰孢分生孢子定量接种是研究作物抗禾谷镰孢的必要手段?本试验利用绿豆(mung bean, MB)与羧甲基纤维素(carboxylmethyl cellulose, CMC)液体培养基在相同条件下培养禾谷镰孢, 比较两种培养基对禾谷镰孢产孢效率的影响; 并用两种培养基诱导分生孢子制成相同浓度孢子悬浮液, 对75个玉米家系进行人工接种鉴定, 检测不同培养基所产分生孢子致病性差异?结果表明, CMC培养基诱导分生孢子增长率(k2=1.125)大于MB培养基诱导增长率(k1=0.844)?在培养第14天, MB培养基诱导获得的分生孢子平均浓度为2.51×105个/mL, CMC培养基诱导的为3.62×105个/mL, 比MB培养基诱导多44.22%?用两种培养基诱生的孢子进行田间接种, 玉米穗腐病发病程度无明显差异?CMC培养基具有产孢快?孢子浓度高的优点, 是一种适宜禾谷镰孢分生孢子诱生的高效液体培养基?     

Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (<Emphasis Type="Italic">F. culmorum</Emphasis>, <Emphasis Type="Italic">F. pseudograminearum</Emphasis> and <Emphasis Type="Italic">F. graminearum)</Emphasis> on hard red spring and durum wheat

Fusarium species involved in the Fusarium crown rot (FCR) complex affect wheat in every stage of development from seedling to grain fill. This study was designed to compare the aggressiveness of the FCR complex members including F. culmorum, F. pseudograminearum and F. graminearum in causing seedling blight, decreased plant vigour and crown rot. To assess their relative pathogenicity, two hard red spring wheat cultivars and two durum wheat cultivars were inoculated in the field with five isolates from each of the three species for two years. Significant differences in patterns of pathogenicity were identified. In particular, F. culmorum caused greater seedling blight while F. pseudograminearum and F. graminearum caused greater crown rot. Greatest yield reductions were caused by F. pseudograminearum. Cultivar differences were identified with respect to seedling disease and late season crown rot. No interactions were identified between cultivar performance and isolates or species with which they were challenged.     

Management strategies and distribution of Aphanomyces root rot of alfalfa (Medicago sativa), a continuing threat to forage production in the United States

Alfalfa (Medicago sativa) is one of several legumes that is affected by Aphanomyces root rot (ARR) caused by Aphanomyces euteiches. Symptoms of ARR on alfalfa seedlings include a yellow-grey discolouration of roots, rotting and loss of lateral roots, stunted growth, chlorotic foliage and reduction of nitrogen-producing nodules on roots. Infection can also occur on adult plants leading to loss of lateral roots and nodules. At the seedling stage, ARR decreases alfalfa stand establishment, and field longevity is reduced when adult plants are infected. A. euteiches is an oomycete pathogen that has motile zoospores and thick-walled oospores that can survive for many years in soil. Two races are currently recognized by pathogenicity on differential alfalfa check cultivars. Most alfalfa cultivars contain race 1 resistance, but there is an increasing development of cultivars with resistance to race 2. Management strategies include planting resistant cultivars, avoiding planting in fields with poor drainage and rotating crops with nonhost plants.     

Diversity and pathogenicity of Fusarium graminearum species complex from maize stalk and ear rot strains in northeast China

The Fusarium graminearum species complex (FGSC) is an important group of pathogens distributed in maize‐producing areas worldwide. This study investigated the genetic diversity and pathogenicity of 40 FGSC isolates obtained from stalk rot and ear rot samples collected from 42 locations in northeastern China during 2013 and 2014. A phylogenetic tree of translation elongation factor (EF‐la) sequences designated the 40 isolates as F. graminearum sensu stricto (67.5%) and F. boothii (32.5%). By using inter‐simple sequence repeat analysis (ISSR), it was shown that the isolates were divided into two clades, which corresponded to the species identity of the isolates. However, the isolates from the two different diseases could not be distinguished in pathogenicity. The disease severity index of seedlings inoculated with stalk isolates was slightly higher than that of seedlings inoculated with isolates from infected ears, whereas the pathogenicity of the stalk and ear isolates were identical.     

Genetic Analysis of the Role of Trichothecene and Fumonisin Mycotoxins in the Virulence of Fusarium

The phytotoxicity of the Fusarium trichothecene and fumonisin mycotoxins has led to speculation that both toxins are involved in plant pathogenesis. This subject has been addressed by examining virulence of trichothecene and fumonisin-nonproducing mutants of Fusarium in field tests. Mutants were generated by transformation-mediated disruption of genes encoding enzymes that catalyze early steps in the biosynthesis of each toxin. Two economically important species of Fusarium were selected for these studies: the trichothecene-producing species Fusarium graminearum, which causes wheat head blight and maize ear rot, and the fumonisin-producing species F. verticillioides, which causes maize ear rot. Trichothecene-non-producing mutants of F. graminearum caused less disease than the wild-type strain from which they were derived on both wheat and maize, although differences in virulence on maize were not observed under hot and dry environmental conditions. Genetic analyses of the mutants demonstrated that the reduced virulence on wheat was caused by the loss of trichothecene production rather than by a non-target mutation induced by the gene disruption procedure. Although the analyses of virulence of fumonisin-non-producing mutants of F. verticillioides are not complete, to date, the mutants have been as virulent on maize ears as their wild-type progenitor strains. The finding that trichothecene production contributes to the virulence of F. graminearum suggests that it may be possible to generate plants that are resistant to this fungus by increasing their resistance to trichothecenes. As a result, several researchers are trying to identify trichothecene resistance genes and transfer them to crop species.     

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。     

Susceptibility of Broccoli Cultivars to Bacterial Head Rot: In Vitro Screening and the Role of Head Morphology in Resistance

Head rot is a major disease of broccoli caused by the soft rot pathogens Pseudomonas fluorescens and Erwinia carotovora. Two in vitro pathogenicity tests were evaluated as a methods to identify broccoli cultivars susceptible or resistant to bacterial head rot. One test used mature heads excised from the plant and inoculated with squares of cotton lint which had been soaked in a bacterial suspension. The other test involved stab-inoculating axenically grown seedlings. With the excised head test, susceptible cultivars showed a black soft rot, whilst less susceptible or moderately resistant cultivars showed only watersoaking, or browning and slight softening of the tissue. No cultivar was completely resistant. Ten cultivars were tested, and their susceptibility ratings corresponded with previously recorded field data, with one exception. This laboratory test could be used to screen for susceptibility to head rot in broccoli breeding programmes. The seedling test distinguished differences in aggressiveness among bacterial isolates but not cultivar susceptibility. Increasing head size correlated negatively with disease resistance. Head shape, i.e. cultivars which showed a domed shape rather than a flat shape, was positively correlated with disease resistance. Thus small domed heads are more resistant to head rot than large flat heads. Other morphological characteristics, viz. floret prominence and number, and sepal stomatal number were not correlated with host resistance.     

黄淮海夏玉米主产区穗腐病病原菌的分离鉴定

为明确我国黄淮海夏玉米主产区玉米穗腐病的病原菌种类、优势种群及虫害、年度、省份对病原菌的影响,以形态学为基础,结合分子生物学方法对2013、2015年随机采自河南、河北、山东3省的155份玉米穗腐病样品进行分离鉴定。结果表明,引起黄淮海夏玉米主产区玉米穗腐病的主要致病菌为镰孢菌Fusarium spp.,包括拟轮枝镰孢F.verticillioides、禾谷镰孢F.graminearum、层出镰孢F.proliferatum、木贼镰孢F.equiseti及藤仓镰孢F.fujikuroi,分离频率分别为49.7%、28.4%、12.3%、3.9%和1.3%;其次为木霉菌Trichoderma spp.,包括哈茨木霉T.harzianum、绿色木霉T.viride和棘孢木霉T.asperellum,分离频率分别为8.4%、3.2%和5.2%;青霉菌Penicillium spp.分离频率较低,为14.2%;曲霉菌Aspergillus spp.包括黑曲霉A.niger和黄曲霉A.flavus,分离频率分别为2.6%和1.9%。研究表明,黄淮海主产区玉米穗腐病优势病原菌为拟轮枝镰孢、禾谷镰孢和木霉菌,不同省份不同年度间病原菌种类及优势病原菌存在差异,虫害能加重玉米穗腐病的发生。     

Management of phytophthora root rot in radiata pine seedlings

Chemical and biological agents were evaluated for their ability to suppress root rot, caused by Phytophthora cactorum , in field-grown radiata pine seedlings in New Zealand. Trials were conducted over two seasons in an area of a forest nursery with a natural infestation of P. cactorum , and a history of root rot. In each season, symptoms of root rot developed during April, one month after root pruning, when seedlings were approximately six months old. In trial one, root rot incidence by mid July 2007 was 9·1% in untreated plots and 8·4% in plots that had been treated with metalaxyl-M/mancozeb (14 kg ha⁻¹) at seedling emergence. Disease incidence was lowest (2·1%) in plots that received seven monthly applications of phosphorous acid (6·5 L ha⁻¹). Other treatments, including seed coating with thiram or Trichoderma spp., and foliar applications of methyl jasmonate, did not control disease. In trial two, effects of treatment timing relative to root pruning were investigated. By late June 2008, three months after root pruning, root rot incidence was 22·2% in the untreated plots. Phosphorous acid was the most effective treatment and almost completely suppressed disease (0·1% incidence) when applied fortnightly from February until May (seven applications). Metalaxyl-M/mancozeb (15 kg ha⁻¹) was not effective (21·4% incidence) when applied five months before root pruning. However, disease incidence was reduced when the chemical was applied one week after root pruning (14·9% incidence) and greater control was achieved (8·2% incidence) when the application rate was increased to 50 kg ha⁻¹.     

抗、感大豆品种对尖镰孢根腐病菌侵染及毒素的反应

为了进一步明确大豆抗感品种对根腐尖镰孢的抗病机制,通过透射电镜和形态观察分析了尖镰孢(M38)侵染及产生的毒素对抗感大豆品种的影响。研究表明大豆幼苗胚根经粗毒素处理后,抗病品种‘东农56’幼苗胚根生长没有显著差别,但显著抑制了感病品种‘黑农53’胚根的伸长和侧根的生长;尖镰孢毒素高浓度和低浓度(V粗毒素∶V无菌水=1∶0和1∶15)对抗感品种的致萎能力无显著差异,高浓度处理平均萎蔫指数为100,低浓度处理为17.9~18.1左右;但中等浓度(V粗毒素∶V无菌水=1∶1,1∶5,1∶10)对于感病品种的致萎作用更大;同时,发现尖镰孢侵染感病大豆品种根内侵染量要明显多于抗病品种,且感病品种组织中菌丝的直径明显大于抗病品种,都出现很明显的质壁分离和细胞壁加厚的现象。     

Trichothecenes and aggressiveness of Fusarium graminearum causing seedling blight and root rot in cereals

Greenhouse trials conducted in 2003 and 2004 investigated the impact of trichothecenes on the severity of seedling blight and root rot in common wheat ( Triticum aestivum ), durum wheat ( Triticum turgidum var. durum ), barley ( Hordeum vulgare ) and triticale (× Triticosecale 6x ) using two trichothecene-producing and two trichothecene-nonproducing Fusarium graminearum strains. In 2003 seedling emergence and survival following soil infestation of the trichothecene-producing strain (Gz3639) were significantly reduced compared with the trichothecene-nonproducing strain (GzT40), while root-rot incidence and severity were increased significantly. In 2004, two trichothecene-producing strains (Gz3639 and GzT106) reduced seedling emergence and survival ( P  ≤ 0·01) in eight of 10 crops/cultivars based on single-degree-of-freedom contrasts. However, when results from all strains were combined no significant differences were observed between two trichothecene-producing and two trichothecene-nonproducing F. graminearum strains. Inoculation with GzT106, a trichothecene-producing 'add-back' strain, resulted in more severe root rot symptoms in eight of 10 cultivars ( P  ≤ 0·01–0·05) and lower seedling emergence and survival in seven of 10 cultivars ( P  ≤ 0·01–0·10), compared with the wild-type parental strain Gz3639. The presence of trichothecenes may play an important role in the aggressiveness of F. graminearum .