小麦赤霉病菌多菌灵抗性群体的扩散路径分析——基于致病菌种类及所产毒素化学型鉴定和抗药性检出的时序性

通过对江苏、安徽、山东、河南、湖北、河北和四川7省小麦赤霉病菌对多菌灵抗性及敏感菌株Fusarium asiaticum和F.graminearum的鉴定、所产生毒素的化学型及多菌灵抗性菌株检出时序性的分析,初步推测了小麦赤霉病菌对多菌灵抗性群体在中国麦区的扩散路径。结果表明:江淮流域的江苏、安徽、湖北3省和四川省小麦赤霉病菌对多菌灵的抗性或敏感菌株优势群体均是F.asiaticum,而黄淮流域的山东、河南2省及河北省小麦赤霉病菌对多菌灵的敏感菌株优势群体为F.graminearum,抗性菌株优势群体则为F.asiaticum。江苏、安徽、山东和河南抗多菌灵菌株F.asiaticum产生毒素的化学型为3-AcDON和NIV,并以3-AcDON为主。江苏省连续使用多菌灵防治小麦赤霉病长达20多年后才检测到田间抗性菌株,而近年来检测到田间抗性菌株的山东、河南2省用多菌灵防治赤霉病的历史较短,且为偶尔使用,药剂的选择压力相对较小,因此推测山东和河南麦区出现的小麦赤霉病菌抗多菌灵菌株可能是通过种子调运及联合收割机跨区作业等方式从抗药性发生较早的江淮麦区流入的。     

2017年-2021年江苏省小麦赤霉病菌群体对4种杀菌剂的抗药性监测

为明确江苏省小麦赤霉病菌群体对常用杀菌剂的抗药性情况,本研究于2017年-2021年连续5年分别从苏南、苏北及苏中小麦种植区采集病穗,分离培养获得赤霉病菌菌株共计4 055株,采用区分剂量法测定了供试菌株对4种杀菌剂(多菌灵、氰烯菌酯、戊唑醇和咪鲜胺)的抗性频率。结果表明:2017年-2021年江苏省田间小麦赤霉病菌对多菌灵的抗性频率分别为58.44%、56.30%、44.68%、48.28%、46.39%,呈逐年下降的趋势;不同地区小麦赤霉病菌对多菌灵的抗药性呈现地区差异性,苏北地区对多菌灵的抗性频率最高,其次是苏中地区,抗性频率最低的是苏南地区。2021年首次在田间采集到对氰烯菌酯、戊唑醇和咪鲜胺的抗性菌株,其抗性频率分别为0.45%、0.89%和0.09%,在以上的抗性菌株中同时具有多菌灵抗性的双重抗性菌株。表明江苏省小麦赤霉病菌对氰烯菌酯、戊唑醇和咪鲜胺产生了抗药性,虽抗性频率较低,仍需尽快制定田间抗性治理方案,延缓田间病菌对常用杀菌剂的抗性发展。     

江苏小麦赤霉病综合防控关键技术研究

近年来气候变暖、秸秆还田和赤霉病菌对多菌灵的抗性上升使江苏省小麦赤霉病流行频率提高。本研究通过田间小区试验,对小麦赤霉病的综合防控关键技术进行了比较。试验结果表明,目前赤霉病菌对多菌灵抗药性频率已达60%以上,多菌灵的防效明显下降,低于60%。研究明确了赤霉病的防控适期和防治次数,对淮南、淮北主要小麦品种进行赤霉病自然发病鉴定,并阐述了品种抗性和药剂防治间的互作效应。研究结果揭示,选育推广对小麦赤霉病具有抗性的品种是小麦赤霉病防控的基础,把握适期于小麦扬花期施用非多菌灵类的戊唑醇、氰烯菌酯、丙硫菌唑等其他杀菌剂可有效控制病害的发生。     

河南省小麦赤霉病发生规律与综合防治关键技术

为明确河南省小麦赤霉病发生规律,对1989年以来河南省小麦赤霉病历史资料进行了分析。结果表明,进入21世纪以来,赤霉病在河南省流行频率提高、为害损失增大,已经成为小麦的常发性病害。调查发现,麦田病残体带菌率10%以上,能满足大发生的菌源量需要。引起河南省小麦穗腐的优势病原菌为禾谷镰刀菌、假禾谷镰刀菌和链格孢,分离频率分别为29.69%、12.89%和42.19%。对赤霉病病菌多菌灵抗性监测结果表明,豫南麦区多菌灵抗性菌株平均比率达10.99%,河南省尚未检测到对戊唑醇、咪鲜胺、氰烯菌酯产生抗性的菌株。43个主栽小麦品种在赤霉病自然发病条件下,普遍表现为不抗侵染。不同杀菌剂对小麦赤霉病的防效试验结果表明,25%氰烯菌酯悬浮剂1 500 mL/hm~2、15%丙唑·戊唑醇悬浮剂900～1 500 mL/hm~2、40%丙唑·戊唑悬浮剂450～600 g/hm~2、20%叶菌唑悬浮剂300～600 g/hm~2及0.3%四霉素水剂1 000 mL/hm~2对小麦赤霉病的防效较好。     

黑龙江和云南葡萄产区灰霉病菌对多菌灵和异菌脲的抗性检测

为明确黑龙江省和云南省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率, 从两省分别采集?分离到葡萄灰霉病菌 Botrytis cinerea 39株和38株, 利用菌丝生长速率法测定其对多菌灵和异菌脲的敏感性, 并分别检测上述菌株两种药剂靶标基因 β-tubulin 和 Bos1 的突变情况?结果表明, 黑龙江省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率以及对两种杀菌剂的多药抗性频率分别为100.0%?51.3%和51.3%; 云南省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率以及对两种杀菌剂的多药抗性频率均为97.4%?两地区的多菌灵抗性菌株的 β-tubulin 基因上均发生了E198A或E198V突变?异菌脲抗性菌株的 Bos1 基因存在I365N?I365S?Q369P+N373和Q369P等4种突变类型?研究结果表明, 在上述两个地区不宜单独使用多菌灵和异菌脲防治葡萄灰霉病, 应使用其他作用机制不同且抑菌效果好的杀菌剂?     

戊唑醇与叶菌唑对小麦赤霉病的防治效果

小麦赤霉病是我国小麦产区的主要病害。种植抗病品种是防治该病最经济有效的方法,但由于小麦对赤霉病的抗性属水平抗性,受环境影响比较大,难以抵抗病菌的侵染危害。因此,防治赤霉病最有效的方法是化学防治。我国目前防治赤霉病的药剂以多菌灵为主,已有报道赤霉病菌对多菌灵产生了抗药性,所以筛选新型的杀菌剂用于小麦赤霉病     

东台市小麦赤霉病病菌抗药性监测与病害防控

通过监测东台市小麦赤霉病病菌对多菌灵的抗药性消长动态,总结出抗性菌株分布广泛、抗性频率上升迅速、区域分布极不平衡、抗性消长存在自然调节等抗性发展特点。分析发现多菌灵长期大量使用、小麦种子远距离调运、抗性菌株竞争力下降等是赤霉病病菌抗药性发展和变化的主要影响因素。在此基础上,提出了引进种植抗病品种、合理安排品种布局、规范种子生产管理、开发生物防控技术、调整化学药剂施用配方等防治小麦赤霉病、控制赤霉病病菌抗药性发展的对策。     

长江中下游地区亚洲镰刀菌NX-2毒素群体的检测

由禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的麦类赤霉病,是农业生产上的重要病害。为明确中国长江中下游冬小麦主产区小麦赤霉病菌种的构成及其地理分布,对2008年从江苏、浙江和湖北3省采集的656株小麦赤霉病菌株进行了分类鉴定。结果显示,其中558个菌株为亚洲镰刀菌(Fusarium asiaticum),98个菌株为禾谷镰刀菌(Fusarium graminearum sensu stricto),表明中国长江中下游冬小麦主产区小麦赤霉病的主要致病菌是亚洲镰刀菌。选择亚洲镰刀菌(F.asiaticum)为研究对象,通过PCR-RFLP的方法对其进行产NX-2毒素菌株的检测。结果没有检测到产NX-2毒素菌株,表明中国长江中下游冬小麦主产区并未出现NX-2毒素群体。本研究旨在了解NX-2毒素群体在中国长江中下游地区的地理分布,为进一步研究麦类赤霉病菌群体遗传多样性和演化趋势奠定基础,为麦类赤霉病的防治和毒素污染的控制提供理论依据。     

江苏垦区小麦赤霉病菌对多菌灵的抗性监测及其替代杀菌剂的防病效果

近年来的调查结果表明,江苏垦区小麦赤霉病菌群体中抗多菌灵菌株的频率逐年上升,多菌灵防效逐年下降,部分农场采用多菌灵防治几乎失效。为了筛选替代多菌灵的药剂,我们在白马湖等6个农场对几种新杀菌剂进行了田间药效试验。结果表明,25%氰烯菌酯SC、40%叶菌唑SC和25%丙硫菌唑WP在所有农场都有较好的防治效果,其中25%丙硫菌唑WP对小麦赤霉病的防治效果最好,在所有农场其防效都超过80%,防治后乳熟期和成熟期病穗率变化不大。40%叶菌唑SC、25%丙硫菌唑WP以及25%氰烯菌酯SC和43%戊唑醇SC混用有显著增产作用,并能大幅度降低籽粒中DON含量。作者认为赤霉病的防治应立足种植抗(耐)病品种,扬花期选用有效杀菌剂适期进行防治。氰烯菌酯、叶菌唑和丙硫菌唑可作为多菌灵的替代药剂。     

北京市及河北省小麦赤霉病菌群体遗传结构及生物学特性

本文分析了北京市与河北省小麦赤霉病菌群体遗传结构以及基础生物学特性。结果表明所有菌株均为禾谷镰刀菌(Fusarium graminearum),属于一个大的单一群体,群体内具有较高的遗传多样性。毒素化学型测定表明,北京与河北地区小麦真菌毒素污染的主要风险为DON与15ADON毒素。表型测定显示,与F.asiaticum群体相比,F.graminearum具有较高的产孢能力,而生长速率和产毒能力较低。该群体对主要杀菌剂多菌灵、戊唑醇和氰烯菌酯均无抗药性。     

禾谷镰孢菌对苯醚甲环唑的敏感性基线及与其他杀菌剂敏感性的相关性分析

由禾谷镰孢菌引起的赤霉病是小麦上的重要病害，可严重影响小麦的产量并降低小麦的品质。苯醚甲环唑属于三唑类杀菌剂，是甾醇脱甲基化抑制剂，具有较高的抑菌活性。采用菌丝生长速率法测定了于2016—2017采集自河南省的107株禾谷镰孢菌对苯醚甲环唑的敏感性。结果表明:苯醚甲环唑对禾谷镰孢菌菌丝的生长具有较强的抑制效果，其有效抑制中浓度(EC₅₀)值范围为0.0128~0.6079 mg/L，符合正态分布，平均EC₅₀值为(0.2239 ± 0.1192) mg/L。因此，这些敏感性数据可以作为河南省禾谷镰孢菌对苯醚甲环唑的敏感性基线。通过对苯醚甲环唑与其他7种杀菌剂氟环唑、多菌灵、氰烯菌酯、氟唑菌酰羟胺、戊唑醇、丙硫菌唑及叶菌唑对20株禾谷镰孢菌的log₁₀ EC₅₀值之间的Spearman’s rho (ρ) 相关性分析发现:苯醚甲环唑与叶菌唑之间具有较低水平的相关性，与其他供试杀菌剂之间无相关性。本研究可为监测河南省禾谷镰孢菌对苯醚甲环唑的抗药性发展和防控小麦赤霉病合理用药提供依据。     

禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈的抗性机制

由禾谷镰刀菌引起的小麦赤霉病是世界小麦生产上的重要真菌病害。为了进一步明确禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈产生抗性的机制,本文以前期室内通过药剂驯化方式得到的4株禾谷镰刀菌对咯菌腈的高水平抗性突变体 (其抗性倍数在318.2~782.9之间) 为主要研究材料,采用生物测定及分子生物学等方法开展了禾谷镰刀菌对咯菌腈的抗性机制研究。结果表明:供试禾谷镰刀菌抗咯菌腈突变体对小麦幼穗的致病力降低了约50%,部分菌株 (2XZ-4R) 甚至完全丧失了对小麦的致病能力;抗性突变体对渗透胁迫 (0.5 mol/L NaCl, 1.0 mol/L MgCl₂, 1.0 mol/L葡萄糖或1.0 mol/L甘露醇) 高度敏感,且菌丝生长抑制率较敏感菌株降低约50%以上,表明其环境适合度显著下降。同时,抗性突变体中苯丙氨酸解氨酶 (PAL)、过氧化物酶 (POD) 和多酚氧化酶 (PPO) 活性较敏感菌株均升高2倍以上。分子生物学分析发现,供试抗性突变体中候选靶标基因 (FgOs1和FgOs5) 的表达量显著下调 (P<0.05),推测FgOs1和FgOs5可能参与了禾谷镰刀菌对咯菌腈抗性的形成过程。总之,该研究探究了禾谷镰刀菌抗咯菌腈突变体的生物学特性,并为深入揭示禾谷镰刀菌对咯菌腈的抗性分子机制提供了新的思路。     

禾谷镰孢菌对戊唑醇抗药性的诱导及抗性菌株特性研究

通过紫外线诱导获得了8株对戊唑醇具有不同抗性水平的禾谷镰孢菌Fusarium graminearum抗药突变体,其抗性倍数均≥30倍,最高达到186.03倍。将抗药突变体在无药培养基上继代培养9代后,其抗性倍数逐渐下降,抗药性可能不能稳定遗传。与亲本菌株相比,抗药突变体在菌落生长速率、菌丝干重、对温度和pH值的敏感性方面均有所下降,推测禾谷镰孢菌对戊唑醇可能具有中等或高抗药性风险。室内交互抗性测定结果表明,戊唑醇对苯醚甲环唑、多菌灵、异菌脲、百菌清及福美双均无交互抗性。     

Within-field variation of Fusarium graminearum isolates for aggressiveness and deoxynivalenol production in wheat head blight

Fusarium head blight (FHB), caused by Fusarium graminearum sensu stricto (s.s.), causes tremendous annual yield losses in wheat worldwide. Variation of aggressiveness of isolates from individual field populations in terms of FHB infection and deoxynivalenol (DON) concentration in the host are important population parameters reflecting parasitic ability. Our main objective was to estimate the variation of both traits within three populations of F. graminearum s.s., each consisting of 30 single-spore isolates collected from small wheat fields in Germany, and to compare it with 11 isolates of a collection (F. graminearum collection) from four countries. The same isolates were characterized using 19 single-sequence repeat markers. All isolates were spray inoculated on a moderately resistant spring wheat cultivar at two field locations over 2 years (i.e., in four environments). The genotypic proportion of phenotypic variance (σ(2)(G)) within populations was significant (P < 0.01) for both traits, and the σ(2)(G) × environment interaction was even more important for mean FHB severity. Ranges in mean FHB severity and DON concentration in the host were only slightly smaller for the field populations than for the F. graminearum collection. Both traits were significantly (P < 0.05) correlated within and across populations. A further partitioning of σ(2)(G) revealed 72% of σ(2)(G) within and 28% of σ(2)(G) across populations for both traits. Molecular variance of the three populations was similarly distributed (73.6% within versus 26.4% between populations). In view of this high within-field variation for traits of parasitic ability and selection, neutral molecular markers, multiple resistance genes of different origin should be employed in wheat breeding programs to obtain a long-term stable FHB resistance.     

2013年湖北省小麦赤霉病菌对多菌灵和戊唑醇的敏感性

2013年从湖北省7个小麦主产区分离获得106株禾谷镰刀菌,测定了其对戊唑醇和多菌灵的敏感性。结果表明:戊唑醇和多菌灵对所有供试菌株的EC50值分别为0.064~0.778和0.090~0.858 mg/L。采用SAS软件的W法对EC50分布进行了正态性检验,表明106株菌株对戊唑醇和多菌灵敏感性的频率分布符合正态分布,其EC50平均值分别为(0.383±0.129)和(0.526±0.151)mg/L。不同地区来源的菌株对两种药剂的敏感性存在显著差异,其中襄阳的菌株对两种药剂的敏感性显著低于其他6个地区的。研究结果显示:湖北省小麦赤霉病菌未出现对戊唑醇和多菌灵抗性菌群,两种药剂用于防治小麦赤霉病仍具有使用价值。     

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.     

Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China

A large number of Fusarium graminearum and F. asiaticum isolates were collected from wheat spikes from all regions in China with a history of fusarium head blight (FHB) epidemics. Isolates were analysed to investigate their genetic diversity and geographic distribution. Sequence characterized amplified region (SCAR) analyses of 437 isolates resolved both species, with 21% being F. graminearum (SCAR type 1) and 79% being F. asiaticum (SCAR type 5). AFLP profiles clearly resolved two groups, A and B, that were completely congruent with both species. However, more diversity was detected by AFLP, revealing several subgroups within each group. In many cases, even for isolates from the same district, AFLP haplotypes differed markedly. Phylogenetic analyses of multilocus DNA sequence data indicated that all isolates of SCAR type 1, AFLP group A were F. graminearum , whilst isolates of SCAR type 5, AFLP group B were F. asiaticum , demonstrating that it is an efficient method for differentiating these two species. Both species seem to have different geographic distributions within China. Fusarium graminearum was mainly obtained from wheat growing in the cooler regions where the annual average temperature was 15°C or lower. In contrast, the vast majority of F. asiaticum isolates were collected from wheat growing in the warmer regions where the annual average temperature is above 15°C and where FHB epidemics occur most frequently. This is the first report of the distribution of, and genetic diversity within, F. graminearum and F. asiaticum on wheat spikes throughout China.