Different Sweet Orange–Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Scion

The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.

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Different Sweet Orange‒Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Roots

HortScience ◽

10.21273/hortsci16206-21 ◽

2022 ◽

Vol 57 (1) ◽

pp. 56-64

Author(s):

Shahrzad Bodaghi ◽

Gabriel Pugina ◽

Bo Meyering ◽

Kim D. Bowman ◽

Ute Albrecht

Keyword(s):

Root Growth ◽

Imaging System ◽

Sweet Orange ◽

Field Performance ◽

Field Trials ◽

Relative Distribution ◽

Production Environment ◽

Citrus Diseases ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Grafting a scion onto a rootstock results in physical and physiological changes in plant growth and development, which can affect tree vigor, productivity, and tolerance to stress and disease. Huanglongbing (HLB) is one of the most destructive citrus diseases and has become endemic in Florida since its introduction in 2005. It is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), which cause severe metabolic disruptions in affected plants. Although most scion cultivars are highly susceptible, some rootstock cultivars are tolerant and allow the grafted tree to cope better with the disease. The objectives of this study were to identify rootstock traits that can be used to assess cultivars under controlled greenhouse conditions in advance of longer-term field trials. We used 10 commercially important rootstocks with different genetic backgrounds and known field performance in graft combination with ‘Valencia’ sweet orange scion. Trees were graft-inoculated with CLas and compared against mock-inoculated trees. Tree health and CLas populations were assessed regularly, and root growth was monitored using a minirhizotron imaging system. Plants were excavated and destructively sampled 21 months after inoculation to assess biomass distributions and other CLas-induced effects. We found significant differences between healthy and infected trees for most variables measured, regardless of the rootstock. In contrast to leaf CLas titers, root titers were significantly influenced by the rootstock, and highest levels were measured for ‘Ridge’ sweet orange and sour orange. Root growth and root biomasses were reduced upon infection but differences among rootstocks did not always agree with reported field performances. Despite severe biomass reductions plants maintained their relative distribution of biomass among different components of the root system, and no dead roots were observed. Root respiration was reduced by CLas infection and was overall higher in tolerant cultivars suggesting its potential as a physiological marker. This study improves our knowledge about the strengths and weaknesses of assessing rootstock traits of grafted trees in a controlled greenhouse setting. Results from the study suggest that in addition to HLB tolerance, other rootstock traits will ultimately have major contributions to field survival and productivity of the grafted trees in an HLB endemic production environment.

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Identification and Characterization of the Huanglongbing Bacterium in Pummelo from Multiple Locations in Guangdong, P. R. China

Plant Disease ◽

10.1094/pdis-92-4-0513 ◽

2008 ◽

Vol 92 (4) ◽

pp. 513-518 ◽

Cited By ~ 23

Author(s):

X. Deng ◽

J. Chen ◽

Z. Feng ◽

Z. Shan ◽

H. Guo ◽

...

Keyword(s):

Sweet Orange ◽

Nucleotide Polymorphisms ◽

Sequence Comparisons ◽

Research Attention ◽

Republic Of China ◽

Citrus Greening Disease ◽

Citrus Production ◽

History Of ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Huanglongbing (HLB, yellow shoot disease, ex. citrus greening disease), caused by Candidatus Liberibacter spp., is highly destructive to citrus production in Asia, Africa, and South America. Although primarily affecting sweet orange and mandarin, HLB has long been observed in pummelo in Guangdong Province, People's Republic of China; however, the disease in pummelo has received little research attention. Accordingly, it remains unclear how closely related the strains of Ca. Liberibacter in pummelo are to those in other citrus species. In this study, the loci of 16S rDNA, rplAJ (β-operon of ribosomal protein), and an outer membrane protein (omp) gene were analyzed and characterized among strains of Ca. Liberibacter in pummelo samples from six different locations in Guangdong. Sequence comparisons indicated that ‘Candidatus Liberibacter asiaticus’, but not ‘Ca. Liberibacter africanus’ or ‘Ca. Liberibacter americanus’, was exclusively associated with HLB symptoms in pummelo. The pummelo strains of ‘Ca. Liberibacter asiaticus’ from Guangdong were highly homogeneous. Analyses of single-nucleotide polymorphisms in the omp locus showed that the Guangdong pummelo strains grouped with ‘Ca. Liberibacter asiaticus’ strains from Thailand, Nepal, and an unspecified location in China but differed from the Philippine and China-Behai strains. Based on the sequence homogeneity at the omp locus, the history of pummelo culture and the means by which HLB is known to be spread, we believe that, likely, the pummelo strain of ‘Ca. Liberibacter asiaticus’ recently was spread to pummelo in the study areas from infected sweet orange or mandarin trees by insect vectors or by propagation of pummelo infected elsewhere.

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Genome Sequence Resource of ‘Candidatus Liberibacter asiaticus’ strain 9PA From Brazil

Plant Disease ◽

10.1094/pdis-05-20-1018-a ◽

2021 ◽

Vol 105 (1) ◽

pp. 199-201

Author(s):

P. A. Silva ◽

J. Huang ◽

N. A. Wulff ◽

Z. Zheng ◽

R. Krugner ◽

...

Keyword(s):

Genome Sequence ◽

Sweet Orange ◽

Whole Genome Sequence ◽

Whole Genome ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

And Control

‘Candidatus Liberibacter asiaticus’, an unculturable α-proteobacterium, is associated with citrus huanglongbing (HLB), a devastating disease threatening citrus production in Brazil and worldwide. In this study, a draft whole-genome sequence of ‘Ca. L. asiaticus’ strain 9PA from a sweet orange (cultivar Pera) tree collected in São Paulo State, Brazil, is reported. The 9PA genome is 1,231,881 bp, including two prophages, with G+C content of 36.7%. This is the first report of a whole-genome sequence of ‘Ca. L. asiaticus’ from Brazil or South America. The 9PA genome sequence will enrich ‘Ca. L. asiaticus’ genome resources and facilitate HLB research and control in Brazil and the world.

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First Report of Dodder Transmission of Huanglongbing from Naturally Infected Murraya paniculata to Citrus

Plant Disease ◽

10.1094/pdis-91-2-0227b ◽

2007 ◽

Vol 91 (2) ◽

pp. 227-227 ◽

Cited By ~ 30

Author(s):

L. J. Zhou ◽

D. W. Gabriel ◽

Y. P. Duan ◽

S. E. Halbert ◽

W. N. Dixon

Keyword(s):

Nested Pcr ◽

Sweet Orange ◽

The United States ◽

Retail Trade ◽

Murraya Paniculata ◽

Pcr Products ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Infection Source ◽

Liberibacter Asiaticus

Huanglongbing (HLB) or “greening” disease of citrus is caused by phloem-limited, uncultured bacteria in the genus “Candidatus Liberibacter”. HLB is one of the most destructive diseases of citrus worldwide and is considered so dangerous to a U.S. citrus production that the USDA has listed “Ca. Liberibacter species” as a Select Agent. HLB is spread by the Asian citrus psyllid, Diaphorina citri, which was intercepted 40 times by APHIS/PPQ at U.S. ports between 1985 and 1998, became established in Florida by 1998, and more recently in Texas (1). HLB was first detected in the United States near Miami, FL during August 2005, and to date has been confirmed to have spread to 12 Florida counties. In addition to citrus, Murraya paniculata (orange jasmine) is a preferred host of D. citri, and retail trade in this ornamental shrub is strongly implicated in the distribution of D. citri (1). M. paniculata is reported to be a cryptic or largely asymptomatic host of “Ca. Liberibacter” (4), but another report concludes that the bacteria cannot replicate in M. paniculata (2). The epidemiological significance of murraya as a host for the HLB pathogen is therefore unclear. We report here the transmission of “Ca. Liberibacter asiaticus” from M. paniculata to citrus. Two M. paniculata plants, suspected of harboring “Ca. Liberibacter” because of their proximity to HLB-infected citrus and infested with D. citri, were removed from the field, treated with insecticide, and transferred to a quarantine facility. Both plants tested positive for “Ca. Liberibacter” by nested PCR using primers OI1 and OI2 (3) as the first set and primers CGO3F (RGG GAA AGA TTT TAT TGG AG) and CGO5R (GAA AAT AYC ATC TCT GAT ATC GT) as the second set. Two, young, sweet orange plants (Citrus sinensis) grown and maintained in psyllid-free greenhouses in Gainesville, FL were infected by dodder (Cuscuta pentagona) grown from seed. After the dodder had become well established on the orange plants, the orange plants were moved adjacent to the two murraya plants and the dodder from the citrus was draped over the murraya. Coinfection of murraya by dodder occurred within a few days. Sixty days later, both murraya plants, both sweet orange plants, and the connecting dodder all repeatedly tested positive for “Ca. Liberibacter” by nested PCR. Beginning 2 weeks later, the orange plants tested positive by standard PCR using primer set OI1 and OI2 or CGO3F and CGO5R, but remained without typical greening symptoms. Sequencing of the PCR products confirmed amplification of “Ca. L. asiaticus” DNA. We conclude that M. paniculata can serve as an infection source of a Select Agent since it can host the HLB pathogen for at least 2 months and the HLB pathogen can be transmitted to sweet orange during this time. References: (1) S. E. Halbert and K. L. Manjunath. Florida Entomol. 87:330, 2004. (2) T. H. Hung et al. J. Phytopathol. 148:321, 2000. (3) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996. (4) T. Li and C. Ke. Acta Phytophylacica Sin. 29:31, 2002.

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Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽

10.1094/pdis-08-15-0940-re ◽

2016 ◽

Vol 100 (6) ◽

pp. 1080-1086 ◽

Cited By ~ 10

Author(s):

Greg McCollum ◽

Mark Hilf ◽

Mike Irey ◽

Weiqi Luo ◽

Tim Gottwald

Keyword(s):

The United States ◽

Insect Vector ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Germplasm ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Florida Citrus ◽

Citrus Macrophylla ◽

Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

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Historical Perspectives, Management, and Current Research of Citrus HLB in Guangdong Province of China, Where the Disease has been Endemic for Over a Hundred Years

Phytopathology ◽

10.1094/phyto-07-18-0255-ia ◽

2018 ◽

Vol 108 (11) ◽

pp. 1224-1236 ◽

Cited By ~ 16

Author(s):

Zheng Zheng ◽

Jianchi Chen ◽

Xiaoling Deng

Keyword(s):

Southern China ◽

Candidatus Liberibacter Asiaticus ◽

Sequencing Technology ◽

Next Generation Sequencing Technology ◽

Historical Perspectives ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Generation Sequencing

Citrus huanglongbing (HLB) is a highly destructive disease currently threatening citrus production worldwide. In China, the disease is exclusively associated with ‘Candidatus Liberibacter asiaticus’, a nonculturable proteobacterium. HLB was observed in Guangdong of China over a hundred years ago. Researchers and citrus growers have been battling with the disease through vigorous research and have exercised various control practices. Much of the early work was not well known outside China. This review is intended to fill in gaps of historical information by reviewing selected literature records. Along the way, the HLB system within southern China was evaluated. Emphases were on comparison of symptomatology, evolution of etiology, control practices, and impacts of using next-generation sequencing technology for ‘Ca. L. asiaticus’ research and detection.

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A Review of Huanglongbing (Citrus Greening) Management in Citrus Using Nutritional Approaches in China

Plant Health Progress ◽

10.1094/php-2010-1003-01-rv ◽

2011 ◽

Vol 12 (1) ◽

pp. 24 ◽

Cited By ~ 7

Author(s):

Yulu Xia ◽

Gecheng Ouyang ◽

Ronald A. Sequeira ◽

Yu Takeuchi ◽

Ignacio Baez ◽

...

Keyword(s):

Nutrient Management ◽

Field Survey ◽

Cultural Practices ◽

Sweet Orange ◽

Asian Citrus Psyllid ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

Candidatus Liberibacter ◽

Citrus Maxima ◽

Liberibacter Asiaticus

The Asian form of huanglongbing (HLB) is caused by ‘Candidatus Liberibacter asiaticus (Las),’ a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama. Nutrient management, together with other cultural practices such as pruning and irrigation, for mitigation of the disease has been practiced in China for many years. Our literature review, field survey, and interviews with Chinese scientists and growers indicate that these cultural practices were generally ineffective for the disease management. However, a nutritional approach in conjunction with other cultural practices such as irrigation can maintain grove productivity for a certain time depending on the type of citrus species/cultivars, the age of the trees, the propagation method of the plants, the Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) population, and other factors. Symptomatic mature pommelo (Citrus maxima Merr) and sweet orange (C. sinensis L. Osbeck) plants can commonly survive and maintain a certain level of productivity for an additional 4 to 5 years, even longer assuming vigorous ACP control. Accepted for publication 27 June 2011. Published 3 October 2011.

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Screen Mesh Size for Exclusion of Diaphorina citri (Hemiptera: Liviidae) in Citrus Production

Journal of Economic Entomology ◽

10.1093/jee/toaa095 ◽

2020 ◽

Vol 113 (4) ◽

pp. 2026-2030

Author(s):

Timothy A Ebert ◽

Laura Waldo ◽

Daniel Stanton ◽

Arnold W Schumann

Keyword(s):

Tree Mortality ◽

Mesh Size ◽

Diaphorina Citri ◽

Short Side ◽

Vector Management ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Pass Through ◽

Individual Trees

Abstract Huanglongbing is a citrus disease that reduces yield, crop quality, and eventually causes tree mortality. The putative causal agent, Candidatus Liberibacter asiaticus (Rhizobiales: Rhizobiaceae), is vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama. Disease management is largely through vector control, but the insect is developing pesticide resistance. A nonchemical approach to vector management is to grow citrus under screen cages either as bags over individual trees or enclosures spanning many acres. The enclosing screen reduces wind, alters temperature relative to ambient, and excludes a variety of pests that are too large to pass through the screen. Here we evaluated the potential of six screens to exclude D. citri. We conclude that screens with rectangular openings need to limit the short side to no more than 384.3 µm with a SD of 36.9 µm (40 mesh) to prevent psyllids from passing through the screen. The long side can be at least 833 µm, but the efficacy of screens exceeding this value should be tested before using in the field.

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Reaction of sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' infection

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2013001100004 ◽

2013 ◽

Vol 48 (11) ◽

pp. 1440-1448 ◽

Cited By ~ 6

Author(s):

Rafaella Teles Arantes Felipe ◽

Francisco de Assis Alves Mourão Filho ◽

Silvio Aparecido Lopes ◽

Beatriz Madalena Januzzi Mendes ◽

Maurel Behling ◽

...

Keyword(s):

Sweet Orange ◽

Pcr Analysis ◽

Candidatus Liberibacter Asiaticus ◽

Randomized Design ◽

Transgenic Lines ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Polymerase Chain ◽

Attacin A ◽

Leaf Symptoms

The objective of this work was to evaluate the reaction of four sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' (Las) infection, a bacterium associated to huanglongbing (HLB) disease. Transgenic sweet orange plants of Hamlin, Natal, Pêra, and Valência cultivars, as well as nontransgenic controls received inocula by grafting budwood sections of HLB-infected branches. Disease progression was evaluated through observations of leaf symptoms and by polymerase chain reaction (PCR) analysis, eight months after inoculation. A completely randomized design was used, with four experiments (one for each cultivar) performed simultaneously. Bacteria title was estimated by quantitative PCR (qPCR). HLB symptoms and Las titers were present in nontransgenic and transgenic plants expressing the attacin A gene of the four sweet orange cultivars, eight months after bacteria inoculation. Five transgenic lines (transformation events) of 'Pêra' sweet orange expressing the attacin A gene have significantly lower Las titers in comparison with nontransgenic plants of this cultivar.

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Functional and Comparative Genomic Analysis of Integrated Prophage-Like Sequences in “Candidatus Liberibacter asiaticus”

mSphere ◽

10.1128/msphere.00409-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 2

Author(s):

Marian Dominguez-Mirazo ◽

Rong Jin ◽

Joshua S. Weitz

Keyword(s):

Causative Agent ◽

De Novo ◽

Evolutionary Relationship ◽

Genomic Analysis ◽

Sequence Type ◽

Comparative Genomic ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Prediction Approach

ABSTRACT Huanglongbing disease (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogen “Candidatus Liberibacter asiaticus” is an alphaproteobacterium of the Rhizobiaceae family that has been identified as the causative agent of HLB. The virulence of “Ca. Liberibacter asiaticus” has been attributed, in part, to prophage-carried genes. Prophage and prophage-like elements have been identified in 12 of the 15 available “Ca. Liberibacter asiaticus” genomes and are classified into three prophage types. Here, we reexamined all 15 “Ca. Liberibacter asiaticus” genomes using a de novo prediction approach and expanded the number of prophage-like elements from 16 to 33. Further, we found that all of the “Ca. Liberibacter asiaticus” genomes contained at least one prophage-like sequence. Comparative analysis revealed a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 “Ca. Liberibacter asiaticus” strain that had previously been reported as lacking prophages. Our findings provide both a resource for data and new insights into the evolutionary relationship between phage and “Ca. Liberibacter asiaticus” pathogenicity. IMPORTANCE Huanglongbing (HLB) disease is threatening citrus production worldwide. The causative agent is “Candidatus Liberibacter asiaticus.” Prior work using mapping-based approaches identified prophage-like sequences in some “Ca. Liberibacter asiaticus” genomes but not all. Here, we utilized a de novo approach that expands the number of prophage-like elements found in “Ca. Liberibacter asiaticus” from 16 to 33 and identified at least one prophage-like sequence in all “Ca. Liberibacter asiaticus” strains. Furthermore, we identified a prophage-like sequence type that is a remnant of an integrated prophage—expanding the number of prophage types in “Ca. Liberibacter asiaticus” from 3 to 4. Overall, the findings will help researchers investigate the role of prophage in the ecology, evolution, and pathogenicity of “Ca. Liberibacter asiaticus.”

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