不同品种柑橘叶际真核生物多样性及 青苔病原差异分析

柑橘青苔病是一种普遍发生且危害严重的病害?本试验选择3个甜橙品种和3个杂柑品种为研究对象, 在重庆忠县和开州分别采集有病症和无病症的叶片样品共24份, 利用Illumina Miseq高通量测序技术对两地不同品种柑橘叶际真核生物的群落组成?结构及差异进行分析?结果表明, 24份柑橘叶片样本共检测到491个OTUs, 多样性较为丰富?柑橘叶表真核生物的优势属为枝孢菌属 Cladosporium, 橡胶藻属 Heveochlorella, 杯梗孢属 Cyphellophora, Parastagonospora 等?有病症组中丰度显著高于无病症组的物种包括不可培养的球藻uncultured Apatococcus?黄绿异小球藻 Heterochlorella luteoviridis?海南橡胶藻 Heveochlorella hainangensis?无柄杯梗孢 Cyphellophora sessilis?椭圆球藻 Chloroidium ellipsoideum 和胶孢炭疽菌 Colletotrichum gloeosporioides, 它们可能与柑橘青苔的发生密切相关?不同品种和不同区域的12份有病症组叶样中优势菌群的组成结构及丰度均存在较大差异?柑橘青苔病的发生可能是由多种绿藻及病原真菌复合侵染造成的, 青苔侵染对柑橘叶际真核生物群落结构有很大影响, 不同品种及地区间柑橘叶片受青苔侵染后的叶际优势菌群特征有差异?     

黄龙病对柑橘叶际微生物组的影响

为明确柑橘叶际微生物组对黄龙病发生的响应规律，采用荧光定量PCR技术检测采集柑橘叶片感染黄龙病菌的情况，通过扩增子测序分析法对PCR检测结果为阴性和阳性叶片的微生物群落进行比较，并通过相关性分析和网络分析对黄龙病菌和叶际微生物的相关性进行解析。结果显示，夏季采集的柑橘叶片中黄龙病菌的检出率为7.5%，而到秋季黄龙病菌的检出率上升到32.3%。黄龙病的发生显著改变了柑橘叶际细菌群落的组成和结构，对于细菌类群的丰度也有一定影响；而对叶际真菌群落的组成和结构无显著影响。黄龙病菌与叶际细菌存在普遍的负相关关系，正相关关系相对较少但相关性更强。黄龙病菌可能通过与厌氧棍状菌Anaerotruncus sp.、梭菌Clostridiales sp.、假单胞菌Pseudomonas sp.、鞘脂单胞菌Sphingomonas sp.、毛螺菌Lachnospiraceae sp.和链球菌Streptococcus sp.的负向互作对柑橘叶际整个细菌群落产生负影响。表明黄龙病发生对柑橘叶际真菌和细菌群落的影响规律不同，同时黄龙病菌可能通过与几种主要细菌的负向互作来实现对叶际微生物组的影响。     

植物抗病性分子机制

 植物对病原物的反应有抗病和感病两大类。从寄主-病原物相互作用角度,抗病反应又叫非亲和性反应,这一系统以寄主抗病和病原物无毒性为特征,寄主植物对病原物有抑制、排斥或减毒作用,病害不发生或受到限制;感病反应又叫亲和性反应,其特征是寄主感病和病原物毒性,结果严重发病。     

夏橙绿斑病病原鉴定及其化学防治

对重庆夏橙基地夏橙发生的绿斑病症状和病原藻形态进行了观察,并进行了病原藻分离及致病性测定.结果表明,该病是由绿藻门虚幻球藻属虚幻球藻Apatococcus lobatus所致.室内毒力测定表明,50%新灵（多菌灵＋代森锰锌）WP、62.25%仙生（腈菌唑＋代森锰锌）WP、10%世高WG、25%炭特灵WP与53.8%可杀得DF对病原藻的生长都具有一定的抑制作用,其EC5o分别为2.3531、2.6234、9.3815、13.8816、28.9808 mg/kg.田间小区试验结果表明,50%新灵WP 600倍和62.25%仙生WP 600倍液的防治效果较好,其次为10%世高WG 1000倍和53.8%可杀得DF1000倍液,而25%炭特灵WP600倍液的防治效果较低.经方差分析,处理间存在显著差异.     

黄瓜绿粉病病原鉴定及其发生规律研究

黄瓜绿粉病是温棚蔬菜上一种新病害,由绿藻门集球藻(Palmellococcus sp.)引起。藻孢卵圆形、长椭圆形、淡绿色、单胞、大小3.39～12.38μm×1.06～10.82μm,表面有山脊状隆起,是一种依附于体表的气生性自养生物。该病害多在1～3月份发生,与湿度密切相关。每日温棚中RH不低于80%,RH100%在12h以上,藻孢生长旺盛;当中午有1h以上RH低于70%时,藻孢的发育受到抑制。凡能增大温棚湿度的栽培措施如灌水、密植等因素均能加重发病。黄瓜品种抗病性无明显差异。     

浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因的PCR检测及其序列比对

长期的观察发现,不同柑橘品种感染黄龙病后出现的病症也不尽相同,针对这种现象本文检测了浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并进行了序列比对,BLAST比对结果说明所扩增的基因序列之间差异性为0,与基因库(NCBI)中黄龙病亚洲韧皮杆菌DNA序列相似性为100％,由此说明浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并未发生变异,出现上述现象可能是其他基因发生变异或者与品种的抗病性有关。     

中国柑橘黑腐病和褐斑病病原菌的系统发育分析

 近些年链格孢属真菌的分类有了长足的进展,但柑橘褐斑病和黑腐病的病原却还是存在着一些争议。本研究从中国柑橘果实上收集了49株黑腐病菌菌株,并选取了8株从柑橘叶片上分离的具代表性的褐斑病菌菌株,基于Alta1 、endoPG、LSU、OPA10-2、OPAl-3和OPA2-1 等6个基因位点串联序列构建系统发育树。结果表明,柑橘黑腐病和褐斑病都可由不止一种链格孢菌引起,均以Alternaria alternata为主。两种病害的病原菌之间不能通过该系统发育树区分,但在致病性上存在差异,且能通过扩增ACT毒素合成基因进行区分。为了使两种病害的病原更加方便阐述,作者建议以它们的主要类群对其进行命名,柑橘褐斑病病原学名还是遵循前人的称呼,为the tangerine pathotype of A. alternata,即链格孢菌橘致病型,而柑橘黑腐病病原应为A. alternata,即链格孢菌。     

中国柑橘黑腐病和褐斑病病原菌的系统发育分析

 近些年链格孢属真菌的分类有了长足的进展,但柑橘褐斑病和黑腐病的病原却还是存在着一些争议。本研究从中国柑橘果实上收集了49株黑腐病菌菌株,并选取了8株从柑橘叶片上分离的具代表性的褐斑病菌菌株,基于Alta1 、endoPG、LSU、OPA10-2、OPAl-3和OPA2-1 等6个基因位点串联序列构建系统发育树。结果表明,柑橘黑腐病和褐斑病都可由不止一种链格孢菌引起,均以Alternaria alternata为主。两种病害的病原菌之间不能通过该系统发育树区分,但在致病性上存在差异,且能通过扩增ACT毒素合成基因进行区分。为了使两种病害的病原更加方便阐述,作者建议以它们的主要类群对其进行命名,柑橘褐斑病病原学名还是遵循前人的称呼,为the tangerine pathotype of A. alternata,即链格孢菌橘致病型,而柑橘黑腐病病原应为A. alternata,即链格孢菌。     

植物病理学 第六章 植物抗病性(二)

三、抗病性的表现 是寄主抗病性和病原物致病性相互作用的结果 病原物与寄主相遇后,寄主表现抗病、感病,还是处于某种中间程度?并不决定于寄主单方面,要看寄主与病原物双方的相互作用。“抗病性”与植物的其他性状有所不同,是两者结合一起,才能表现出来的一种性状,它是寄主病原物“结合体”(或作     

湖北柑橘主产区主要病虫害种类及防治技术

为摸清湖北柑橘主产区主要病虫害种类及发生特点,2017年～2018年通过实地调查,并采集标本进行种类鉴定。结果表明,湖北柑橘主产区主要病害有11种,以柑橘炭疽病、沙皮病、绿斑病(青苔病)、根腐病为害最严重;主要害虫有19种,以柑橘红蜘蛛、潜叶蛾、柑橘大实蝇为害最严重。本文对几种主要病虫害提出了防控措施。     

湖南省永州地区柑橘慢衰病与黄龙病病原鉴定及分布调查

为明确湖南省柑橘黄化症状与柑橘慢衰病和柑橘黄龙病的相关性,应用形态学和分子生物学方法对湖南省永州地区表现黄化症状的柑橘进行了两种病原的鉴定及分布调查。结果表明,造成该地区柑橘黄化现象的主要原因为柑橘半穿刺线虫和黄龙病菌对柑橘的侵染。所检样本中,柑橘慢衰病平均发生率为82.1%,土壤中的半穿刺线虫群体密度最高达到3 077条/100mL。永州地区柑橘黄龙病病菌属于韧皮部杆菌属类细菌亚洲种,平均检出率为64.3%。柑橘半穿刺线虫和黄龙病菌在柑橘园存在混合侵染现象,混合侵染率为53.6%。柑橘半穿刺线虫在永州地区柑橘产区分布广泛,是造成柑橘黄化症状的重要病因,重视并有效防控柑橘半穿刺线虫已迫在眉睫。     

黄龙病菌在柑橘枝条上的分布和多样性分析

 柑橘黄龙病是由“Candidatus Liberibacter asiaticus”(CLas)引起的毁灭性病害,在染病植株的叶片和果实上表现出不同症状。但目前还未见在同一植株或枝条上表现不同症状的叶片或果实中的CLas是否存在多样性的报道。本研究分析来自美国佛罗里达与中国广东两地染病柑橘样品,发现佛罗里达的CLas遗传多样性较低。以3对位于CLas短串联重复位点两端的序列为引物,对广东柑橘黄龙病病株的样品进行PCR扩增和PAGE检测,结果发现:同一植株上的CLas多样性高于同一枝条;同一病枝上显不同症状的叶片和果实中的CLas浓度分布不均匀,但成熟病果中CLas的浓度最高。以同一病枝条为一个组,PCR扩增和PAGE检测53个枝条的样品,发现不同组间CLas多样性不一:3对引物扩增条件下分别有4组(7.55%)、7组(13.21%)和11组(20.75%)样品存在组间多样性,推测同一枝条中含有不同的CLas菌株。以上结果说明了CLas在柑橘枝条上的分布规律,为监测我国CLas的多样性提供了科学依据。     

Association of ‘Candidatus Liberibacter asiaticus’ root infection,but not phloem plugging with root loss on huanglongbing‐affected trees prior to appearance of foliar symptoms

Huanglongbing (HLB) is a systemic disease of citrus caused by phloem‐limited bacteria ‘Candidatus Liberibacter’ spp. with ‘Ca. Liberibacter asiaticus’ (Las) the most widespread. Phloem‐limited bacteria such as liberibacters and phytoplasmas are emerging as major pathogens of woody and herbaceous plants. Little is known about their systemic movement within a plant and the disease process in these tissues. Las movement after initial infection was monitored in leaves and roots of greenhouse trees. Root density, storage starch content, and vascular system anatomy in relation to Las presence in field and greenhouse trees, both with and without symptoms, showed the importance of root infection in disease development. Las preferentially colonized roots before leaves, where it multiplied and quickly invaded leaves when new foliar flush became a sink tissue for phloem flow. This led to the discovery that roots were damaged by root infection prior to development of visible foliar symptoms and was not associated with carbohydrate starvation caused by phloem‐plugging as previously hypothesized. The role of root infection in systemic insect‐vectored bacterial pathogens has been underestimated. These findings demonstrate the significance of early root infection to tree health and suggest a model for phloem‐limited bacterial movement from the initial insect feeding site to the roots where it replicates, damages the host root system, and then spreads to the rest of the canopy during subsequent leaf flushes. This model provides a framework for testing movement of phloem‐limited bacteria to gain greater understanding of how these pathogens cause disease and spread.     

福建省柑橘褐斑病菌的分离、鉴定和系统进化分析

为明确在福建省南平市的橘柚和三明市的温州蜜橘上发现的疑似柑橘褐斑病的病原菌种类,采用组织分离法获得纯化菌株,通过回接法验证菌株的致病性,利用形态学特征对病原菌进行初步鉴定,并采用最大似然法以多聚半乳糖醛酸酶基因endoPG为靶标对本研究以及国内外已报道的链格孢菌株构建系统发育树,分析其遗传多样性。结果表明,从病组织中共分离获得26株纯培养菌株,经形态学鉴定均为链格孢菌Alternaria spp.。利用分生孢子液接种橘柚离体嫩叶发现,有22株菌株能侵染橘柚叶片并产生与田间相似的褐斑病症状,确认该病害为链格孢引起的柑橘褐斑病。系统发育树分析结果显示,分离所得的26株菌株均聚在已报道的4个柑橘链格孢进化分支Clade1～Clade4中,其中21株菌株聚在国内特有的分支Clade4中,有3株菌株和1株菌株分别聚在国内外兼有的分支Clade3和Clade1中,1株菌株聚在国外特有的分支Clade2中,表明在福建省采集的这些柑橘褐斑病菌均为链格孢菌,且遗传多样性较丰富。     

柑桔溃疡病菌存活期的研究

本文对鄂东南地区生态条件下柑桔溃疡病菌在不同场所的存活期进行了研究 ,并对其能否作为侵染源进行了评价。证实在病株病斑内的病菌存活时间可达一年以上 ,是此病发生最主要的侵染源。病菌在田间条件下的土壤、落叶、落果、果皮及自然水中的存活期均相当有限 ;其中以冬季病落叶中的病菌存活期最长 ,也不超过 3个月 ;故年前存在于这些场所的病菌均不能成为第 2年的初侵染源     

Anatomical distribution of abnormally high levels of starch in HLB-affected Valencia orange trees

The citrus disease Huanglongbing (HLB or citrus greening) is characterized, among other symptoms, by extraordinary levels of starch accumulation in leaves. This condition denotes imbalances in carbohydrate source sink relationship which in turn may have direct implications in the overall health of HLB-trees and in future strategies to manage the disease. Using light, scanning, and transmission electron microscopy we investigated the extent of carbohydrate partitioning imbalances throughout the tree. In all aerial tissues, starch accumulation in HLB-affected trees far exceeded that of HLB-negative control trees. Starch accumulated extensively in photosynthetic cells as well as phloem elements and vascular parenchyma in leaves and petioles. In stems, starch was commonly observed in xylem parenchyma and in the phelloderm of HLB-affected trees but absent from control samples. In contrast, roots from HLB-affected trees were depleted of starch whereas roots from control trees contain substantial starch deposits. The data supports the notion that the substantial changes in carbohydrate partitioning observed throughout the citrus tree may not only be a result of HLB infection, but in itself, a cause for the rapid decline and death of infected trees.     

重庆市草坪草病害种类调查及病原鉴定初报

为了明确重庆草坪草主要病害及其病原菌的种类,作者调查了重庆草坪草病害种类,并分离鉴定了病原。结果表明：重庆草坪草常见病害有锈病、结缕草炭疽病、褐斑病、镰刀枯萎病、结缕草弯孢霉叶枯病和结缕草根结线虫等病害,分别由柄锈菌（Puccinia spp.）、尾状炭疽菌（Colletotrichum caudatum）、立枯丝核菌（Rhizoctonia solani）、镰刀菌（Fusarium sp.）、弯孢霉（Curvularia spp.）和根结线虫（Meloidogyne sp.）危害所致。     

A cultivation independent,PCR-based protocol for the direct identification of plant pathogens in infected plant material

A protocol involving PCR, shot-gun cloning and sequencing was developed as a pre-diagnostic screening tool working directly on disease symptoms. The method was used to show the presence of biotrophic and non-biotrophic eukaryotic plant pathogens in leaves and fruits.     

Variation in Occurrence of Dematiaceous Hyphomycetes on Forage Bermudagrass over Years, Sampling Times, and Locations

ABSTRACT Leaf samples of forage bermudagrass with symptoms of infection by species of Bipolaris, Curvularia, and Exserohilum (dematiaceous hyphomycetes) were collected from three swine waste application sites in Mississippi at eight sampling times during each of 3 years. Samples were assayed for pathogens by observing sporulation on plated leaf tissue. Among 3,600 leaves assayed, eight species of the three genera were observed. Features and criteria for the practical identification of species on plated leaf samples are described. Sporulation by dematiaceous hyphomycetes was observed on 97% of leaves; a single pathogen was observed on 20% and two to five pathogens were observed on 77% of leaves. Distributions of leaves among classes with one to five pathogens per leaf, for sites within years, always differed significantly (P = 0.01) from a Poisson distribution and usually included fewer leaves than expected with four or five pathogens. Significant (P = 0.05) variation in frequencies of occurrence of pathogens among 72 samples of 50 leaves each was attributed to pathogen species, sampling times, and species-time interactions. Exserohilum rostratum, Curvularia lunata, and Bipolaris cynodontis were the most frequent pathogens across years and sites; B. spicifera and C. geniculata were intermediate; and B. hawaiiensis, B. sorokiniana, and B. stenospila were least frequent. For the five most common pathogens, significant differences in frequency among locations were commonplace. Six pathogens exhibited significant (P = 0.05) positive and negative correlations with others in overall frequencies of occurrence across years, sampling times, and sites. However, chi(2) tests of dual occurrence indicated that interactions between specific pairs of pathogens in or on leaves are not likely to be major causes for overall frequency correlations. Results indicate that dematiaceous hyphomycetes typically infect forage bermudagrass on swine waste application sites in complexes rather than as individual species; that E. rostratum, C. lunata, and B. cynodontis are the predominant pathogens; and that frequencies of pathogens often differ significantly between locations.