Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean C_t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean C_t value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C_t value in mature leaves = 33·6).     

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.     

Up‐regulation of PR1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘Candidatus Liberibacter asiaticus’

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las), is a devastating disease of citrus trees in Florida. Previous work showed that the rootstock cultivar Cleopatra mandarin (Citrus reticulata) has a higher population of Las in roots than Swingle citrumelo (C. paradisi × Poncirus trifoliata). Las reduced fibrous root biomass and sucrose content in Cleopatra mandarin more than in Swingle citrumelo. To understand the mechanisms for susceptibility to Las infection, sucrose and hormone metabolism status were evaluated in Cleopatra mandarin and Swingle citrumelo. In fibrous roots of Cleopatra mandarin, higher expression of genes related to sucrose cleavage was consistent with lower sucrose content compared to noninoculated seedlings at 5 weeks post‐root trimming (wpt). In fibrous roots of Swingle citrumelo, both sucrose content and gene expression related to sucrose cleavage were less disrupted by Las infection compared to Cleopatra mandarin at 5 wpt. Genes associated with salicylic acid (SA), ethylene (ET) and abscisic acid (ABA) synthesis, and ABA signalling, phospholipases D (PLD), and phospholipase A2 (PLA2) were activated by Las infection at 5 wpt in Cleopatra mandarin. Expression of downstream effectors of SA, i.e. NPR1, WRKY70 and PR1, did not change in Cleopatra mandarin, suggesting inhibition of the response to SA by the elevation of ABA, ET and PLD. In contrast, the up‐regulation of PR1, lower response of sucrose metabolism genes and down‐regulation of biosynthesis of phytohormones indicates that Swingle citrumelo activates a more effective defence against this biotrophic pathogen than Cleopatra mandarin.     

Contrasting canopy and fibrous root damage on Swingle citrumelo caused by ‘Candidatus Liberibacter asiaticus’ and Phytophthora nicotianae

Huanglongbing (HLB), associated with the phloem‐limited bacterium ‘Candidatus Liberibacter asiaticus’ (Las), is devastating trees in citrus orchards of Florida. Additionally, Phytophthora nicotianae, omnipresent in citrus soils, causes root rot that reduces water and nutrient uptake by fibrous roots. To investigate fibrous root damage and replacement and canopy size in relation to infection of fibrous roots by Las and P. nicotianae, rootstock seedlings of Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) were inoculated with Las or P. nicotianae in two greenhouse pot trials. Phytophthora nicotianae caused root damage within 5 weeks post‐inoculation, which led to greater reduction of canopy size than for Las‐infected seedlings by the end of the experiment. Las increased accumulation of fibrous root biomass at 5 weeks post‐root trimming (wpt) in the 2014 trial and at 11 wpt in the 2015 trial. New root length was not consistently increased by Las. Reduced total leaf area of symptomless Las‐infected seedlings compared to noninoculated controls might be due to the combined effect of altered carbohydrate allocation between shoots and roots and altered leaf morphology.     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

基于短串联重复和PAGE的柑橘黄龙病菌‘Candidatus Liberibacter asiaticus’种间遗传多样性分析

 韧皮部杆菌亚洲种(‘Candidatus Libriacter asiaticus’,‘Ca. L. asiaticus’)是目前流行范围最广、危害最严重的柑橘黄龙病致病菌。本研究利用‘Ca. L. asiaticus’基因组中全套串联重复基因(short tandem repeat, STR)开发了一套系统的方法用于‘Ca. L. asiaticus’遗传多样性和黄龙病分子流行学研究。PCR和PAGE分析筛选到的36个STR位点中,有33个获得了有效扩增。其中20对引物对不同来源的32个样品的扩增产物多态性较好,最多的可扩增出9种条带类型。采自我国6省的32个‘Ca. L. asiaticus’阳性样品之间的Shannon’s信息指数为0.14~1.90,平均为0.68;Nei’s基因多样性指数为0.06~0.81,平均为0.38,各省的菌株表现出较高的遗传多样性,特别是来自福建、广西和云南三省的。聚类分析发现可能为中国黄龙病发源地的广东省的黄龙病菌株具有一定的遗传特异性;其余邻省之间存在由木虱传播引起的菌株交流的可能性可以解释该病害在省际间传播。研究表明,开发的STR标记结合PAGE的方法可作为今后菌株遗传多样性和病害分子流行学分析的高效方法。     

Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector–pathogen relationships

Citrus huanglongbing (HLB), previously called greening, is a serious citrus disease in Asia, eastern and southern Africa. It is caused by Candidatus Liberibacter asiaticus (Las), a phloem-limited, nonculturable bacterium transmitted by the Asian citrus psyllid ( Diaphorina citri ) in Asia. A PCR-based assay was developed for monitoring Las in vector psyllids using a rapid DNA extraction from psyllid bodies and PCR amplification. The entire procedure for Las detection in psyllids can be completed within 5 h. Using this method, Las can be accurately detected in psyllid adults as well as nymphs in different instar stages. The assay is sensitive enough for Las detection in single-psyllid extract from adult, fifth, fourth and third instars. In a transovarial transmission experiment, Las was not detected in eggs or in offspring produced by Las-carrying psyllid females. In a retention test, the Las-carrying psyllids remained Las-positive for 12 weeks after they were moved to common jasmine orange, a Las-immune plant. From these experimental results it was concluded that Las persists in the Asian citrus psyllid vector, but is not transovarially transmitted by the vector. These data help in understanding epidemiological characteristics of Las and psyllids in citrus HLB.     

Phoma leaf spot of wasabi (Wasabia japonica) caused by Leptosphaeria biglobosa

A leaf spot disease on wasabi plants grown in commercial greenhouses in the Fraser Valley of British Columbia was characterized. Mycelial growth and pycnidial formation were observed within lesions when leaves were incubated under conditions of high humidity. Isolation from diseased tissues consistently yielded colonies of a Phoma species. Sequence analysis of the rDNA internal transcribed spacer (ITS1‐5.8S‐ITS2) region of eight isolates showed 100% nucleotide sequence identity with Phoma wasabiae and Leptosphaeria biglobosa subspecies ‘occiaustralensis’ and 99.2% identity with L. biglobosa ‘canadensis’. Pathogenicity studies on wasabi leaves showed that wounding greatly facilitated infection and enhanced lesion development for most isolates but was not required for all isolates. Chlorotic areas appeared around the inoculation sites within 4 days, followed by necrosis. Isolates displayed a range of virulence, from weakly to highly virulent, on wasabi leaves. Similar results were observed on leaves of canola cultivar Westar, i.e. wounding significantly increased lesion size and isolates displayed a range of virulence. An isolate of Leptosphaeria maculans ‘brassicae’ from canola was highly virulent on wasabi and canola leaves, causing lesions similar to those of L. biglobosa ‘occiaustralensis’ while an isolate of L. biglobosa ‘canadensis’ from canola was weakly virulent on both hosts and required wounds to infect. These results demonstrate that isolates of L. biglobosa ‘occiaustralensis’ from wasabi are as virulent as L. biglobosa ‘canadensis’ on wasabi and canola leaves but in some cases were comparable in virulence to L. maculans ‘brassicae’.     

Effects of huanglongbing on fruit quality of sweet orange cultivars in Brazil

Citrus huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus and Ca. L. americanus and transmitted by the Asian psyllid Diaphorina citri, is the most serious disease of citrus worldwide because of crop devastation and difficulty to control. Since 2004, approximately 3 million trees were eliminated in attempts to limit its spread in Brazil. Where HLB becomes endemic, the disease progression in the orchard and the increasing symptom severity throughout the tree canopy can be relatively fast, greatly reducing the economic life of affected orchards because of tree decline and yield reduction. Although the majority of the fruit from symptomatic branches drop before harvest, a significant amount of affected fruit remain attached, are available for harvest, and can affect juice quality. To quantify and compare the effects of HLB on fruit quality of the most important sweet orange cultivars grown in São Paulo State, 4–6 year-old sweet orange trees from 26 blocks (two of ‘Valencia Americana’, eight of ‘Hamlin’, four of ‘Westin’, seven of ‘Pera’, and five of ‘Valencia’) were selected prior to harvest. In each block, 14–21 HLB-symptomatic trees were chosen. In each tree, the quality of 20 fruit normal in appearance from asymptomatic branches and 20 symptomatic fruit from symptomatic branches were assessed. In general, compared to normal fruit, the symptomatic fruit were small, light, more acidic, and had lower juice percentage, Brix, total soluble solids per box, total soluble solids per fruit, and Brix/acidity ratio. These effects of fruit quality were less pronounced on early and mid season sweet orange cultivars than on late season cv. Valencia.     

中国不同地区亚洲韧皮杆菌遗传多样性分析

 柑桔黄龙病是最严重的柑桔病害之一,现已威胁世界柑桔产业。已被证实在中国南部该病害通过嫁接和木虱传播。因此黄龙病的流行病学研究就显得尤为重要。本研究中,基于外膜蛋白(omp)基因的PCR-RFLP的方法被用来研究中国南部各柑桔主产区的亚洲韧皮部杆菌的遗传多样性。研究采用不同的限制性内切酶消化各地区的亚洲韧皮部杆菌的omp基因产生了不同的RFLP指纹图谱,并对各地亚洲韧皮部杆菌的omp基因进行克隆测序然后构建系统发育树。结果显示:中国不同地理区域的亚洲韧皮部杆菌具有一定的遗传多样性,即便在某一特定的地区也有一定的差异。序列分析显示中国地区的亚洲韧皮部杆菌有很高的同源性。这些结果对揭示中国柑桔黄龙病的流行病学及制定科学有效的防治策略具有一定的指导意义。     

Effects of pymetrozine, an antifeedant of Hemiptera, on Asian citrus psyllid, Diaphorina citri, feeding behavior, survival and transmission of Candidatus Liberibacter asiaticus

BACKGROUND: Vector‐borne plant disease management can be enhanced by deployment of antifeedants in addition to the use of broad‐spectrum neurotoxic insecticides. The effects of pymetrozine on Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, feeding behaviour, survival and transmission of Candidatus Liberibacter asiaticus (Las), the presumed causal pathogen of huanglongbing, were investigated. RESULTS: Pymetrozine applied at 52 and 104 µg mL^?1 to citrus plants [Swingle citrumelo (X Citroncirus webberi Ingram and Moore)] modified the feeding behavior of ACP and increased the amount of time spent performing non‐penetration behaviors while decreasing the time spent performing ingestion behaviors compared with the controls 1 day after treatment. However, the antifeedant effect of pymetrozine subsided 5 days after application. Pymetrozine reduced the survival of both adults and nymphs on treated plants compared with the control. However, it had a greater impact on survival of nymphs than on survival of adults. Pymetrozine applied at 52 and 104 µg mL^?1 on Las‐infected ‘Valencia’ sweet orange plants [Citrus sinensis L. (Osbeck)] reduced acquisition (12 and 21% respectively) and transmission (11 and 18% respectively) of Las by feeding ACP adults compared with the controls; however, these reductions were not statistically significant. CONCLUSIONS: Pymetrozine exhibited moderate antifeedant effects by modifying the feeding behavior of ACP adults with short residual activity. The impact of pymetrozine on survival of nymphs was greater than on adults at the higher concentrations tested. Pymetrozine also reduced the acquisition and transmission of Las by feeding ACP adults up to 21 and 18%, respectively, compared with untreated controls. Copyright © 2010 Society of Chemical Industry     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

Frequency and occurrence of the carrot pathogen ‘Candidatus Liberibacter solanacearum’ haplotype C in Finland

Occurrence of ‘Candidatus Liberibacter solanacearum’ (CLso) was studied in field‐grown carrots (Daucus carota) in different regions of Finland. In addition, the frequency of CLso in carrots and in field populations of its vector, the carrot psyllid (Trioza apicalis), was studied in southwestern Finland. CLso was detected in six of the seven regions where the main carrot cultivation areas are located. The highest disease incidence was found in southwestern Finland, in the area where this carrot pathogen was originally found. In the Tavastia Proper and Southwest Finland regions, CLso was detected in 26 out of 30 randomly chosen fields inspected in 2013 and 2014, and in a third of those fields more than 10% of plants showed symptoms. Of those carrots showing both psyllid feeding‐associated leaf curling and CLso infection‐associated leaf discolouration symptoms, 77% were CLso positive in the PCR test. Some symptomless carrots from the affected fields also tested positive. Of the carrot psyllid individuals collected from the same area, 60% were CLso positive. Elsewhere, disease incidence was variable in South Ostrobothnia in western Finland and low but established in South Savonia in eastern Finland. CLso was not detected in the North Ostrobothnia region. Sequencing of the amplified DNA fragments confirmed that the bacteria in the carrot samples from different areas within Finland all represented CLso haplotype C. The frequent occurrence and wide distribution of this pathogen, transmitted by a psyllid that does not migrate over long distances, suggest that it is persistent in Finland.     

应用多重PCR技术同步检测柑橘4种重要嫁接传播病原

 柑橘衰退病毒(Citrus tristeza virus,CTV),柑橘碎叶病毒(Citrus tatter\|leaf virus,CTLV),柑橘裂皮病类病毒(Citrus exocortis viroid,CEVd)和柑橘黄龙病(Huanglongbing, HLB)亚洲种病原(Candidatus liberobacter asiaticus)是重要的柑橘嫁接传播病原。本文建立了同时检测HLB病菌、CTV、CEVd 和CTLV 4种柑橘嫁接病原的一步法、双温多重PCR检测技术体系,同时在体系中设置内参基因。应用该体系快速评价了4种嫁接传播病原在田间侵染情况,结果表明28个田间样品CTV、CEVd、CTLV和HLB感染率分别为89.3 %、17.9 %、10.7 %和28.6 %,接近半数样品为混合感染。并且将该方法应用于快速评价茎尖嫁接苗病毒的脱除情况。     

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print-based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR-based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE-qPCR) was compared to DNA extraction of plant tissue followed by PCR (X-PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t-test; P = 0.009). All extracts prepared for TPE-qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE-qPCR and immunoassay. Although the detection rate by TPE-qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE-qPCR. Thus, a higher detection rate would be achieved by combining TPE-qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.     

Quantification of viable <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management. The authors P. Trivedi and U. S. Sagaram contributed equally to this work.