贝莱斯芽胞杆菌HN-Q-8菌株发酵液稳定性测定及抑菌活性成分分析

【背景】贝莱斯芽胞杆菌(Bacillus velezensis) HN-Q-8菌株是本实验室前期获得的能有效拮抗立枯丝核菌(Rhizoctonia solani)的生防细菌。【目的】明确贝莱斯芽胞杆菌HN-Q-8菌株的抑菌活性物质。【方法】采用菌丝生长速率法检测其发酵液对5种马铃薯病原菌的拮抗能力以及稳定性,利用基质辅助激光解吸电离飞行时间质谱技术对HN-Q-8菌株活性物质进行鉴定。【结果】HN-Q-8菌株对立枯丝核菌(Rhizoctonia solani)、尖镰孢菌(Fusarium oxysporum)、茄链格孢(Alternaria solani)和致病疫霉(Phytophthora infestans)具有良好的抑菌活性,抑菌率可达50%-90%。发酵液分别经紫外照射35min、自然光照射10 h以及100°C高温处理后,相对抑菌率分别为74%、92%和98%;发酵液的抑菌活性不受胰蛋白酶和蛋白酶K的影响;但不耐强酸、强碱,其适宜pH为4.0-10.0,表明HN-Q-8菌株活性物质具有良好的稳定性。活性成分能使立枯丝核菌菌丝形态畸形扭曲,从而抑制立枯丝核菌的生长,经鉴定活性物质...     

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。     

藓类提取物对植物病原真菌的抑菌性研究

以立枯丝核菌(Rhizoctonia)、尖孢镰孢菌(Fusarium oxysporum)、苹果轮纹病菌(Macrophoma kawatsukai)、梨黑星病菌(Fusicladium pirina)、草莓灰霉菌(Botrytis cinerea)等七种植物病原真菌为供试菌,通过生长速率法测定了24种藓类的乙醇提取物对病原真菌的抑制作用.结果表明,在提取物浓度为1.2mg干样/mL,大镰刀藓提取物对立枯丝核菌的抑制作用最强,抑菌率为67.8%.藓类提取物不仅对植物病原真菌有抑菌活性,而且还有多种藓类提取物对病原真菌菌丝有促进生长活性,如锐尖匍灯藓对立枯丝核菌菌丝生长的促进率为95.1%.选出大镰刀藓、扁灰藓、紫萼藓、山地水灰藓、疣小金发藓、橙色净口藓、锐尖匍灯藓和羊角藓对立枯丝核菌和灰霉菌的抑菌性或菌丝生长促进性进行了进一步研究,藓类提取物对病原菌的抑菌性或菌丝生长促进性在一定范围内随浓度的增加而增强,并且抑菌性随时间的延长呈下降趋势.     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

立枯丝核菌营养菌丝多型性观察

采用了2种不同的方法对立枯丝核菌营养菌丝的形态进行了观察和比较,观察到2种不同的营养菌丝的形态,即菌核类和假分生孢子类。为以后进一步研究立枯丝核菌营养菌丝的多型性奠定了一定的基础。     

三种苔藓植物提取物对植物病原菌的抑菌性研究

以立枯丝核菌(Rhizoctoniasolani)、棉花枯萎病菌(Fusariumoxysporum)、苹果轮纹病菌(Physalosporapiricolanosa)、草莓灰霉病菌(Botrytiscinerea)四种植物病原菌为供试病原菌,对大镰刀藓Drepanocladusexannulatus、锐尖匍灯藓Plagiomniumacutum和疣小金发藓Pogonatumurnigerum三种藓类醇提液进行抑菌活性筛选,结果表明,大镰刀藓提取液对立枯丝核菌有较好的抑制作用,对立枯丝核菌的EC50为0.878mg/mL;而锐尖匍灯藓的提取液对立枯丝核菌的生长却有促进作用。     

立枯丝核菌对高粱幼苗活性氧及抗氧化酶活性的影响北大核心CSCD

以立枯丝核菌AG1-IA接种‘龙杂19号’高粱幼苗,通过盆栽试验在不同侵染时间观测分析高粱幼苗生长及生理代谢相关指标,揭示立枯丝核菌侵染对高粱生长、渗透调节物质及其抗氧化酶活性的影响机理。结果表明,(1)高粱幼苗株高、根长、地上(茎和叶片)鲜质量和干质量、地下(根)鲜质量和干质量均随接种时间延长呈下降趋势,且在接菌72 h时较对照依次分别显著下降了41.0%、29.2%、50.0%、50.0%、53.3%和50.0%。(2)幼苗叶片叶绿素a含量在接菌72 h时较对照显著下降45.3%,叶片PSⅡ最大光化学效率(Fv/Fm)值随接菌时间增加而逐渐下降。(3)幼苗叶片内MDA、O^(-·)_(2)和H_(2)O_(2)含量随接菌时间增加均呈逐渐上升趋势,在接菌72 h时较对照分别显著上升244.6%、140.4%和137.0%;叶片SOD、POD、APX和CAT的活性在接菌后变化趋势不尽相同,但在接菌72 h时较对照分别显著上升了16.5%、60.3%、50.0%和36.5%。(4)幼苗叶片可溶性蛋白和可溶性糖含量随接菌时间增加均呈逐渐上升趋势,并在接菌24~72 h增幅均达到显著水平。研究发现,接种立枯丝核菌可致高粱幼苗植株产生病斑,体内活性氧过量积累,膜质发生显著氧化损伤;侵染前期高粱植株主要以积累更多的渗透调节物质来抵御立枯丝核菌带来的伤害,侵染后期随植株受到伤害加重,同时诱导植株抗氧化酶类活性显著增强,以维持高粱体内活性氧代谢稳态平衡,减少植物膜进一步氧化损伤。     

草坪禾草丝核菌的核相研究

2003～2005年从上海市、浙江省、山东省、河南省和陕西省草坪褐斑病的97份病株标本中,分离得到了73个丝核菌分离物,其寄主包括多年生黑麦草Lolium perenne、高羊茅Festuca arundinacea、草地早熟禾Poa pratensis、匍匐翦股颍Agrostis palustris、结缕草Zoysia japonica和狗牙根Cynodon dactylon。用载玻片培养法和DAPI染色法对各分离物菌丝进行了染核观察。结果73个分离菌株可区分为单核丝核菌、双核丝核菌和多核丝核菌三大类,可能属于多个物种。单核丝核菌在中国为首次报道,同时也是其侵染草坪禾草的首次报道。     

丝核菌的分类系统：现状及存在问题

以重要植物病原菌为特征的丝核菌是一类在土壤中广泛分布的丝状真菌,通常不产孢,以菌丝或菌核的形式存在,多样性非常丰富。本文基于国内外最新研究进展,对依据菌丝体的细胞核数目、菌丝融合、有性生殖和系统进化等方面的基本特征展开的丝核菌分类体系及分类现状进行了综述。基于菌丝的细胞核数目,丝核菌被分为单核、双核和多核丝核菌三大类群。自然界中单核丝核菌数量极少,多核和双核丝核菌在全球分布广泛,占丝核菌的绝大多数。基于菌丝融合试验的结果,目前多核丝核菌被分为13个菌丝融合群,双核丝核菌被分为18个菌丝融合群。部分融合群内又根据一些稳定的特征分了亚群,但亚群的建立标准并不统一。目前的分子系统学研究结果基本支持丝核菌的菌丝融合群及亚群的分类。基于部分有性世代被发现的菌株的形态特征,多核和双核丝核菌分别被鉴定为亡革菌属和角担菌属。此外,目前已有分属重要植物病原菌和兰科菌根菌类群的至少9个融合群或亚群的17个菌株完成了基因组测序,比较基因组学和线粒体组学开始在丝核菌分类和进化研究中发挥作用。丝核菌分类系统特殊且复杂,作者在文末提出了目前丝核菌分类学研究面临的问题和今后研究的趋势,期待更多的学者参与到这个重要菌...     

江苏大麦纹枯病菌酯酶同工酶的测定

对立枯丝核菌(Rhizoctonia solani giihn)和禾谷丝核菌(R．E erealis Vande r Hoeven)的16个菌丝融合群或亚群的标准菌株及来自}工苏大麦纹枯病的9个菌丝融合群或亚群共68个菌株进行了聚丙烯酰胺凝胶电泳,测定酯酶同工酶。结果表明：1．立枯丝核菌主酶带数的变幅范围比禾谷丝核菌大;2．无论禾谷丝核菌,还是立枯丝核菌,各菌丝融合群或亚群之间的电泳图谱都有显著差异,但与各自对应的融合群或亚群的标准菌株的图谱则相似;3．44个大麦禾谷丝核菌CAG一1群菌株间的图谱也有差异,但都有一条共同的主酶带(E．);4．据主副酶带数目和位置,将禾谷丝核菌CAG一1群菌株再分为2个类型(1型和II型)和5个亚型(1a、Ib、Ic和Iia、Iib);酶谱类型与菌株的采集品种和致病性无关,但与采集地点似有一定的联系。     

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. TAXONOMY: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. IDENTIFICATION: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 μm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 μm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. DISEASE SYMPTOMS: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. RESISTANCE: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. MANAGEMENT: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.     

Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers

AIMS: The aim of this study was to develop a specific and sensitive identification method for Rhizoctonia solani AG 1-IB isolates based on phylogenetic relationships of R. solani AG-1 subgroups using rDNA-internal transcribed spacer (rDNA-ITS) sequence analysis. METHODS AND RESULTS: A neighbour-joining tree analysis of 40 rDNA-ITS sequences demonstrated that R. solani AG-1 isolates cluster separately in six subgroups IA, IB, IC, ID, IE and IF. A molecular marker was generated from a random amplified polymorphic DNA fragment (RAPD). After conversion into a sequence-characterized amplified region (SCAR), a specific primer set for identification of subgroup AG 1-IB was designed for use in a polymerase chain reaction (PCR). The primer pair amplified a single DNA product of 324 bp. CONCLUSIONS: R. solani AG-1 subgroups were discriminated by sequence analysis of the ITS region. The designed SCAR primer pair allowed an unequivocal and rapid detection of R. solani AG 1-IB in plant and soil samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence analysis of the rDNA-ITS region can be used for differentiation of subgroups within AG-1. The use of the developed SCAR primer set allowed a reliable and fast identification of R. solani AG 1-IB and provides a powerful tool for disease diagnosis.     

Amendment with peony root bark improves the biocontrol efficacy of Trichoderma harzianum against Rhizoctonia solani

We tested Trichoderma harzianum as a biocontrol agent for Rhizoctonia solani AG2-1, using six natural antifungal materials to improve its efficacy. Among the six materials tested, peony (Paeonia suffruticosa) root bark (PRB) showed the strongest antifungal activity against R. solani AG2-1, and was not antagonistic to T. harzianum. Scanning electron microscopy showed that treatment with PRB extract resulted in shortened and deformed R. solani AG2-1 hyphal cells. The control of radish damping-off caused by R. solani AG2-1 was greatly increased by combined treatments of T. harzianum and PRB, as compared with either of the two treatments alone, with the control effect increased from 42.3-51.5% to 71.4-87.6%. The antifungal compound in PRB, which was isolated in chloroform and identified as paeonol by mass spectrometry, 1H NMR, and 13C NMR analyses, inhibited the growth of R. solani AG2-1 but not that of T. harzianum. Thus, PRB powder or extract may be used as a safe additive to T. harzianum to improve the control of the soil borne diseases caused by R. solani AG2-1.     

Highly polymorphic microsatellite loci in the rice- and maize-infecting fungal pathogen Rhizoctonia solani anastomosis group 1 IA

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.     

Study of the aggressiveness of Rhizoctonia solani isolates

This paper presents an in vitro test to screen the pathogenicity of different Rhizoctonia solani isolates on a host range. The level of aggressivity of the different isolates was different for several host plants tested. There were significant differences between the crops and the isolates tested. In general, the disease level was higher on beans, lettuce and cabbage. In carrot and rye grass the level of infection was lower for the isolates of R. solani tested. The potato isolates of R. solani were less aggressive than the isolates coming from maize, fodder beet and sugar beet. The R. solani isolates were also biochemically characterized by pectic zymograms: the isolates Rs0401 (from maize) and Rs0504 (from sugar beet) belong both to the anastomosis group AG2-2.     

丝核菌菌丝融合群种类,寄生专化性及与温度的关系

丝核菌（Ｒｈｉｚｏｃｔｏｎｉａｓｐｐ．）是最重要的土传植物病原菌之一,分布世界各地。１９２１年Ｍａｔｓｕｍｏｔｏ用菌丝融合群（ＡｎａｓｔｏｍｏｓｉｓＧｒｏｕｐ,简称ＡＧ）的方法来划分多核立枯丝核菌（Ｒ．ｓｏｌａｎｉ）在生理上特异的菌株［６］,Ｐａｒ...     

Genetic diversity of Rhizoctonia solani associated with potato tubers in France

The soilborne fungus Rhizoctonia solani is a pathogen of many plants and causes severe damage in crops around the world. Strains of R. solani from the anastomosis group (AG) 3 attack potatoes, leading to great yield losses and to the downgrading of production. The study of the genetic diversity of the strains of R. solani in France allows the structure of the populations to be determined and adapted control strategies against this pathogen to be established. The diversity of 73 French strains isolated from tubers grown in the main potato seed production areas and 31 strains isolated in nine other countries was assessed by phylogenetic analyses of (i) the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA), (ii) a part of the gene tef-1α and (iii) the total DNA fingerprints of each strain established by amplified fragment length polymorphism (AFLP). The determination of the AGs of R. solani based on the sequencing of the ITS region showed three different AGs among our collection (60 AG 3 PT, 8 AG 2-1 and 5 AG 5). Grouping of the strains belonging to the same AG was confirmed by sequencing of the gene tef-1α used for the first time to study the genetic diversity of R. solani. About 42% of ITS sequences and 72% of tef-1α sequences contained polymorphic sites, suggesting that the cells of R. solani strains contain several copies of ITS and the tef-1α gene within the same nucleus or between different nuclei. Phylogenetic trees showed a greater genetic diversity within AGs in tef-1α sequences than in ITS sequences. The AFLP analyses showed an even greater diversity among the strains demonstrating that the French strains of R. solani isolated from potatoes were not a clonal population. Moreover there was no relationship between the geographical origins of the strains or the variety from which they were isolated and their genetic diversity.