基于Dual RNA-seq分析的褪黑素影响萝卜与链格孢菌互作的作用研究

由芸薹链格孢菌(Alternaria brassicae)严重影响萝卜(Raphanus sativus)生产,外源喷施褪黑素可减轻病症,但褪黑素同时存在于植物和微生物中,外源喷施时可对感病植株上寄主和病原物的生命活动均有影响,其介导寄主-病原菌互作的作用模式。为解析褪黑素参与调控病叶上萝卜和链格孢菌互作的生理和基因转录机制,本研究通过对感黑斑病萝卜叶面外施0~1 500 μmol·L^-1浓度褪黑素,检测病情指数变化,同时利用互作转录组分析0、500和1 500 μmol·L^-1褪黑素处理下萝卜和链格孢菌转录水平变化。结果显示,50~500 μmol·L^-1褪黑素可分别显著提高萝卜和链格孢菌双方的生长势和抗逆性,且500 μmol·L^-1表现最优,1 000和1 500 μmol·L^-1与500 μmol·L^-1相比作用较小,呈明显剂量依赖型模式,但500 μmol·L^-1褪黑素处理下,萝卜对链格孢菌抗性明显提升。Dual RNA-seq检出萝卜基因组reads比对率>88%,链格孢菌基因reads比对率≤0.06%,萝卜差异表达基因数显著高于链格孢菌检出数。对萝卜及链格孢菌生长及抗逆性调控相关基因表达模式进行验证,基因表达模式基本与表型反应一致。综上所述,褪黑素介导萝卜与链格孢菌的作用存在低浓度促进、高浓度抑制的剂量依赖性效应,500 μmol·L^-1浓度下褪黑素对双方均具生长促进作用,但褪黑素对寄主的影响超过对链格孢菌的影响,最终表现为诱导萝卜对链格孢菌抗性增强。本研究为褪黑素在萝卜黑斑病抑制中的应用提供了数据支撑和理论基础,并在转录水平上初步解析了褪黑素参与萝卜对链格孢菌抗性变化的作用模式。     

鲜枣贮藏期致腐病原菌种类研究

以7个鲜枣品种为试材,采用植物病理学方法,研究了鲜枣在贮藏期主要致腐病原菌的种类。试验共分离得到6种病原菌:链格孢Alternaria alternata(Fr.∶Fr.)Keissler,细极链格孢Alternaria tenuissima(Fr.)Wilt-shire,桔青霉Penicillium citrinumThom,顶青霉Penicillium corylophilum Dierckx,镰孢Fusarium sulphureum Schlecht,芽枝孢Cladosporium sp.,发现其中5种病原菌是目前鲜枣贮藏期发生腐烂的主要病原菌。     

万寿菊叶斑病的发生及病原鉴定

2006年对万寿菊叶斑病在甘肃主要栽培区的武威黄羊镇、古浪、景泰等地发生情况的调查结果表明,发病率在33%～85%,病情指数在5.7～48.0。通过室内分离鉴定,确定引起当地万寿菊叶斑病的病原为真菌类的链格孢Alternaria sp.。     

谷物中链格孢毒素的研究进展

链格孢毒素是由链格孢霉产生的广泛污染谷物和饲料的真菌毒素,其中最主要的几种链格孢毒素有交链孢酚(AOH)、交链孢酚单甲醚(AME)、交链孢烯(ALT)、交链孢菌酮酸(Te A)和交链孢毒素(ATX),本文系统的总结了谷物中链格孢霉毒素的污染来源、毒性作用机制、检测方法和降解研究进展,旨在为谷物中链格孢毒素的消解提供技术基础,从而减少谷物的污染损失,保障粮食质量安全。     

橄榄黑斑病病原菌的分离鉴定及防治药剂筛选

为明确引起潮州市橄榄黑斑病的病原菌种类，筛选有效防治药剂，对发病果实采用组织分离法进行了病原菌的分离与纯化，并结合形态学和分子生物学技术对病原菌进行鉴定。鉴定结果表明，引起橄榄黑斑病的病原菌为链格孢菌(Alternaria alternata)。通过菌丝生长速率法测定了14种杀菌剂对病原菌的抑菌效果，结果表明14种杀菌剂对链格孢菌均有抑制作用，其中3%中生菌素可湿性粉剂、80%克菌丹水分散粒剂、8%氟硅唑微乳剂和40%苯甲·嘧菌酯悬浮剂的抑菌效果较好，EC50分别为1.267、1.207、1.100、0.944 mg·L^-1。田间防效测定结果表明，40%苯甲·嘧菌酯悬浮剂、8%氟硅唑微乳剂的防治效果显著，防效为68.58%、65.44%；而30%王铜、2%春雷霉素的防效较差，仅为10%左右。综上，推荐将8%氟硅唑微乳剂和40%苯甲·嘧菌酯悬浮剂2种药剂作为防控橄榄黑斑病的首选药剂。本研究可为橄榄黑斑病高效杀菌剂的筛选提供数据参考，并为其防控工作提供科学依据。     

甘肃玉米镰孢茎腐病病原菌种群多样性分析

为了解甘肃玉米镰孢茎腐病致病菌的种群结构和数量,于2015和2017年在甘肃省10个市(州)采集玉米茎腐病样品42份,根据形态学特征和EF-1α (tef)基因序列分析进行病原菌的种类鉴定。结果表明,共获得10种镰孢菌,分别为禾谷镰孢菌复合种(Fusarium graminearum species complex, FGSC, 59.3%)、拟轮枝镰孢(F.verticillioides,11.5%)、木贼镰孢(F. equiseti,10.3%)、胶孢镰孢(F. subglutinans,5.9%)、层出镰孢(F. proliferatum, 4.7%)、变红镰孢(F. incarnatum, 4.0%)、三线镰孢(F. tricinctum, 1.9%)、温带镰孢(F. temperatum, 1.2%)、锐顶镰孢(F. acuminatum,0.8%)和尖孢镰孢(F. oxysporum, 0.4%),其中三线镰孢和锐顶镰孢作为玉米茎腐病新病原,属国内外首次发现。禾谷镰孢菌复合种、拟轮枝镰孢、木贼镰孢和胶孢镰孢在甘肃四大生态区(陇东地区、陇南地区、陇中地区和河西走廊)均有分布,其余种仅在1~3个生态区分布。利用镰孢菌的特异性引物EF-1α (tef)对甘肃玉米镰孢茎腐病优势病原菌禾谷镰孢复合种进行种群检测,共鉴定出布氏镰孢(F. boothii)和禾谷镰孢2个种群,其比例为2.75∶1。选用玉米品种甘宇301按照柯赫氏法则进行致病性测定,结果发现10种镰孢菌均可致病。本研究结果为甘肃玉米茎腐病的综合防控提供了科学依据。     

2,4-表油菜素内酯结合氯化钙处理对采后杏果实黑斑病及苯丙烷代谢的影响

为探究减轻杏果实采后黑斑病的方法,以赛买提杏果实为试验材料,经3.0 mg·L^-1 2,4-表油菜素内酯(EBR)结合2%氯化钙(CaCl₂)减压渗透处理后,于温度1.0±1℃、相对湿度90%~95%条件下放置48 h后,进行交孢链格孢(Alternaria alternata)损伤接种,并进行A. alternata离体抑菌试验。结果表明,3.0 mg·L^-1 EBR结合2% CaCl₂对A. alternata的菌落直径和孢子萌发没有直接抑制作用。EBR结合CaCl₂处理可通过诱导苯丙氨酸解氨酶(PAL)、肉桂酸-4-羟化酶(C4H)、香豆酸辅酶A连接酶(4CL)活性上升,增加总酚、类黄酮和木质素含量来抑制杏果实接种发病率与病斑直径的上升。综上,EBR结合氯化钙处理可通过诱导苯丙烷代谢途径来增强杏果实对黑斑病的抗性。本研究结果可为减轻杏果实采后黑斑病提供技术参考与理论依据。     

十字花科植物黑斑病的研究进展

十字花科植物黑斑病是由死体营养型致病菌链格孢属(Alternaria spp.)真菌侵染引起的重要病害之一。链格孢菌侵染植物的过程,首先通过释放毒素诱导侵染处植物细胞的程序性死亡,再通过细胞壁降解酶分解死亡的细胞。同时,植物也会通过激发先天性免疫反应,调节植物内源激素和植物抗毒素等分子抵御病菌的侵染。本文重点综述了链格孢菌侵染十字花科植物过程中,双方在基因组(致病和抗病基因)、转录组(调控因子)、代谢组(毒素和抗毒素)和蛋白组(酶类)等方面的相关变化及其作用机制,以期为培育抗黑斑病的十字花科蔬菜提供有价值的参考。     

二苯-α-吡喃酮类链格孢霉毒素和人血清白蛋白的相互作用及机理探究

为探究链格孢酚单甲醚(AME)和链格孢酚(AOH)这两种二苯-α-吡喃酮类链格孢霉毒素与人血清白蛋白(HSA)之间的分子相互作用及机理,本研究模拟血液生理pH值,采用稳态荧光光谱、同步荧光光谱、3D荧光光谱以及圆二色光谱等方法研究两者之间的相互作用。结果表明,AME和AOH与HSA相互作用均发生了静态猝灭,具有较高亲和力,以氢键和范德华力(ΔH<0,ΔS<0)结合形成1:1复合物,互作过程中这两种类毒素破坏了人血清白蛋白中稳定二级结构的氢键网络,使蛋白二级结构展开,且AME和AOH使人血清白蛋白中的α-螺旋结构含量从48.93%分别减少至39.41%和44.01%,并使色氨酸残基极性降低、疏水性增加,但酪氨酸残基极性变化不大,即结合位点可能位于色氨酸残基所在的空腔(即结合位点I);AME对HSA的猝灭程度、猝灭常数(K_sv)及结合常数(K_a)均大于AOH,结合距离也更小(r_AME=2.56 nmAOH=2.60 nm)。本研究结果为进一步探究二苯-α-吡喃酮类链格孢霉毒素的毒代动力学和药代动力学,完善丰富其毒理学相关资料,以及进行风险评估和在食品中限量标准的制定提供了参考依据。     

链格孢ATMT转化体系的构建及弱毒突变株验证

本文利用来自质粒pUCATPH的构巢曲霉色氨酸合成基因trpC的启动子(PtrpC)、终止子(TtrpC)和潮霉素磷酸转移酶抗性基因(hph)以及植物表达载体pROKⅡ成功构建了适用于丝状真菌的根癌农杆菌介导转化的双元载体pROKIIHPH,并转化根癌农杆菌Agrobacterium tumefaciens LBA4404,建立了根癌农杆菌LBA4404介导的链格孢Alternaria alternata分生孢子转化体系;再从乙酰丁香酮(AS)浓度、不同的共培养时间、受体菌分生孢子浓度和农杆菌菌液浓度对A. alternata转化效率的影响对体系进行优化。结果确定了根癌农杆菌菌液体积为200mL(OD600=0.15),A. alternata分生孢子浓度为106个/mL,在A. tumefaciens的预培养时期以及与A. alternata共培养时期分别加入200μmo1/LAS,共培养时间48h,转化率达120～200个/105分生孢子。在所筛选的约800个转化子中获得了1株毒性明显低于野生菌sd1的弱毒突变株t108,通过PCR验证推测其毒力降低可能由于T-DNA插入阻断了基因的表达。该实验结果为与突变相关基因的研究以及弱毒株t108的进一步研究和利用提供了实验材料,也为深入研究链格孢sd1菌株的基因功能奠定了基础。     

梨果黑斑病菌磷酸二酯酶PDE基因克隆及其在侵染结构分化中的表达分析

磷酸二酯酶(PDE)能够特异地催化cAMP/cGMP 3', 5'环磷酸的3'环磷酸键水解,调节胞内cAMP水平,进而调控致病真菌的生长发育,但PDE基因在梨果黑斑病菌中的具体调控机制有待研究。本研究采用反转录PCR(RT-PCR)克隆了梨果黑斑病菌Alternaria alternata中的PDEL和PDEH基因;通过TMHMM、ProtScale、SOPMA等在线分析工具对AaPDEL和AaPDEH基因及其编码蛋白进行生物信息学分析;采用实时荧光定量PCR(qRT-PCR)技术分析了PDEL和PDEH基因在梨果黑斑病菌侵染结构分化过程中的表达特性。克隆得到梨果黑斑病菌PDEL和PDEH,分别命名为AaPDEL和AaPDEH,蛋白编码区(CDS)长度分别为3 132和2 886 bp,分别编码1 043和961个氨基酸。生物信息学分析表明AaPDEL和AaPDEH均属于不稳定亲水蛋白,不含信号肽也无跨膜结构,均具有多个磷酸化位点,分别定位于线粒体和细胞核上;AaPDEL和AaPDEH分别具有Class Ⅱ型和Ⅰ型保守催化结构域。系统分析结果表明,AaPDEL和AaPDEH与小麦褐斑病菌(Pyrenophora tritici-repentis)和番茄匍柄霉(Stemphylium lycopersici)亲缘关系最近,同源性分别高达79.25%和88.80%。qRT-PCR结果显示,疏水性诱导,A. alternata侵染结构分化过程中,AaPDEL和AaPDEH基因在附着胞形成阶段(6 h)表达量显著提高,较萌发初期阶段(2 h)分别上调1.19和1.97倍,表明AaPDEL和AaPDEH在A. alternata侵染结构分化中具有重要的调控作用。本研究为进一步从分子水平揭示PDE调控机制奠定了基础。     

Ureide production by N2-fixing and non-N2-fixing leguminous trees

Xylem-sap and stem and leaf extracts from 35 species, comprising 14 genera, of leguminous trees were analyzed for ureides, nitrate and -amino acids. Trees were either inoculated with Rhizobium or fertilized with NH₄NO₃. The dominant form of soluble N in stem and leaf extracts and xylem sap was 2-amino acids. Certain non-N₂-fixing species, i.e. Tamarindus indica and Adenantherapavonina, produced significant amounts of ureides. Several N₂-fixing species. Mimosa scabrella, Sesbania grandiflora. Acacia mearnsii and Gliricida sepium, grown on mineral-N had higher absolute amounts of ureides in both extracts and exudates than did most nodulated species. Nodulated A. meamsii and S. grandiflora, had the highest amounts of ureides in xylem sap. The relative abundance of ureides in stem and leaf extracts was lower than in xylem sap, but was correlated. Results indicated that the presence of ureides, per se, was not a reliable indicator of N₂-fixing activity. Moreover, the relative abundance of ureides in most of the species tested was too low to use as a presumptive test for, or as a means of, estimating N₂ fixation.     

接触辉光放电等离子体对Fusarium solani的杀菌作用机制及动力学模型研究

为研究接触辉光放电等离子体（CGDP）对茄病镰刀菌（Fusarium solani）的杀菌作用及机制,本试验以Fusarium solani为试材,考察CGDP处理过程中电压、处理时间、抗坏血酸浓度、孢子初始浓度等因素对杀菌效果的影响;并对Fusarium solani生长、孢子形态、细胞膜完整性及过程中所产生的活性粒子进行分析。通过Linear、Weibull和Log-Logistic 3种数学模型分析不同电压下CGDP杀菌动力学特性,以相关系数（R2）、精确因子（Af）、偏差因子（Bf）和均方根方差（RMSE）4个参数评价拟合效果。结果表明,CGDP对Fusarium solani孢子有着明显的杀菌效果;升高电压、延长处理时间、降低抗坏血酸浓度和孢子初始浓度均能有效抑制孢子生长,使杀菌效率提高（P<0.05）;CGDP处理30 min,活性物质（·OH、H2O2、NO3-）浓度分别增加至1.57 mg·L-1、73.89 mmol·L-1和12.72 mg·L-1,pH值由7.07降至4.66;孢内核酸和蛋白质的渗漏量以及PI染色和扫描电镜结果表明,CGDP对Fusarium...     

Arbuscular mycorrhizal fungi in northern Greece and influence of soil resources on their colonization

Abundance of arbuscular mycorrhizal fungi (AMF) in the roots of plant species was assessed in two areas in Greece in a 4-year study (2004–2007). The field experiment was conducted in a mountainous and herbaceous grassland in Greece in which both nitrogen (N)- and phosphorus (P)-limited plant community productivity. In 2006, data were also collected from a pot experiment in which 14 herbaceous plant species were grown as monocultures in P-limited soil. A factorial design of two levels of N and P was established in the mountainous field to test plant response to nutrient additions with respect to AMF colonization levels. Effects of fungicides were also investigated over year in the pot experiment and over three years in the field experiment. In addition, the effect of irrigation on AMF colonization was determined in a 1-year field study. Measurements included estimating the level of plant species specific hyphal colonization of roots according to the McGonicle et al. [McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, D.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesicular–arbuscular mycorrhizal fungi. New Phytol. 115, 495–501] method. AMF colonization was highest in the leguminous species, intermediate in the forbs and lowest in the grasses. AMF responses to N and P additions were not uniform. P addition in the field experiment increased the colonization level of the high P demanding annual forb (non-leguminous dicot) Galium lucidum, decreased hyphal abundance of the forb Plantago lanceolata and the grass Agrostis capillaris, and appeared to have a negligible effect on the forb Prunella vulgaris and on leguminous species. Effects of N addition were influenced by P addition and were only significant in plots not enriched with P where N addition increased the AMF colonization. Irrigation increased colonization of the tested species A. capillaris and P. lanceolata but only significantly increased that of P. lanceolata. There was interannual variation in the effects of fungicides on AMF colonization, which was partly due to differences in the active ingredient and formulation used. Among the tested species, A. capillaris was the most susceptible to fungicides.     

Soil fungal population in preharvest maize ecosystem in different tillage practices in Argentina

The distribution of the soil borne fungal population in maize non-rhizospheric soil in experimental trials were studied during the harvest seasons 2000/2001 and 2001/2002. The effect of different tillage practices was evaluated. Soil samples were collected from three tillage treatments, conventional tillage, reduced tillage and no tillage, with and without grazing. The distribution of fungal propagules in different treatments was compared. Soil samples were collected from the top soil (3 cm) before planting, in the sprong. No tillage without grazing and reduced tillage with grazing had the highest densities of filamentous fungi (5.71 and 5.85 log, respectively). The mycological survey showed the presence of six genera of filamentous fungi. They were Aspergillus, Fusarium, Penicillium, Trichoderma, Cladosporium, Alternaria. The species identified from genus Aspergillus were A. flavus, A. restrictus, A. candidus, A. parasiticus and other Aspergillus spp. The species identified from genus Penicillium were P. pinophylum, P. citreonigrum, P. implicatum, P. purpurogenum, P. minioluteum, P. waksmanii, P. restrictum, P. rugulosum, P. funiculosum and P. variabile. Species from genus Fusarium were identified as F. verticillioides, F. oxysporum, F. proliferatum and others Fusarium species. The isolation frequency of Aspergillus increased in the no tillage and grazing practices treatments. The data indicate that different agricultural practices impact soil inocula of principal maize toxigenic fungi in the production area. To our knowledge, this is the first report on the effect of tillage management practices on fungal populations in preharvest maize agroecosystem in Argentina.     

Suppression of rice blast,cabbage black leaf spot,and tomato bacterial wilt diseases by Meyerozyma guilliermondii TA-2 and the nature of protection

This study was conducted to evaluate the efficacy of yeast strain TA-2 for controlling rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases. Microscopic and phylogenetic analyses based on rDNA-internal transcribed region (ITS) and rDNA-D1/D2 sequences indicated that yeast strain TA-2 is Meyerozyma guilliermondii. Pretreatment with TA-2 by soil drenching significantly reduced the severity of black leaf spot disease caused by Alternaria brassicicola and leaf blast disease caused by Magnaporthe oryzae. Symptom development of tomato bacterial wilt caused by Ralstonia solanacearum in both soil drench and needle inoculation tests was significantly reduced in TA-2-pretreated plants under soil drenching. Disease severity and R. solanacearum growth were significantly reduced in tomato plants pretreated with yeast culture, cell suspension, or culture filtrate of TA-2 under soil drenching. TA-2 does not produce antibiotics. The present study indicates that disease suppression is systemic, as the roots were treated with TA-2 and the pathogens were inoculated onto leaves or stems, thereby separating the two spatially. M. guilliermondii TA-2 could become a promising natural antimicrobial agent against rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases and might be useful as an eco-friendly control measure, contributing to sustainable agriculture.     

Assessment of Verticillium biguttatum in agricultural soils

Two different methods were evaluated for the assessment of Verucillium biguttatum in soil: the pellet sampler (PS) and the modified Andersen air sampler (AS). Both techniques allowed the distribution of soil on selective Rhizoctonia plates (RP) as soil fine particles and cylindrical pellets, respectively. The white speculating colonies of V. biguttatum can easily be recognized on RP.

Compared with the PS, the AS method yielded more colonies of V. biguttatum over a wider range of artificially-produced densities, and with a lower detection threshold. It is assumed that for germination and subsequent development of colonies a more or less close contact is essential. Microscopical observations revealed that the soil particles are often not in immediate contact with hyphae of R. solani and then no outgrowth of V. biguttatum can occur.

Combined with the AS, sampling the soil volumetrically with PS can serve as a more efficient substitute for the time-consuming weighing procedure.

The observed differences for various soils suggest that besides the presence of R. solani, more factors affect the germination process. The value of the AS method is discussed in view of its applicability to studies on population dynamics of V. biguttatum in soil.     

Habitat selection and conservation of an endemic spanish lizard Algyroides marchi (reptilia, lacertidae)

We study the habitat selection of Algyroides marchi, a small lizard endemic to the southeastern mountains of the Iberian Peninsula whose populations are concentrated in a few small localities. Three spatial scales of study were used to analyse the different factors affecting the species distribution patterns: regional scale, local scale and within-habitat use of different substrates. The results obtained at these different spatial scales showed a high consistency, suggesting that the same ecological pressures probably acted at different scales. Localities occupied by A. marchi were characterized by high altitude, high geomorphological complexity, northern aspect, high cover of large rocks, and presence of water. Density of A. marchi in a favourable locality was 213 adult-subadult lizards/ha. Abundance and use of space of A. marchi (at local and individual levels) were directly associated with extent of water (streams and small pools) and the presence of large rocks, and inversely with hours of direct solar radiation and cover of small stones and vegetation. Considering the habitat selection of A. marchi and the management practices within its geographical range, potential risks for the conservation of the species are identified.