Citrus Huanglongbing Nian Wang University of Florida November, 2017
Outline Worldwide distribution of Liberibacters and associated diseases Epidemiology Pathogens Virulence mechanisms Management
Citrus Huanglongbing (HLB) Typical symptoms: reduced plant height, pale yellowing of leaves, blotchy mottle and/or variegated chlorosis of leaves. Infected leaves can become upright, followed by leaf drop at the laminar abscission zone or petiole abscission and twig dieback at later stages; yellowing of new shoots; rotting of rootlets; shortening of internodes; zincdeficiency; enlarged and corky veins; early blossom or offseason flower; fruit not color well and deformed, bitter fruit with aborted seeds.
Citrus huanglongbing pathogen Candidatus Liberibacter asiaticus, americanus, africanus a phloem-restricted, non cultured, Gramnegative bacterium vectored by citrus psyllids Diaphorina citri
Key events of citrus greening in Florida and USA and its worldwide distribution 1998 Diaphorina citri 2005 2008 Brazil: 2004 FL: 2005 LA: 2008 TX: 2012 CA: 2012 Las in South Africa (Ethiopia 2010) Trioza erytreae in Spain 2014
Citrus HLB in Chaoshan, Guangdong, China in 1930 S. Lin was the first to demonstrate by graft inoculation the infectious nature of the disease. For this reason, the International Organization of Citrus Virologists proposed in 1995 at the 13th IOCV that the official name of the disease be huanglongbing. Why does citrus virologists name a bacterial disease? Lin Kung Hsiang
Historical events Reinking (1919) reported yellow shoot disease in southern China, a disease which he thought to be of little importance in those days. However, later surveys showed that by 1936 the disease had spread to become a serious problem. Lin 1956 showed that HLB is a graft-transmissible, infectious disease, and should neither be attributed to physiological disorders such as mineral deficiencies or water logging, nor to soil-borne diseases such as nematode infestation or Fusarium infection. In 1928, South Africa, citrus greening In 1921, Mottle leaf in the Philippines. thought to be related to zinc deficiency (Lee, 1921). The major contribution of the Philippines to HLB was the demonstration, in 1967, that mottle leaf could be transmitted by the Asian citrus psylla, D. citri (Martinez and Wallace, 1967). In the same year, it was also reported in India that HLB could be transmitted by D. citri from trees affected by citrus dieback. In India, citrus dieback. 18 th century Phloem necrosis and Vein phloem degeneration in Indonesia. HLB first appeared in Thailand in the 1960s
HLB pathogen 16S rdna analysis alpha proteobacteria closest relatives: alpha-2 subgroup. Alpha proteobacteria: Agrobacterium, Bradyrhizobium spp. etc Asia Candidatus Liberibacter asiaticus Brazil, FL, China Africa Candidatus Liberibacter africanus South Africa South America Candidatus Liberibacter americanus Brazil Kingdom: Phylum: Class: Order: Family: Genus: Bacteria Proteobacteria Alpha Proteobacteria Rhizobiales Rhizobiaceae Candidatus Liberibacter Jagoueix et al., 1997 Jose Bové and Monique Garnier
Evidences for Las as the causal agent for HLB Koch s postulates have not been fulfilled due to the difficulty in culturing the bacterium Guilty by association: detection of Las in HLB diseased trees using DNA based methods Tyler et al. 2009, metagenomic DNA sequences from the phloem tissue of HLB-diseased citrus trees show that only Las was identified from the phloem tissue. Treatment with antibiotics kills Las and relieves HLB symptoms
Diseases caused by Liberibacters, their hosts, and distributions Pathogen Diseases Plant hosts Insect vector Distribution Ca. L. asiaticus (Las) HLB Citrus and citrus relatives Asian citrus psyllid (Diaphorina citri) Widespread in most citrus areas of Asia, Africa, and Americas, Ca. L. africanus (Laf) HLB Citrus and citrus relatives African citrus psyllid (Trioza South Africa erytreae) Ca. L. africanus subsp. capensis (LafC) Cape chestnut African citrus psyllid (Trioza South Africa erytreae) Ca. L. africanus subsp. clausenae (LafCl) Horsewood (Clausena) African citrus psyllid (Trioza South Africa erytreae) Ca. L. africanus subsp. zanthoxyli (LafZ) Small forest knobwood African citrus psyllid (Trioza South Africa erytreae) Ca. L. africanus subsp. subsp. vepridis White ironwood (Vepris) African citrus psyllid (Trioza South Africa (LafV) erytreae) Ca. L. africanus subsp. tecleae (LafT) Flaky Cherry-orange African citrus psyllid (Trioza South Africa erytreae) Ca. L. americanus (Lam) HLB Citrus and citrus relatives Asian citrus psyllid (D. citri) Brazil Ca. L. solanacearum (Lso) Haplotype A ZC solanaceous crops Bactericera cockerelli Central America, western Mexico western US, and New Zealand Haplotype B ZC solanaceous crops B. cockerelli eastern Mexico, central US Haplotype C Carrot Trioza apicalis Finland, Sweden, France, Norway, Netherlands, and Germany Haplotype D Carrot B. trigonica Spain, Morocco Haplotype E celery and carrots B. trigonica (likely) Spain, France, Morocco Ca. L. europaeus (Leu) Rosaceae plants including Cacopsylla pyri (L.) Italy and Hungary apple (Malus domestica), blackthorn (Prunus spinosa) and hawthorn (Crataegus monogyna), pear. Scotch broom Broom psyllid (Arytainilla New Zealand, Australia (?), Europe (?) spartiophila) L. crescens (Lcr) mountain papaya (Carica Unknown Puerto Rico stipulata x C. pubescens) Ca. L. brunswickensis (CLbr) Eggplant Eggplant psyllid (Acizzia solanicola) Australian
Distribution of Candidatus Liberibacter species Agrobacterium tumefaciens Rhizobium leguminosarum SEMIA 2083 Rhizobium etli CFN 42 13 Lcc Lam Leu 27 Laf 100 Lso 6 23 3 Ca. L. asiaticus, Ca. L. africanus, Ca. L. americanus, Ca. L. solanacearum, Ca. L. europaeus, Liberibacter crescens Ca. L. brunswickensis Haapalainen et al. 2014 2 276 Las Wang et al 2017 ARP Laf was present on the African continent before the introduction of citrus, possibly in an indigenous Rutaceous species (da Graca 2008) such as Calodendrum capense, Clausena anisate, Vepris lanceolate, and Zanthoxylum capense. Lam might have evolved from Liberibacter resembling Leu introduced into Brazil from Europe via infected plants considering ACP has been first reported in Brazil in 1942 while Lam was first found in Brazil in 2004. Major concerns: Establishment of African citrus psyllid Trioza erytreae in mainland Europe (Massimino Cocuzza 2016) Incursion of Las into Africa.
HLB caused by Las, Lam, and Laf Laf: is heat-sensitive, and occurs only in cool areas, with temperatures remaining below 30-32 C. Similarly, the African psyllid vector, T. erytreae, thrives only in cool environments, and is also sensitive to high temperature combined with low relative humidity. In South Africa, HLB and the African psyllid vector, T.erytreae, occur in the cool areas. Las: HLB and the Asian psyllid vector, D. citri, are found in hot low altitude areas. Las is heat tolerant, and symptoms occur even when temperaturesare well above 30 C. The Asian psyllid vector, D. citri, has similar properties, and is also heat-tolerant. Lam: D. citri, was reported in Brazil as early as1942, whereas Lam was reported in 2004. heat-tolerant
Insect host Transmitted by graft inoculation as well as by the two citrus psyllids. Asia (Brazil, FL, China) Las Diaphorina citri Africa (South Africa) Laf Trioza erytreae America (Brazil) Lam and Las Diaphorina citri Adult Asian citrus psyllid, Diaphorina citri Kuwayama. Photograph by: Douglas L. Caldwell, University of Florida Asian citrus psyllid, Diaphorina citri Kuwayama, nymphal instars. Image:David Hall, USDA-ARS, Ft. Pierce, FL Trioza erytreae Adult(s) South Africa Research International S.P. van Vuuren, Citrus
Psyllid transmission of Las How does psyllid transmit Las? Can Las replicates in psyllid?
Alimentary canal Psyllid transmission of Liberibacter LSO+ LSO- Las Salivary gland salivary gland Sengoda et al. 2014 nucleus muscles fat tissue ovary haemolymph male accessory gland
Psyllid transmission of Liberibacter How does psyllid transmit Las? Can Las replicates in psyllid? The latent period: 1 to 8 days post-acquisition. Propagative Circulative
Host range of Ca. L. asiaticus What determine the host range of Las? Asian citrus psyllid Las Artificial host of Las Citrus spp. Family: Rutaceae Murraya paniculata (Orange jasmine) Citrus species Citrus relatives (e.g., Chinese box orange (Severinia buxifolia) ) Periwinkle Tomato Tobacco Solanum lycopersicum Family: Solanaceae Murraya paniculata Family: Rutaceae Periwinkle (Catharanthus Dodder (Cuscuta spp.) roseus) Family: Apocynaceae Family: Convolvulaceae Nicotiana Family: Solanaceae
Response of citrus varieties to HLB Trifoliate orange (P. trifoliata L. Raf.) and some of its hybrids reportedly lack distinct disease symptoms despite infection with the pathogen. US-897 is a hybrid of trifoliate orange and Cleopatra mandarin (C. reticulata Blanco), the latter being highly susceptible to HLB. (Kim D. Bowman USDA ARS) Sugar Belle (Fred Gmitter, UF) Folimonova et al. 2009
The origin and evolution of citrus species Sweet orange (C. maxima x C. reticulata) x C. maxima as egg donor and a male C. reticulata, with some introgression with C. maxima Clonal propagation and nucellar embryony (a form of apomixis) have resulted in low genetic diversity within cultivated citrus types. Three real species. Currently, after thousands of years of cirus cultivation and interbreeding, there are 25 species and around 250 commercial citrus varieties.
Resistance of wild citrus germplasm to HLB Ramadugu et al. 2016 Seeds were collected from 108 accessions of citrus and citrus relatives belonging to the subfamily Aurantioideae, family Rutaceae. Six- to nine-month-old seedlings in HLB endemic areas Observation after six years: two immune, six resistant, and 14 tolerant Pink wampee, immune Orange berry, immune Curry leaf, resistant White sapote, resistant Australian desert lime, resistant Murraya paniculata, resistant Naringi crenulata, resistant Japanese prickly ash, resistant
Resistance of wild citrus germplasm to ACP White sapote, resistant Simmons trifoliate Poncirus trifoliata (L.) Raf. RUTACEAE Westbrook et al. 2011. 87 Rutaceae see-source genotypes, freechoice situation for infestations of natural Florida populations of D. citri. White sapote avoids all life stages (egg, nymph, and adult) of D. citri. Two Poncirus trifoliata (L.) Raf, populations: Simmon s trifoliate and Little-leaf host very low levels of D. citri. However, Citroncirus (from intergenic hybrids of Poncirus with Citrus) such as S-281 citrangelo, Swingle citrumelo, X639, and Rusk did not inherit the resistance of pure P. trifoliata. Late, Richardson and Hall 2013 identified 36% of 81 accessions of P. trifoliata and xcitroncirus spp. host zero eggs. Citrus macrophylla (alemow) and B. koenigii (curry leaf) are among the most susceptibles. Citrus macrophylla Curry leaf, resistant
Reductive evolution of Liberibacters Las How does reductive evolution happen for Las? The genome size of Ca. Liberibacter spp. is much smaller. 1.23 Mb for Las 1.26 Mb for Lso 3.4 Mb for Agrobacterium sp. H13-3 5.7 Mb (Agrobacterium tumefaciens C58), 6.3 Mb (A. vitis S4), 6.5 Mb (Rhizobium etli CFN 42), 6.7 Mb (Sinorhizobium meliloti 1021), and 7.3 Mb (A. radiobacter K84); The reduced genome size of Ca. L. asiaticus and Ca. L. solanacearum are hypothesized to be the result of a stable and nutrient-rich environment, attenuated purifying selection due to small population size, and strong bottleneck effects (Wernegreen 2002; Moran et al. 2008; Moya et al. 2008). Other vascular residing bacteria: Xylella (~2.7 Mb), Phytoplasma (~860-kb), Spiroplasma (~1.8 Mb). Bacteria that have larger genomes tend to be versatile in their lifestyles and are presumably adapted to changing environments, whereas bacteria that have small genomes seems to prefer more stable habitats (Batut et al. 2014).
How Liberibacters genomes were sequenced? DNA extraction Sequencing method Las: from psyllid, whole genome amplification and 454 pyrosequencing (Duan et al. 2009) Lam: periwinkle, pulsed-field gel electrophoresis, 454 pyrosequencing (Wulff et al. 2014) Lso:potato psyllids (Bactericera cockerelli Sulc), 454 pyrosequencing (Lin et al. 2011)
Update on the culturing of Las and other Liberibacters Co-cultivation of Candidatus Liberibacter asiaticus with Actinobacteria from Citrus with Huanglongbing. Davis et al. 2008 Cultivation of 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' associated with huanglongbing. Sechler et al. 2009. A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined.
Can we culture Las in artificial medium? Challenges? Moisture, ph, temperature, osmotic pressure, atmosphere and nutrients are required for bacterial growth. Las has the ability to metabolize sugars such as glucose, fructose, and xylulose, but not mannose, galactose, rhamnose or cellulose. Las encodes for ATP/ADP translocase in addition to its ATP synthase, allowing it to both synthesize ATP as well as uptake ATP from its hosts. Las: aerobic. Las: producing serine, glycine, cysteine, aspartate, lysine, threonine, glutamate and arginine and incapable of making histidine, tyrosine, phenylalanine, tryptophan, asparagine, isoleucine, methionine, alanine, valine, leucine and proline.
What do we know about the virulence mechanism of Candidatus Liberibacter asiaticus Transmission into phloem directly by psyllids, reduces needs for virulence factors for infection and avoids recognition by plants using pattern recognition receptors, which are typically localized in the plant cell membrane. Reductive evolution in the host Ca. L. asiaticus (1.23 Mb). Most flagella genes present, but the structure is not observed. Las contains the rough LPS, which consists of the lipid A membrane anchor and core oligosaccharide based on genome analysis (Duan et al. 2009). The genes responsible for synthesis of O-antigen are missing. http://tamubugworld.wordpress.com
Ca. L. asiaticus has very few weapons Motility? LPS biosynthesis Pilus Sec T1SS Lipopolys accharide Reductive evolution in the host Ca. L. asiaticus (1.23 Mb). Las contains a Sec dependent pathway and many proteins with Sec-dependent signal peptide
HLB causes anatomical aberration, localized pockets of necrotic phloem scattered through the vascular system, massive accumulation of starch in the plastids, aberrations in cambial activity, and excessive phloem formation Wang et al. 2017 Annual Review of Phytopathology
Clues to understand HLB pathogenicity mechanism One of the hallmarks of diseases caused by Liberibacter is the relatively consistent symptomology that occurs among different symptom-expressing hosts. This pattern of symptomology is regularly observed in citrus when infected by other phloem-limited pathogens. For example, citrus stubborn disease caused by Spiroplasma citri and citrus disease caused by Ca. Phytoplasma produce symptoms similar to HLB.
Small RNAs in disease development Zhao et al. 2013 Host small RNAs (srna) play a vital role in regulating host responses to pathogen infection srnas were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new micrornas (mirnas), 76 conserved mirnas, and many small interfering RNAs (sirnas) were discovered. mir399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn a disease with symptoms similar to HLB. a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLBpositive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida
What do we know about the virulence mechanism of Candidatus Liberibacter asiaticus Phloem blockage, phloem necrosis and aberrations Reduced photoassimilate transportation Starch accumulation in infected aerial tissues, but depleted in roots Phloem loading Metabolic imbalances by nutrient depletion Hormones (ethylene/iaa) Root damage Prophages
Bacteriophage Two largely homologous, circular phage genomes, SC1 and SC2. SC2 is lysogenic. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with Ca. L. asiaticus were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.
Bacteriophage Prophages have been suggested to assist Las in suppressing plant defenses (Jain et al. 2015). For example, SC2-gp095 encodes a ROS-scavenging peroxidase, which is a predicted lysogenic conversion factor. SC2-gp095 expression is suppressed in psyllids, but it is expressed at a high level in periwinkle. Expression of the peroxidase in Lcr, but not in E. coli, results in enhanced resistance to H2O2. Nonclassical secretion has been predicted for SC2-gp095, and its secretion from Lcr has been confirmed by enzymatic and western blot analyses. Transient expression of the peroxidase in planta results in strong transcriptional downregulation of RbohB, the key gatekeeper of the H2O2-mediated defense signaling in plants. It has been suggested that Las evades early detection in the phloem by virtue of its secreted peroxidase activity, enabling it to build up a high titer in citrus over several years before manifesting disease symptoms, thus resulting in the long incubation period often associated with HLB progression. However, the lack of a prophage in many Las strains does not appear to relate to the lack of HLB symptoms because Ishi-1 and the Guangdong isolates, which do not contain any prophages, induce similar HLB symptoms as isolates containing prophages. Overall, this evidence suggests that prophages contribute to bacterial virulence but are not required for Las pathogenicity.
Control of citrus HLB Main strategies: Insecticide applications for vector control, removal of HLB diseased trees, Las free seedlings. Other strategies: heat treatment, antimicrobial treatment, horticultural approaches (inducing plant defenses), developing resistant or tolerant varieties.
Landscape Varieties Weather (hurricane) Horticultural practices (organic fertilizer etc) Labor cost Society and organization (tree removal, insect control) New planting area Monoculture and diversity Difference of HLB on citrus production in Florida, China, and Brazil and its implication in HLB management HLB is really an economic problem. Orange production
Las thrives at 22-25 C. Heat treatment Continuous thermal exposure to 40 to 42 C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate Las entirely in HLBaffected citrus seedlings (Hoffman et al. 2013). Tent, hours to days Steam treatment 50-55 C, minutes Limitations Fig. 1. Phenotypes of three citrus varieties before and after exposure to 40 C for 10 days. 1A, Citrus limon before and 1B, 270 days after treatment (DAT); 2A, C. reticulata before and 2B, 150 DAT; and 3A, C. paradisi before and 3B, 150 DAT.
Antimicrobials Effective antibiotics against Las: Ampicillin, Carbenicillin, Penicillin, Cefalexin, Rifampicin and Sulfadimethoxine. They are all highly effective in eliminating or suppressing Las. Noneffective: Amikacin, Cinoxacin, Gentamicin, Kasugamycin, Lincomycin, Neomycin, Polymixin B and Tobramycin, did not eliminate or suppress Las in the tested concentrations, resulting in plants with increased titers of Las. Huanglongbing (HLB)-affected grapefruit ( Duncan ) plants with graft-inoculation of Las-infected lemon scions treated with different antibiotics. (Zhang et al. 2014)
Antimicrobials Tetracycline antibiotics are protein synthesis inhibitors, inhibiting the binding of aminoacyl-trna to the mrnaribosome complex. Naturally produced by soil-dwelling bacterium named Streptomyces. More than 10 tons of OTC are applied annually in several states of the USA (McManus et al., 2002) to control Xanthomonas arboricola on peach and nectarine, Erwinia amylovora on pear, and in particular streptomycinresistant isolates of E. amylovora on apple (McManus et al., 2002;Christiano et al., 2010). Streptomycin is a bactericidal antibiotic. Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rrna of the 30S subunit of the bacterial ribosome, interfering with the binding of formylmethionyl-trna to the 30S subunit. Streptomyces griseus. Streptomycin Pros and cons
Application of HLB tolerant citrus rootstocks Valencia orange (Citrus sinensis [L.] Osbeck) was grown on 17 rootstocks through seven years of age and the first four harvest seasons in a central Florida field trial severely affected by huanglongbing (HLB) disease. All trees in the trial had HLB symptoms and were shown by PCR to be infected with Las. Large differences were noted between rootstocks for many metrics examined, including yield, fruit quality, and tree size. Highest yields in the trial were on US-942 rootstock, which was significantly more productive than trees on the common commercial rootstocks Carrizo, Kuharske, Cleopatra, and Kinkoji. Use of a tolerant rootstock is suggested as an effective means of ameliorating crop losses to HLB. (Bowman et al. 2016)
1. Under cover production system (pros and cons) 2. Protector for young plantings 3. Spray with plant defense inducers 4. Enhanced nutrient program 5. Beneficial microbes Other HLB control methods