波尔多液对烟草叶际微生物群落结构与代谢功能的影响

测定了波尔多液对烟草赤星病菌的毒力,并采用高通量测序与Biolog代谢表型技术分别测定了其对烟叶健康与感病组织叶际微生物群落结构和代谢功能的影响。结果表明:波尔多液对烟草赤星病菌的抑制活性较弱,其抑制菌丝生长和孢子萌发的EC₅₀值分别为450.19和757.17 mg/L。健康与感病烟叶组织叶际细菌均分布于变形菌门 (6.93%和39.07%) 和厚壁菌门 (16.45%和0.65%),优势细菌均有Kosakonia (3.46%和22.38%) 和假单胞菌属 (0.22%和5.95%);真菌均分布于子囊菌门 (63.82%和93.74%) 和担子菌门 (6.82%和2.53%),优势真菌有链格孢属 (36.48%和84.52%) 、Symmetrospora (5.56%和2.27%) 和枝孢霉属 (14.87%和6.66%)。波尔多液1 500 g/hm2处理对健康和感病烟叶叶际细菌和真菌群落结构与代谢功能均有影响,处理5 d时降低了叶际Kosakonia、鞘脂单胞菌属和乳杆菌属的相对丰度,增加了假单胞菌属、劳尔氏菌属等6种细菌菌属的相对丰度;降低了链格孢属、Symmetrospora等6种真菌属的相对丰度,增加了亚隔孢壳属、绿僵菌属等10种真菌属的相对丰度。处理10和15 d时对叶际真菌、细菌的影响逐渐降低。健康与感病烟叶叶际微生物均可高效代谢糖类、氨基酸类、羧酸类、双亲化合物、聚合物和胺/氨基化合物等29种碳源,但对α-丁酮酸的代谢较弱。波尔多液处理对烟叶叶际微生物的代谢抑制活性随时间延长逐渐减弱。研究结果揭示了波尔多液施用不同时期后对烟叶叶际微生物的影响规律,为了解药剂持效期的生态效益提供了参考依据。     

菌核净对烟草靶斑病菌的抑制作用及对烟叶叶际微生物群落结构的影响

烟草靶斑病是烟叶生产上一种主要真菌性病害，为评价菌核净防控烟草靶斑病的潜力，并从微生态层面揭示菌核净施用后对烟叶叶际微生物群落结构的影响，本研究采用菌丝生长速率法，测定了菌核净对烟草靶斑病菌的抑制活性，并利用Illumina Hiseq高通量测序技术，分析了菌核净处理后不同持效期内，健康与感病组织中叶际真菌及细菌群落结构和多样性的变化规律。结果表明：菌核净对靶斑病菌菌丝生长有较强的抑制活性，其EC₅₀值为1.20μg/mL，在6.47μg/mL下即可完全抑制菌丝生长。40%菌核净可湿性粉剂按有效成分4200 g/hm²剂量施用后0～18 d，健康与感病组织的叶际微生物群落结构间均存在显著性差异，其中叶际真菌优势菌属为亡革菌属、链格孢属和镰刀菌属，叶际细菌优势菌属为假单胞菌属、葡萄球菌属和鞘氨醇单胞菌属。施药后3 d，健康与感病组织中亡革菌属相对丰度分别下降12.41%和51.62%，链格孢属相对丰度分别上升0.54%和0.42%，假单胞菌属相对丰度分别下降13.48%和19.17%；施药后9 d，亡革菌属相对丰度分别上升1.38%和47.42...     

烯酰吗啉对葡萄叶际微生物区系影响研究

 为探究施用烯酰吗啉后不同葡萄叶际微生物群落变化,以田间‘红地球’品种葡萄为研究对象,连续施用农药烯酰吗啉,采集不同组别样品,利用 Illumina Hiseq 高通量测序技术分析葡萄叶际真菌、细菌群落结构变化。基于可操作分类单元(operational taxonomic units, OTUs)的物种分类分析,共得到葡萄叶际微生物真菌群落共计6个门,23个纲,60个目,131个科,212个属,296个种;细菌群落共计42个门,91个纲,222个目,398个科,846个属,1 469个种。在云南农业大学砂壤土质、篱架式栽培的‘红地球’葡萄基地中发现:(1)葡萄叶际优势真菌有枝孢菌属(Cladosporium)、白粉菌属(Erysiphe)、unclassified -k_-Fungi属和Symmetrospora属;优势细菌有乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)、拟杆菌属(Bacteroides)、肠球菌属(Enterococcus)、毛螺菌属(unclassified-f-Lachnospiraceae)、norank-f-Muribaculaceae属、芽孢杆属(Bacillus)、Blautia属和肠杆菌属(Kosakonia)。(2)经烯酰吗啉处理后,在健康葡萄叶片中其叶际真菌群落丰富度略降低,多样性显著增加,叶际细菌群落丰富度增加,多样性增加,但零星发病叶片中,叶际真菌、细菌群落丰富度和多样性则显著降低。(3)在零星发病葡萄叶片中,相对健康叶片,葡萄叶际真菌群落之间的差异被增大,而葡萄叶际细菌群落的差异被减小。(4)在健康及发病叶片中,相对丰度均上调的真菌为白粉菌属(Erysiphe)、球腔菌属(Mycosphaerella)、线黑粉酵母属(Filobasidium)、黑孢霉属(Nigrospora)、赤霉属(Gibberella)、被孢霉菌(Mortierella)和镰刀菌属(Fusarium),细菌为巨单胞菌属(Megamonas)、链球菌属(Streptococcus)、葡萄球菌属(Staphylococcus)和弯曲杆菌属(Campylobacter);相对丰度均下调的真菌为枝孢属(Cladosporium)、壳针孢属(Septoria)、链格孢属(Alternaria)和担孢酵母属(Erythrobasidium);细菌为短小杆菌属(Kosakonia)鞘脂单胞菌属(Sphingomonas);推断这些菌对烯酰吗啉较为敏感。(5)施用烯酰吗啉农药对葡萄叶际微生物的影响在健康叶片与零星发病叶片中有差异,且有新的优势菌属出现。     

两类抗生素药剂对‘纽荷尔’脐橙黄龙病菌的抑制作用及根际细菌群落结构的影响

为探究磺胺二甲氧嘧啶钠盐（sulfadimethoxine sodium salt, SDM）和氨苄青霉素（ampicillin, AMP）对柑橘黄龙病（citrus Huanglongbing, HLB）的防效以及对柑橘根际细菌群落结构的影响, 本试验以‘纽荷尔’脐橙为研究对象, 采用0.5 g/L SDM和1 g/L AMP进行灌根, 使用TaqMan qPCR技术监测病树叶内黄龙病菌菌量的动态变化, 并利用宏基因组技术分析根际细菌群落结构的变化。结果显示, AMP连续处理后病树叶片内黄龙病菌菌含量在一定程度上受到抑制, 初步判断AMP对黄龙病有一定的抑制效果, SDM处理对黄龙病菌菌含量抑制效果较差;2类抗生素处理组中根际主要优势类群结构和相对丰度在门、属分类水平上均发生改变, 解磷细菌中慢生根瘤菌属Bradyrhizobium细菌的相对丰度在处理后有明显变化, pstA、pstC等部分磷循环功能基因相对丰度发生改变, 表明2类抗生素药剂均影响了‘纽荷尔’脐橙病树根际细菌群落结构。     

田间柑橘植株不同部位黄龙病菌的PCR检测及发病原因分析

［目的］了解黄龙病菌在柑橘植株不同部位的分布,为深入研究病菌在植株体内的扩散情况奠定基础;明确病害的发生原因为有效防控该病害提供借鉴。［方法］ 调查浙江省台州市柑橘黄龙病（huanglongbing, HLB）的发生情况,通过常规和巢式PCR,检测了发病情形不同的两个果园内柑橘病株不同部位及不同植株中的黄龙病菌,并对其发病原因进行分析。［结果］ 发现一果园内病株的无症状叶片、有症状叶片和枝条中均含有黄龙病菌,而其周围植株不含病菌;另一果园内病株的有症状叶片、枝条、主干和砧木中均含有黄龙病菌,而其周围植株也含菌。［结论］分析认为这两种果园内柑橘植株发病原因不同,一种可能为通过携带黄龙病菌的柑橘木虱所感染,另一种可能为嫁接过程中通过带菌的接穗感染。     

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?     

滋生青苔对柑橘叶际生物多样性的影响

本文利用Illumina Miseq高通量测序技术对重庆地区有青苔病症和无病症的柑橘叶际真核生物的群落组成、结构、多样性及差异进行了研究,旨在找到可能的病原物类群,为更好地防治柑橘青苔病奠定基础。结果表明13个柑橘叶片样本共检测到303个OTU,多样性较为丰富;优势门为链形植物门Streptophyta、子囊菌门Ascomycota、绿藻门Chlorophyta等;通过分析发现可能的病原物为不可培养的虚幻球藻属球藻uncultured Apatococcus sp.、黄绿异小球藻Heterochlorella luteoviridis、无柄杯梗孢Cyphellophora sessilis、海南橡胶藻Heveochlorella hainangensis、Coniochaetales sp. GMG C4、椭圆球藻Chloroidium ellipsoideum、Kalinella bambusicola等;不同区县表现出青苔病症状的柑橘叶片上疑似病原物的种类和丰度都差异很大。柑橘青苔病可能是由多种藻类及真菌复合侵染造成的,病原物和柑橘叶片间的相互作用有待进一步研究。     

桑树青枯病与根际土壤肥力及微生物群落结构特征的研究

本文比较分析了桑树青枯病发病和健康植株根际土壤肥力以及微生物（细菌和真菌）群落结构特征差异，旨在挖掘和利用丰富微生物资源的有益功能，为构建桑树青枯病生物防治技术体系奠定基础。结果表明，桑树青枯病发病植株根际土壤中指示土壤肥力与健康状况的生物学性状指标β-葡糖苷酶和磷酸酶的活性显著降低，微生物生物量碳、磷显著下降；细菌门分类水平上，发病植株根际土壤中酸杆菌门、放线菌门和疣微菌门等优势细菌相对丰度降低，变形菌门、绿弯菌门、粘球菌门、厚壁菌门、拟杆菌门等优势细菌相对丰度增加；细菌属分类水平上，发病植株根际土壤富集了硝化螺菌属和放线菌属等特有细菌属，缺失了诸如链霉菌属等具有分泌抗生素功能的优势细菌属。真菌门分类水平上，发病植株根际土壤中子囊菌门和担子菌门真菌相对丰度增加，unclassified_k_Fungi和霉菌门真菌相对丰度降低；真菌属分类水平上，发病植株根际土壤富集了地霉菌属、腐质霉属和丝孢菌属等腐生真菌，缺失了被孢霉属、枝顶孢属和曲霉属等具有产抑菌化合物功能的优势真菌。研究表明，桑树青枯病与根际土壤β-葡糖苷酶、磷酸酶活性，微生物生物量碳、磷以及微生物群落结构都具有一定的相关性。链霉菌属细菌以及被孢霉属、枝顶孢属和曲霉属真菌有望作为生物防控青枯病的有效备选菌属。     

花生田施用绿僵菌对土壤微生物群落的影响

昆虫病原真菌绿僵菌对地下害虫防治具有良好应用前景，而大量释放绿僵菌可能引起土著微生物群落变化，从而影响其植物保护的综合效果。本文研究了花生播种期施用绿僵菌后，在根际和根围土壤中绿僵菌自身种群及土著细菌、真菌和放线菌种群的消长和群落结构变化。结果表明，绿僵菌种群在30 d内快速下降，之后降速减缓，以低密度持续存活。随花生生长，土壤微生物自身有消长过程，施用绿僵菌对放线菌影响最大、真菌其次、细菌最小，在根际的影响大于在根围的影响。分别地，绿僵菌处理对根际和根围细菌、根围真菌无显著影响；使根际真菌种群初期的下降速度减缓，到达谷底时间由15 d推迟到30 d，但回升速度加快，达峰顶由60 d提前到45 d，峰值降低1/3；绿僵菌明显抑制了根际放线菌种群，使其消长变化周期与对照完全相反，且45 d时回落至低点后不再回升；绿僵菌减缓了根围放线菌种群下降，使达谷底时间由30 d推迟到45 d，且回升幅度减小。土壤微生物群落结构在施菌后15～30 d的幼苗至初花期有明显变化，细菌种群总体占比显著下降，但与对照相比，施菌后的根际细菌占比提高，真菌占比下降，放线菌占比显著地先抑后扬。在根围的变化相对较小。45 d即盛花期后，三类微生物结构逐渐恢复趋近原来状态。     

高通量测序分析高温覆膜对韭菜根际微生物多样性的影响

为明确高温覆膜对韭菜根际土壤微生物群落多样性和组成的影响。本研究在覆膜前后对韭菜根际土壤中总DNA进行提取，采用Illumina Hiseq 2500高通量测序技术对土壤中细菌的16S rDNA和真菌的ITS基因进行了序列测定，分析了高温覆膜前后韭菜根际土壤中微生物群落结构特征。结果表明，高温覆膜前后分别获得细菌OTUs 1849个和1819个，真菌OTUs 148个和151个，无显著差异。覆膜前后物种分类显示，细菌种类均隶属于24门53纲112目218科322属，其中优势类群为蛋白菌门、厚壁菌门和放线菌门，覆膜前后的相对丰度分别为43.79%、21.70%、21.03%和42.36%、17.55%、23.92%。真菌种类均隶属于4门10纲21目27科34属，优势类群为子囊菌门和担子菌门，覆膜前后的相对丰度分别为62.48%、15.28%和51.13%、40.95%。可见，土壤中细菌群落的多样性和丰富度均高于真菌，覆膜前后微生物总量无显著变化，细菌、真菌在多样性指数上无显著差异，而在结构组成上差异显著。     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

Rapid and sensitive detection of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus” by cycleave isothermal and chimeric primer-initiated amplification of nucleic acids

We developed a detection method for “Candidatus Liberibacter asiaticus”, causal agent of citrus huanglongbing, using isothermal and chimeric primer-initiated amplification of nucleic acids combined with cycling probe technology (Cycleave ICAN). With Cycleave ICAN, the reaction was done in one tube in 1 h without the need for electrophoresis, and false positives were not generated. In addition, Cycleave ICAN method was more sensitive than the conventional PCR method. Cycleave ICAN helps shorten the time for the large-scale detection needed to manage huanglongbing.     

Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid,Diaphorina citri

BACKGROUND: Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits the causal bacteria of the devastating citrus disease huanglongbing (HLB). Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. The objective of this laboratory study was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. RESULTS: Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 µg mL^?1) of imidacloprid significantly decreased adult longevity (8 days), fecundity (33%) and fertility (6%), as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7–94%) compared with controls in a concentration‐dependent manner suggesting antifeedant activity of imidacloprid. CONCLUSIONS: Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival and longevity of ACP, which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Copyright © 2009 Society of Chemical Industry     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.     

Biofilm formation and motility of Xanthomonas strains with different citrus host range

Xanthomonas citri subsp. citri (Xcc) strain A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp. and close relatives. Two restricted host range strains of CBC, A^w and A*, from Florida and southwest Asia, respectively, infect Mexican lime. Several studies have linked biofilm formation by Xcc to bacterial colonization prior to and after plant ingress, but none have evaluated connections between biofilm formation and the behaviour of different strains of Xcc on citrus hosts and non‐hosts. In this study biofilm formation and swimming motility were evaluated for citrus pathogenic xanthomonads including wide and restricted host range strains of Xcc, X. alfalfae subsp. citrumelonis (Xac) (the causal agent of citrus bacterial spot) and X. campestris pv. campestris (Xc). Differential biofilm formation was observed in vitro and in planta among the Xanthomonas strains assayed. Minimal medium XVM2 increased biofilm formation, especially for those strains with a host range restricted to Mexican lime. In planta, strains produced more biofilm on leaves or fruits of their host than on non‐hosts. Scanning electron microscopy of biofilms on leaf and fruit surfaces revealed differences in structure of bacterial aggregates with respect to the strain's host range. In addition, swimming motility varied widely depending on the host range of the strain. It was concluded that biofilm formation in vitro and in planta for strains of Xcc and Xac was related to their host range, as these processes affect colonization at the early stages of the infection process.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.     

Functional Characterization of Citrus Polygalacturonase-inhibiting Protein

A cDNA encoding a polygalacturonase-inhibiting protein gene (SaiPGIPA) was identified from the citrus cultivar Sainumphung (Citrus sp.), one of the most popular cultivars in northern Thailand. SaiPGIPA was expressed in Escherichia coli cells, and the functional properties of citrus PGIP were analyzed. The PGIP fusion protein inhibited by a maximum of about 60% of the endopolygalacturonase activity, and a mixture of the PGIP and fungal endopoly-galacturonase released oligogalacturonides from polygalacturonic acid. The mixture containing the oligogalactur-onides, endopolygalacturonase and PGIP induced expression of the PGIP gene and a chalcone synthase gene in citrus leaves. The mixture also induced resistance in cucumber leaves against Colletotrichum lagenarium. Received 5 September 2001/ Accepted in revised form 20 November 2001     

<Emphasis Type="Italic">In planta</Emphasis> multiplication and graft transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’ revealed by Real-Time PCR

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g⁻¹ of tissue, increased 10,000 times from 10³ at 30 dpi to approximately 10⁸ CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10⁵ CN g⁻¹). Blotchy mottle symptoms were observed in trees with 10⁷ CN g⁻¹ of bacteria after 180 dpi. Buds taken from infected, but non-symptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.