植物病原真菌对几类重要单位点杀菌剂的抗药性分子机制

单位点杀菌剂是植物病害管理的重要组成部分,随着单位点杀菌剂的大量、广泛使用,抗性问题也随之产生。目前为止,有植物病原菌对各大类单位点杀菌剂均具抗性的报道。本文作者主要阐述了生产中常用的5类单位点杀菌剂,包括苯并咪唑类杀菌剂（MBCs）、二甲酰亚胺类杀菌剂（DCFs）、14α-脱甲基酶抑制剂（DMIs）、QoIs和琥珀酸脱氢酶抑制剂（SDHIs）的作用机理及抗性分子机制的研究进展,并进一步论述了抗药性产生的机理及抗性治理原则。MBCs作用于β-微管蛋白,抗性主要与靶标蛋白基因的点突变有关,突变造成的氨基酸变化多集中于第50、167、198、200和240等5个位置,主要突变位点为第198位,同一菌株通常只发生一个氨基酸变异,不同位点的点突变甚至同一位点的不同氨基酸替代均会引起抗性水平的差异;DCFs的作用靶标尚不清楚,病原真菌对其抗性可能与双元组氨酸激酶OS基因的点突变有关;DMIs通过抑制14α-脱甲基酶最终影响麦角甾醇的合成,抗性主要与Cyp51的点突变或过量表达或运输体的过量表达相关,Cyp51点突变是抗DMI的主要机制,同一突变对不同的三唑类杀菌剂敏感性表现不尽相同,不同位置的点突变在同一病原菌中对不同三唑类杀菌剂的敏感性影响也不同。点突变数量在不同的真菌中表现不同,有单个发生,也有多个同时发生,且对抗药性具有积累效应;QoIs作用于电子传递链的复合物III,抗性主要与Cytb的点突变有关,与抗性相关的点突变主要发生在Cytb的120-155和255-280两个编码区,其中G143A和F129L为最主要的点突变;SDHIs作用于电子传递链的复合物II,抗性主要与SdhB、SdhC或SdhD的点突变有关,大部分病原真菌对SDHIs的抗性与SdhB点突变有关,SdhB点突变发生位置比较单一,在多种病原菌中突变均发生在相同的组氨酸上即H272, 而SdhC和SdhD突变位点比较多。     

绿海龟背甲坏死病病原真菌的鉴定及其药敏特性

人工饲养的绿海龟(Chelonia mydas)患背甲坏死病的背甲整体呈现"灰白甲"症状,用伍德灯检查有荧光现象,即疑似真菌感染;从绿海龟背甲边缘病变坏死处分离纯化得到1株优势真菌,编号为SDABAI,通过形态学观察、分生孢子形态并结合ITS 18S rDNA序列分析对疑似致病菌株进行鉴定,进而构建系统发育树进行分析。结果表明:SDABAI菌株(登录号为:MK583351)与Fusarium solani(登录号为:KU377470.1)基因相似性达99%,判定SDABAI菌株为腐皮镰刀菌(Fusarium solani),真菌菌丝形态、分生孢子产生和形态也符合该菌。将患病绿海龟背甲病灶处分离得到的SDABAI菌株,接种到4 mL离心管中,斜面培养生长4～5 d长出白色絮状菌苔后,用光固化型树脂封闭在2只健康绿海龟(A1、A2)的背甲上各2只带菌离心管(A1-1;A1-2;A2-1;A2-2)内,攻毒感染第24天,A1出现被感染症状,有荧光现象,产生了和自然发病一样的症状,并于背甲边缘生长纹处分离到了和SDABAI菌株一样的腐皮镰刀菌。A2较A1症状较弱。根据绿海龟背甲整体呈现"灰白甲"症状和腐皮镰刀菌病原,定名为绿海龟背甲坏死病。选用大蒜油、艾叶油和茶树油进行腐皮镰刀菌药敏试验,结果表明,三者均可起到一定抑制该菌生长的效果,还可以抑制次生菌生长,大蒜油的抑菌效果最佳。特比萘芬、克霉唑、咪康唑、制霉菌素、两性霉素5种药对腐皮镰刀菌药敏试验结果显示咪康唑和制霉菌素的抑菌效果较佳。     

特比萘酚及其盐酸盐的农用抗真菌活性

采用菌丝体生长速率抑制法和盆栽喷雾法测定特比萘酚和盐酸特比萘酚对16种植物病原菌的离体杀菌活性及其对9种植物病害的盆栽防效。结果表明,特比萘酚和盐酸特比萘酚对多种植物病原菌具有良好的杀菌活性,其中特比萘酚对8种真菌(山茶灰斑病菌、小麦赤霉病菌、玉蜀黍赤霉病菌、茄链格孢病菌、火龙果溃疡病菌、香蕉炭疽病菌、橡胶炭疽病菌和冬瓜炭疽病菌)的抑制活性较强,8种真菌的EC₅₀为0.03～0.24 mg·L?1,盐酸特比萘酚对山茶灰斑病菌、小麦赤霉病菌、玉蜀黍赤霉病菌、茄链格孢病菌和火龙果溃疡病菌的抑制活性较强,EC₅₀为0.03～0.22 mg·L?1。盆栽活体实验结果表明,特比萘酚和盐酸特比萘酚对多种农作物真菌病害具有良好的防治效果,在浓度为100 mg·L?1时均能100%控制豇豆白粉病;在浓度为400 mg·L?1时均能够有效抑制水稻纹枯病、小麦白粉病、黄瓜靶斑病、黄瓜白粉病和玉米大斑病,防效达到90%～100%。     

Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in the high-quality soft wheat cultivar Yangmai 15

Fusarium head blight(FHB) is one of the most detrimental wheat diseases which greatly decreases the yield and grain quality, especially in the middle and lower reaches of the Yangtze River of China. Fhb1 and Fhb2 are two major resistance loci against Fusarium graminearum. Yangmai 15(YM15) is one of the most popular varieties in the middle and lower reaches of the Yangtze River, and it has good weak gluten characters but poor resistance to FHB. Here we used Fhb1 and Fhb2 to improve the FHB resist...     

An integrated pest management program for managing fusarium head blight disease in cereals

Fusarium head blight (FHB) is a worldwide devastating disease of small grain cereals and Fusarium graminearum species complex (FGSC) is the major pathogen causing the disease. The epidemics of FHB lead to the reduction of grain yield and economic losses. Additionally, mycotoxins produced by the FHB pathogens are hazardous to the health of human and livestock. In this review, we summarize the epidemiology of FHB, and introduce effects of this disease on economy, environment and food safety. We focus on the integrated management approaches for controlling FHB including agronomic practices, resistant cultivars, chemical control, and biocontrol. In addition, we also discuss the potential novel management strategies against FHB and mycotoxin.     

A LAMP-assay-based specific microbiota analysis reveals community dynamics and potential interactions of 13 major soybean root pathogens

Soybean root diseases are associated with numerous fungal and oomycete pathogens; however, the community dynamics and interactions of these pathogens are largely unknown. We performed 13 loop-mediated isothermal amplification(LAMP) assays that targeted specific soybean root pathogens, and traditional isolation assays. A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages(30, 60, and 90 days after planting) in 2016. In LAMP results, we found that pathogen communities differed slightly among locations, but changed dramatically between soybean growth stages. Phytophthora sojae, Rhizoctonia solani, and Fusarium oxysporum were most frequently detected at the early stage, whereas Phomopsis longicolla, Fusarium equiseti, and Fusarium virguliforme were most common in the later stages. Most samples(86%) contained two to six pathogen species. Interestingly, the less detectable species tended to exist in the samples containing more detected species, and some pathogens preferentially co-occurred in diseased tissue, including P. sojae–R. solani–F. oxysporum and F. virguliforme–Calonectria ilicicola, implying potential interactions during infection. The LAMP detection results were confirmed by traditional isolation methods. The isolated strains exhibited different virulence to soybean, further implying a beneficial interaction among some pathogens.     

Method for Isolating Mitochondrial DNA from Etiolated Tissue of Cabbage

Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.     

Characterization of laccase gene StLAC6 and its involvement in the pathogenicity and peroxisome function in Setosphaeria turcica

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. tur...     

FgHAT2 is involved in regulating vegetative growth,conidiation, DNA damage repair,DON production and virulence in Fusarium graminearum

Histone lysine acetylation is catalyzed by acetyltransferases(HATs), which is important in regulating gene expression and physiological function in eukaryotic cells. HATs can be classified into two main types: A-and B-type HATs. Recently, in Fusarium graminearum, it has been reported that A-type HATs are involved in hyphal development, conidiation, sexual reproduction and virulence. However, the biological roles of B-type HATs are unknown. Here we report the identification and characterization of two B-type HATs(FgHat1 and FgHat2) in F. graminearum. Targeted deletion of FgHAT1 did not result in any detectable phenotypes. However, ΔFghat2 mutants were severely defective in vegetative growth, conidia production and morphogenesis, deoxynivalenol(DON) biosynthesis and virulence. Interestingly, deletion of Fg HAT2 resulted in significantly increased sensitivity to the DNA-damaging agent methyl methanesulfonate(MMS). Furthermore, double deletion mutants(ΔFghat1ΔFghat2) displayed similar phenotypes to the ΔFghat2 mutants. Taken together, we conclude that FgHat2 but not FgHat1 plays essential roles in regulating morphogenesis, DNA damage repair, DON production and virulence in F. graminearum.     

水稻稻曲病菌的分离鉴定及室内毒力测定

为优化稻绿核菌的分离方法,筛选防控稻曲病的有效药剂,采用孢子敲落法和火焰灼烧法分别分离不同成熟度的稻曲球,从形态学、致病性、rDNA-ITS序列等方面对HP-7菌株进行鉴定,并通过菌丝生长抑制法测定了HP-7菌株对7种杀菌剂的敏感性。结果表明,采用孢子敲落法分离鲜黄色稻曲球成功率最高,达98.33%,将HP-7菌株的rDNA-ITS序列在NCBI上进行BLAST比对,菌株HP-7与稻绿核菌相似度达99%,结合形态学特征分析确定HP-7为稻绿核菌。离体试验表明,戊唑醇和嘧菌酯对稻绿核菌的菌丝生长抑制作用最强,其EC_(50)值分别为0.109 1、0.158 5mg·L~(-1)。其结果可为稻绿核菌的分离鉴定及稻曲病防控提供理论参考。     

几种复配杀菌剂对小麦赤霉病及DON毒素污染控制作用

为了筛选防治小麦赤霉病和控制毒素的高效杀菌剂,通过田间小区药效试验及小麦样品DON毒素检测,研究了4种杀菌剂对小麦赤霉病的防治效果和控制毒素的能力。结果表明,36%克菌丹·叶菌唑SC、45%戊唑醇·咪鲜胺EW、48%氰稀·戊唑醇SC和40%戊唑·多菌灵SC在小麦抽穗80%,扬花初期施用,对小麦赤霉病均有较好的防治效果,病指防效在84.25%~93.96%,样品DON毒素检测结果在0.12~0.64μg/ml,其中36%克菌丹·叶菌唑SC 900 ml/hm2的病指防效为93.96%,样品平均DON毒素检测结果为0.12μg/ml,控制毒素效果达93.72%。     

几种杀菌剂对黄瓜枯萎病菌的联合毒力

采用室内离体平皿法测定了12种杀菌剂对黄瓜枯萎病的抑菌作用以及福星、世高和福美双不同组分配比的联合毒力.结果表明:40%福星EC、10%世高DG和50%福美双DC对黄瓜枯萎病菌具有优异的抑菌作用,其EC50分别为0.0998 μg/mL、0.2669 μg/mL和0.3795 μg/mL,3个混配制剂对黄瓜枯萎病的共毒系数分别为699.9、241.9和173.1.福星·世高配比(福星:世高为1:49)对黄瓜枯萎病菌的增效程度高于福美双·福星配比(福美双:福星为47:3)和福美双·世高配比(福美双:世高为42:8).     

A 13-kilodalton maize mitochondrial protein in E. coli confers sensitivity to Bipolaris maydis toxin

The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility. Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl. A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli. After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria. The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl. These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.     

SsRSS1 mediates salicylic acid tolerance and contributes to virulence in sugarcane smut fungus

Sugarcane smut caused by Sporisorium scitamineum is a destructive disease responsible for significant losses in sugarcane production worldwide. However, the mechanisms underlying the pathogenicity of this fungus remain largely unknown. In this study, we found that the disruption of the SsRSS1 gene, which encodes a salicylic acid (SA) sensing regulator, does not affect phenotypic traits such as the morphology, growth rate, and sexual mating ability of haploid basidiospores, but rather reduces the tolerance of basidiospores to SA stress by blocking the induction of SsSRG1, a gene encoding a SA response protein in S. scitamineum. SsRSS1 deletion resulted in the attenuation of the virulence of the fungus. In addition to a significant reduction in whip formation, a portion of plantlets (18.3%) inoculated with the ΔSsRSS1 strains were found to be infected but failed to produce whips for up to 90 days post-inoculation. However, the development of hyphae and teliospore from the ΔSsRSS1-infected plants that formed whips seemed indistinguishable from that in the wild-type-infected plants. Combined, our findings suggested that SsRss1 is required for maintaining fungal fitness in planta by counteracting SA stress.     

Comparsion of the Effects of Succinate and NADH on Postmortem Metmyoglobin Redcutase Activity and Beef Colour Stability

In two experiments, the effects of succinate and NADH（reduced nicotinamide adenine dinucleotide） on metmyoglobin reductase activity and electron transport chain-linked metmyoglobin reduction were investigated and compared. In experiment 1, metmyoglobin（MetMb）, substrate and inhibitors were incubated with mitochondria. Comparsion of the effects of succinate and NADH on MetMb reduction was investigated. The MetMb percentage in sample treated with 8 mol L-1 succinate decreased by about 69% after 3 h incubation, and the effect was inhibited by the addition of 10 mol L-1 electron transfer chain complex II inhibitor malonic acid; the MetMb percentage in samples treated with 2 mol L-1 NADH decreased by 56% and the effect was inhibited by the addition of 0.02 mol L-1 electron transport chain complex I inhibitor rotenone. These results indicated that electron transport chain played an important role in MetMb reduction. Both complex II and complex I take part in the MetMb reduction in mitochondria through different pathways. NADH-MetMb reduction system was less stable than succinateMetMb system. In experiment 2, the beef longissimus dorsi muscle was blended with different concentrations of succinate or NADH. Enhancing patties with higher concentration of succinate or NADH improved colour stability in vacuum packaged samples（P〈0.05）. These results verified that mitochondria electron transport chain is related to the MetMb reduction in meat system.     

In-vitro assessment for the control of Fusarium species using a lactic acid bacterium isolated from yellow pitahaya(Selenicereus megalanthus(K. Schum. Ex Vaupel Moran))

The fungistatic activity of a lactic acid bacterium, which had been isolated from yellow pitahaya cultures, against fungi associated with basal rot(Fusarium oxysporum and Fusarium fujikuroi) was measured in the present study. Its activity was assessed in three fractions: fermented(S1), metabolic products(S2), and biomass(S3), using two fermentation substrates: Man Rogosa Sharpe agar(MRS) and potato dextrose agar(PDA). The bacterium was molecularly identified as Lactobacillus plantarum. S3 reduced F. fujikuroi growth by 100% over 48 h of fermentation, which occurred during the stationary phase of bacterial growth. The three fractions' fungistatic activity against F. fujikuroi depended on the substrate employed. The fermentation kinetic parameters for L. plantarum indicated that its specific growth rate was 0.46 h~(–1), with 93.63% substrate consumption, 0.045 kg kg~(–1) cell yield, and 0.54 kg kg~(–1) product yield. The kinetic parameters calculated will allow for bacteria production scaling. These in-vitro trials reveal L. plantarum's possible application as a biocontrol agent for diseases associated with Fusarium. However, further ex-vivo and in-vivo researches are required to demonstrate its behavior in crops.     

观赏百合根腐病病原鉴定及药剂筛选

从百合典型根腐病症状组织中分离到了4种镰刀菌。经过对其病原菌形态、培养性状和致病性等方面的研究表明:尖孢镰刀菌(F. oxysporum)和茄病镰刀菌(F. solani)是引起观赏百合根腐病的主要致病菌,其分离频率分别为73.33 % 和16.67 %;其它两种镰刀菌F3、F4没有致病性。药剂筛选结果表明:供试药剂中50 % 多菌灵可湿性粉剂、70 % 甲基托布津可湿性粉剂、30 % 抗枯宁、多菌灵与甲基托布津复配剂(1 : 1)、多菌灵与代森锰锌复配剂(1 : 3)和甲基托布津与代森锰锌复配剂(1 : 3)6种药剂对百合根腐病的防治效果最好。     

香蕉枯萎病的发生及防控研究现状

香蕉是世界第2大水果作物,也是世界贸易量最大的水果。由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp.cubense,Foc)引起的香蕉枯萎病是香蕉生产上最重要的病害之一,极大地限制了世界香蕉产业的健康和可持续发展。近年来国内外学者在其病害发生、病原生物学、侵染过程、病害流行、基因组测序、香蕉与Foc互作以及病害防控等方面开展了大量研究,但鲜见较为系统和全面的评述。本文就香蕉枯萎病的发生历史和危害现状、病原小种和遗传多样性以及防控方法等进行较为全面的梳理和总结,以期为该病害的研究和防控提供一定的参考。     

戊唑醇与羟菌唑对小麦赤霉病的防治效果及对小麦产量的影响

通过室内和田间药效试验,研究了新型三唑类杀菌剂戊唑醇与羟菌唑对小麦赤霉病的防治效果。温室盆栽试验结果表明:25%的戊唑醇(3.15mL/L)、6%的羟菌唑(3.75mL/L)可有效控制赤霉病菌在小麦穗部小穗之间的扩展,防效高于多菌灵常规用量。田间药效测定结果表明:与对照相比,接种前2d喷药与接种后2d喷药,2001年,戊唑醇可使小麦赤霉病病情指数分别减少80.32%和70.64%,产量增加12.95%与8.74%;羟菌唑可使小麦赤霉病病情指数减少80.5%和68.0%,产量增加11.41%和11.45%;2002年,在小麦赤霉病严重发生的情况下,戊唑醇能使小麦赤霉病病情指数减少76.47%和78.67%,羟菌唑能使小麦赤霉病病情指数减少82.06%和81.89%,产量增加极显著。说明这2种药剂可以作为防治小麦赤霉病的替代品种。