Detection and characterization of Ralstonia pseudosolanacearum infecting Eucalyptus sp. in Brazil

Ralstonia solanacearum sensu lato causes bacterial wilt in many agronomic crops and tree species economically important worldwide. It is a species complex that has been divided into phylotypes and sequevars, commonly related to geographic distribution. Knowledge of the phylotype composition and genetic variability in populations of this phytopathogenic bacterium is useful for implementing effective control measures. In a survey conducted in 2019, six bacterial strains were obtained from wilted Eucalyptus urophylla trees in plantations located in the municipality of Dom Eliseu, Pará state, Brazil. Multiplex PCR based on the internal transcribed spacer (ITS) indicated that the bacterial strains belonged to two different species, namely R. pseudosolanacearum (phylotype I) and R. solanacearum (phylotype II). In a phylogenetic analysis, the nucleotide sequence of the endoglucanase (egl) gene from eucalypt strains of phylotype I clustered together with sequevar 18 sequences from GenBank. Separation of the strains into two different species was confirmed by repetitive element palindromic PCR (rep‐PCR). Pathogenicity tests demonstrated that the R. solanacearum and R. pseudosolanacearum strains recovered from E. urophylla cause disease in both tomato and eucalypt plants. Until now, only R. solanacearum (Phylotype II) has been reported causing wilt symptoms on Eucalyptus spp. in Brazil. Therefore, the presence of R. pseudosolanacearum and a need for better understanding of its genetic and aggressiveness variability as well as possible differences between the two species should be considered in breeding programmes aimed at the deployment of host resistance.     

Ralstonia solanacearum decreases volumetric growth of trees and yield of kraft cellulose of Eucalyptus spp.

Ralstonia solanacearum is currently one of the most important plant pathogenic bacteria worldwide, with a wide geographical distribution and host diversity. The pathogen infects more than 200 plant species belonging to approximately 50 plant families, including Eucalyptus spp. Although, high losses have been reported in nurseries, little is known on the negative impact of the disease in the field. In this study, we evaluated the incidence of R. solanacearum and its effect on volumetric growth and cellulose yield of discoloured wood chips obtained from infected trees of one clone of Eucalyptus urophylla and two hybrid clones of Eucalyptus urophylla × E. grandis. The average incidence of bacterial wilt ranged between 60.6% and 72.4%. Volumetric growth of infected trees disease decreased 78.6% and 81.7% at 18 and 30 months, respectively. The pulp screen yield of three clones decreased between 3.2 and 6.4%, with an average 4.3%. The results of this work provide useful information on the losses of volumetric growth and pulp yield of eucalypt caused by R. solanacearum.     

First report of bacterial wilt caused by Ralstonia solanacearum on eucalypts in South Africa

Ralstonia solanacearum, the causal agent of bacterial wilt, has one of the widest host ranges of all phytopathogenic bacteria. This pathogen was first reported on Eucalyptus spp. in the late 1980s in Brazil. Since then, there have been reports of its occurrence on this host in Australia, China and Venezuela. Early in 1997, an 18‐month‐old clonally propagated Eucalyptus grandis × Eucalyptus camaldulensis (GC) hybrid in Zululand, KwaZulu/Natal, showed signs of wilting. The vascular tissue of infected trees was dicoloured and bacterial exudation was produced from cut surfaces. The bacterium was consistently isolated from diseased tissue, purified and identified as R. solanacearum biovar 3 race 1, using the BioLog bacterial identification system. Inoculation trials were conducted on three E. grandis × E. camaldulensis clones (GC515, GC550 and GC505). Clone GC550 displayed wilting after 3 days and all cuttings subsequently died. Clones GC515 and GC505 appeared to be less susceptible with cuttings not showing signs of disease until 7 days after inoculation. After 14 days, 90 and 80%, respectively, of cuttings of these two clones had died. This is the first report of bacterial wilt on Eucalyptus in South Africa.     

First report of bacterial wilt caused by Ralstonia solanacearum on fig trees in China

This article presents the first report of bacterial wilt on fig (Ficus carica) trees in China. In 2014, fig trees with typical bacterial wilt symptoms were observed for the first time in China. The causal pathogen was determined to be Ralstonia solanacearum. Identification was based on symptomatology, morphology, BioLog carbohydrate utilization, 16S rDNA sequence analyses and phylotype‐specific multiplex‐PCR assays.     

Antimicrobial activity of heartwood components of sugi (Cryptomeria japonica) against several fungi and bacteria

Methanol extract of sawdust of sugi (Cryptomeria japonica) heartwood was fractionated with toluene and n-hexane to give solvent-soluble and solvent-insoluble fractions. The n-hexane-soluble fraction showed the most inhibition activity among the fractions against phytopathogenic microorganisms, namely Fusarium oxysporum, Phytophthora capsici, Pythium splendens, and Ralstonia solanacearum. Sandaracopimarinol and ferruginol, isolated from the n-hexane-soluble fraction, showed moderate antifungal activity against the three fungi and strong antibacterial activity against R. solanacearum. The content of sandaracopimarinol (7.07 g/kg based on the dried sawdust) in the heartwood was about twice that of ferruginol. Sandaracopimarinol and ferruginol strongly inhibited the growth of Gram-positive bacteria but did not show inhibitory action against Gram-negative bacteria except for R. solanacearum. The antibacterial effect of sandaracopimarinol was first found in the present study and was stronger than that of ferruginol.     

我国桉树青枯病研究进展

桉树青枯病害是危害桉树生产的主要病害,近年来桉树青枯病的流行和发生对我国南方桉树产业造成很大经济损失.本文对当今主要桉树青枯病病害特点,青枯病的发生和流行因素,青枯假单胞杆菌对寄主桉树的致病机制及对抗病的差异和抗病机理等研究成果和现状作一概述,并就存在的问题提出相应的建议.     

桉树抗青枯病的鉴定技术

采用菌液浸泡法、顶尖接种法对不同的桉树无性系盆栽苗和生根组培苗进行抗青枯病性能测定,并结合林间调查筛选桉树抗青枯病树种和无性系,并从根系分泌物和组织研磨液的角度研究其对桉树青枯病菌生长的抑制作用,探索不同抗性桉树无性系抗病性强弱与其根系分泌物和组织研磨液之间的关系。结果表明:供试的18个桉树无性系中,bd1、bd2、赤桉和南宁巨尾桉为抗病无性系;U6、南宁尾叶桉、雷9、钦32-29为中抗无性系;DH32-27、钦9、南宁广9、钦8、邓恩桉、钦32-22、雷2、巨桉、尾叶桉和钦广9为感病无性系。不同抗性桉树无性系根系分泌物和组织研磨液对青枯菌没有直接拮抗作用,但随着接种时间的延长,青枯病菌在抗病性强的无性系的根系分泌物及组织液中的增殖显著低于感病无性系,验证通过茎段浸泡接种筛选出的桉树无性系的准确性,同时表明根系分泌物和组织研磨液可以反映桉树不同无性系的抗病性强弱。     

木麻黄青枯病菌的分离及强致病菌株的筛选

[目的]筛选出强致病菌株用于木麻黄抗病育种研究工作。[方法]在广东沿海木麻黄青枯病发病区采集病根,开展病原菌两种不同分离方法的比较研究,对分离出的31个病株进行16s rRNA测序鉴定及致病性测定。[结果]采用稀释分离法及根系溢出法在TTC培养基上共分离出了31个病原菌株,根系溢出法操作简便,杂菌含量低,分离率在60%左右,可作为常规稀释分离法的补充。31个菌株进行分子鉴定,只有22个菌株扩增出了特异性条带,经测序比对确定这22个菌株为青枯菌。青枯菌株致病性测定结果显示菌株致病性在无性系间、菌株间及菌株与无性系间的交互作用均具有极显著差异(P0.01),不同接种方法间菌株致病性相关系数值较小,介于0.496 6~0.731 0之间,即室内水培接种与小苗盆栽接种不存在密切的直线相关关系。[结论]综合选择在不同无性系及不同接种方法中均具有较强致病性的GL-2、H、M、TC-1、F、Q菌株作为下一步木麻黄种质资源抗性鉴定及抗病育种研究试验菌株。     

Screening for resistant clones of Casuarina equisetifolia to bacterial wilt and the analysis of AFLP markers in resistant clones

Bacteria wilt (BW) of Casuarina equisetifolia L. Johnson, caused by Ralstonia solanacearum (E. F. Smith) Yabuuchi, is one of the most important diseases of C. equisetifolia L. Johnson, the main coastal protective forest tree in southeast China. The resistance of 33 C. equisetifolia L. Johnson clones to BW was determined by means of hydroponic and pot inoculation methods with the suspension of R  solanacearum (E. F. Smith) Yabuuchi as inocula in the two‐step procedure. The results showed that clones FJ11 and FJ13 were highly resistant to BW with the lowest disease incidences of 10.8 and 15.8%, respectively, whereas clones FJ9 and GD15 were the most susceptible ones with the highest disease incidences of 100.0%. The analysis of AFLP markers was performed on clones FJ11, FJ13, FJ9 and GD15, and the results indicated that three DNA fragments E‐AAA/M‐CCG, E‐AAG/M‐CGC and E‐AGG/M‐CAA were found to be linked to the BW resistance. BLAST searches revealed that the first two fragments (GenBank accession numbers: KC111411 and KC135871) had no identities to any sequences, and the third one showed a high similarity to some sequences which code for heat shock protein. Taken together, the two resistant clones screened in our research were undoubtedly the valuable resistant resources of C. equisetifolia L. Johnson, and the three AFLP markers could be used in marker‐assisted selection of C. equisetifolia L. Johnson in the future.     

桉树青枯病病原菌的分子鉴定及其致病性测定

对华南4个地区的桉树青枯病病原菌进行了分子鉴定和致病性测试研究,以期为有效预防其发生提供依据.分别对来源于不同区域的11株桉树青枯病病原菌进行分子鉴定和致病力强弱测试比较,11株病原菌与Ralstonia solanacearum序列形成支持率为86％分枝,表明其病原菌16S rRNA基因序列与Ralstonia solanacearum具有高度同源性.以桉树无性系DH32-29组培苗为材料,采用伤根法对11个菌株进行致病力测试,不同菌株间在致病性上差异显著(P＜0.01或P＜0.05),以发病高峰期、高峰期发病率和平均发病率进行K-均值聚类分析(K=3),结果表明来源于广东、广西、海南的各菌株间致病性存在差异,HY01、HY02、DA01、HP04菌株与HP05分为1类,DA02和DA03为第2类,而YJ01、HP01、HP02和HP03为第3类.对4个不同地区(采样点)的青枯病菌株平均发病率进行方差分析,结果发现来源广东阳江菌株致病性显著地低于其他来源菌株.不同地理来源菌株之间在致病性上无明显相关性,同一地区内同时存在着致病力强弱菌株,HP03的生物型4-1菌株与其他生物型3菌株在致病性亦无明显的差别.     

Bacteria wilt of Casuarina equisetifolia in India

Pseudomonas solanacearum (Smith) was consistently isolated from the wilted plants of Casuarina equisetifolia. The isolate gave positive reaction to oxidase, catalase and gas production from nitrate and negative reaction for arginine dihydrolase and acid was produced from various carbohydrates. Wilt produced on tomato, egg plant and capsicum indicated that the pathogen belongs to Race I and Biovar III of P. solanacearum which is a new record from India.     

Effect of some nursery practices on incidence and severity of diseases,and growth of Eucalyptus grandis seedlings*

Nursery practices influenced incidence and severity of diseases of Eucalyptus grandis viz. damping-off caused by Pythium sp., Rhizoctonia solani and Cylindrocladium quinqueseptatum, web might by R. solani, seedling blight by C. quinqueseptatum and shoot wilt by Sclerotium rolfsii. Growth of seedlings of E. grandis and microclimatic conditions were also affected by nursery practices. Shading with coconut leaf thatch led to low light intensity (av. 1463 lux) with high soil water potential, low soil and ambient temperatures, and high severity of damping-off and web blight diseases and poor shoot:root ratio of seedlings. Seed beds under coirmat had dispersed light (av. 22 299 lux), apparently high severity of seedling blight and shoot wilt, and good growth (snoot:root ratio) of seedlings. In both the types of shading high soil moisture regime and high seed rate contributed to high disease severity as well as low shoot:root ratio of seedlings.     

拟南芥AtFBDL 1 在植物顶端生长调节中的作用

拟南芥 AtFBDL1 基因是FBD-like基因家族的一员,其编码蛋白含有类似于F-box的结构域。表达模式网络预测结果显示该基因在茎顶端分生组织中高丰度表达,但对于FBD-like基因家族的研究还很少,其功能目前尚不明确。为此,本研究通过组织半定量表达分析和GUS染色显示 AtFBDL1 基因在拟南芥中具有时空表达特异性。结果表明:在真叶形成前和形成初期,该基因主要在茎顶端分生组织和下胚轴区域表达;真叶形成后,该基因在下胚轴的表达明显减少,而主要集中在茎顶端分生组织表达。遗传转化显示:与野生型植株相比,过表达 AtFBDL1 基因的植株生长发育缓慢,抽薹时间推迟3 4 d,莲座叶叶片面积减小,叶片数目平均增多10片,并且伴随有变态叶出现;过表达植株株高比野生型矮,株高最大差值达到12 cm。共表达网络预测 AtFBDL1 与多个与生长素和花发育相关的基因具有共表达关系。以上研究结果表明: AtFBDL1 基因在拟南芥的生长发育过程中,特别是在顶端分生组织分化过程中起重要作用。     

Spatiotemporal dynamics of bacterial wilt in Eucalyptus

Bacterial wilt (caused by Ralstonia spp.) is one of the most damaging diseases of Eucalyptus species, and is responsible for substantial losses to producers. For efficient, scientifically-based management of this disease, it is necessary to understand the various factors involved in its development, including greater knowledge of the epidemiology of the pathogen on Eucalyptus spp. This study aimed to determine the spatialtemporal dynamics of bacterial wilt in Eucalyptus under natural infection conditions. An experiment was conducted in a commercial plantation in the municipality of Itinga, state of Maranhão, Brazil, using the clone FGCA0385 (Eucalyptus urophylla var. platyphylla). The study comprised of four plots composed of 450 plants each, subdivided into nine rows with 50 plants per row with a spacing of 3 × 3 m. Disease incidence was quantified over 1 year. The spatial dynamics of the disease was determined using the dispersion index, a modified Taylor law, and the analysis of the dynamics and structure of foci. For temporal dynamics, the curve of the disease incidence progress was plotted, and the data were analysed by simple linear regression analysis fitted to three empirical models: logistic, monomolecular, and Gompertz. The distribution pattern of Eucalyptus bacterial wilt was random, as confirmed by the Index of dispersion. Analysis of the dynamics and structure of the foci, showed that 69 disease foci occurred, 44 of which were unitary, with an average number of plants per focus of 1.63. Foci had greater length in the direction of the planting line. The epidemics were best described by the monomolecular model, with an estimated incidence of Eucalyptus bacterial wilt of 27.77% in the fourth year of the study. According to the spatiotemporal dynamics of this work, management strategies such as eliminating symptomatic plants and crop renovation can be used based on economic viability.     

Pseudomonas syringae pv. aesculi: foliar infection of Aesculus species and temperature–growth relationships

Since 2001, the incidence of bleeding canker of horse chestnut (Aesculus hippocastanum) has increased markedly in western Europe. The causal agent, the bacterium Pseudomonas syringae pv. aesculi, originally isolated from foliar lesions on Indian horse chestnut (Aesculus indica) in India, is a bark killing pathogen on A. hippocastanum. In this study, P. syringae pv. aesculi was found as a foliar epiphyte on both A. hippocastanum and A. indica trees growing in the UK. When Aesculus leaves were challenged with cell suspensions (10⁹ CFU ml^?1) of Pseudomonas syringae pv. aesculi, a high level of asymptomatic infection occurred in all the species tested. The degree of re‐isolation of the bacterium after surface sterilization of leaves ranged from 33% (A. pavia) to 84 and 97% for A. hippocastanum and A. chinensis, respectively. The studies suggest both epiphytic and intrafoliar populations of P. syringae pv. aesculi could play a role in the incidence and spread of bleeding canker of horse chestnut. Growth–temperature responses of P. syringae pv. aesculi indicated a minimum of approximately ?4°C and a maximum of approximately 35°C, with an optimum of approximately 25°C. These findings show that P. syringae pv. aesculi is not restricted to bark lesions but is likely to be widespread in the environment. It is also capable of causing foliar infection of several Aesculus species and could persist under extremes of weather in the UK.     

Xyleborus glabratus attacks and systemic colonization by Raffaelea lauricola associated with dieback of Cinnamomum camphora in the southeastern United States

Laurel wilt, caused by Raffaelea lauricola, is responsible for extensive mortality of redbay and other American members of the Lauraceae in the southeastern United States. Raffaelea lauricola is a mycangial symbiont of the redbay ambrosia beetle (Xyleborus glabratus), and the beetle and fungus were accidentally introduced from Asia. Branch dieback of camphortree (Cinnamomum camphora), an Asian member of the Lauraceae, has been occasionally observed in areas where laurel wilt has decimated redbay populations, and R. lauricola was isolated from such camphortrees. However, the role of X. glabratus and R. lauricola in this branch dieback remains unclear. Examination of camphortrees on Jekyll Island, Georgia showed that healthy‐appearing trees and those with branch dieback had been attacked by X. glabratus, but the trees with branch dieback had four times as many beetle attacks. Raffaelea lauricola was routinely isolated from discoloured xylem near beetle tunnels in healthy trees and those with dieback. Single‐point inoculations with R. lauricola on stems of mature, healthy camphortree trees failed to induce wilt‐like symptoms or branch dieback, although areas of discoloration were scattered throughout the xylem, and R. lauricola was reisolated irregularly at various heights in some inoculated trees. In growth chamber experiments, single‐point inoculations with R. lauricola resulted in systemic colonization but no wilt symptoms or branch dieback in camphortree saplings. In contrast, inoculations at multiple points along the stem (simulating multiple attacks by the vector) caused branch dieback and wilt‐like symptoms, including a brownish, diffuse discoloration of the xylem. Camphortree appears to be more resistant than American species of Lauraceae to the vascular wilt caused by R. lauricola. The fungus does colonize camphortrees systemically, however, and can apparently cause branch dieback. This suggests that the fungus may provide brood material for X. glabratus in Asia as it does in the southeastern United States.     

Wilt and die‐back of Eucalyptus spp. caused by Erwinia psidii in Brazil

A new disease of unknown bacterial aetiology has been observed in eucalyptus stands since 2009. It is characterized by die‐back, wilting and lesions on the branches, petiole and midrib in association with macroscopic and microscopic bacterial ooze. To date, this disease has been observed in stands of clonal Eucalyptus saligna, E. grandis and E. urophylla x E. grandis hybrids and in E. dunnii seedling plantations in the states of São Paulo, Rio Grande do Sul and Mato Grosso do Sul. Considering the economic importance of eucalyptus plantations and the potential losses caused by this disease, this study aimed to identify and characterize the causal agent. Thirty‐four strains were obtained from infected plants, which were collected in the field from four locations. The inoculation of detached leaves and intact rooted cuttings supported pathogenicity in eucalyptus. The phylogenetic analysis of four housekeeping genes (16S rDNA, gapA, recA and rpoB) as well as biochemical tests confirmed the identity of strains belonging to the species Erwinia psidii. This is the first report of E. psidii as the cause of wilt and die‐back in Eucalyptus spp. in Brazil.     

Pseudomonas syringae pv. aesculi associated with horse chestnut bleeding canker in Germany

Bacteria were isolated from necrotic lesions on a horse chestnut tree (Aesculus hippocastanum) with bleeding canker in Hamburg, Germany. Sequencing of the rDNA-ITS region revealed great similarity to pathovars of Pseudomonas syringae. Pseudomonas syringae pv. aesculi was identified by sequence homology of the gyrase B gene. This is the first report of P. syringae pv. aesculi in Germany. Phytophthora was not detected.     

Wound response of Eucalyptus clones after inoculation with Cryphonectria cubensis

Twenty-five different Eucalyptus grandis clones were artificially wounded and inoculated with a virulent isolate of Cryphonectria cubensis. The capacity of wounds to close through callus production was correlated with the relative susceptibility of these clones to infection by C. cubensis. Clones with the greatest capacity to close wounds were those that were also most tolerant to C. cubensis infection. Those with a lower capacity to close wounds were most susceptible to Cryphonectria canker.