Managing aboveground pests: Arthropod vectors of citrus pathogens Part II February 17, 2021 Lecture Overview
• Bacteria and bacteria-like microorganisms • Mollicutes: Spiroplasmas and phytoplasmas • Proteobacteria • Vector-borne diseases of citrus • Case study: Citrus greening disease Types of vector-borne plant pathogens • Viruses • Bacteria • Mollicutes: Spiroplasmas and phytoplasmas • Proteobacteria Spiroplasmas and phytoplasmas
Class: Mollicutes • Bacteria lacking cell walls • Often obligate parasites of plants and insect vectors • Dual host transmission cycles: Acquired by insect vectors that feed on infected plants. • Persistently transmitted (usually propagative) • Latent period in vector is approximately 15–20 days Spiroplasmas
• Restricted to the phloem sieve tubes and transmitted by phloem sap-feeding insects • Citrus Stubborn Disease (Spiroplasma citri): leafhoppers Citrus stubborn disease
Causal agent: Spiroplasma citri Citrus Stubborn Disease
• Stubborn disease does not kill trees, but stunts growth and inhibits fruit production • In young trees: the entire tree may remain small and unproductive • In mature trees: a single branch may show symptoms, and the disease may or may not spread slowly throughout the tree • low yield of small, lopsided fruit • Stunted, feathery canopy growth • Leaves are small and grow upright close to the stems • Trees usually develop unseasonal growth flushes and blossoms Citrus Stubborn Disease: small, lopsided fruit Rosettes of cup-shaped leaves and the presence of off season flowers and fruit at all stages of maturation Spiroplasma citri (Citrus Stubborn Disease) Citrus Stubborn Disease: Vectors
Neoaliturus haematoceps
Circulifer tenellus Citrus Stubborn Disease: Vectors
• Naturally transmitted by leafhoppers: • Circulifer tenellus, Scaphytopius nitridus and S. acutus delongi in California (Oldfield, 1988) • Neoaliturus haematoceps and C. tenellus in the Mediterranean area (Bové, 1986, Klein et al., 1988) • Leafhoppers do not have a host preference for citrus and may acquire S. citri from other hosts • Insects become infective after a latent period of 10-20 days (Liu et al. 1893) • Persistant, propagative transmission • Infective throughout lifetime • No transovarial transmission Phytoplasmas
• Phytoplasmas originally named mycoplasma-like organisms or MLOs after their discovery in 1967 • Phloem-limited • Cannot be cultured in vitro in cell-free media • Spread by insects in the families Cicadellidae (leafhoppers), Fulgoroidea (planthoppers) and Psyllidae (jumping plant lice) • Once established, phytoplasmas will be found in most major organs of an infected insect host • Witches broom: leafhopper Witches broom disease of lime (WBDL)
Causal agent: Candidatus Phytoplasma aurantifolia has been observed in the phloem of infected plants • A non-citrus host reservoir for the causal agent is suspected • Kills lime trees in less than 5 years WBDL: Symptoms
• Witches' brooms of thin proliferating twigs that have small, pale leaves • Additional witches' brooms appear as the disease progresses and extensive die-back occurs • Trees die within 3-5 years • Symptoms may appear within 6 months in graft- inoculated plants and warm conditions favor symptom expression. Witches' Broom Disease of Lime (WBDL)
Severe die-back of lime caused by witches' broom disease indicated by arrows Witches' Broom Disease of Lime (WBDL)
Witches' brooms of lime with small and pale leaves indicated by arrows. WBDL: Vector
• Putatively vectored by the leafhopper, Hishimonus phycitis • Reproduces on lime trees • Hishimonus phycitis is common on citrus in Oman, UAE, and Iran is suspected, but has not been confirmed experimentally. • Phytoplasma occurs in leafhoppers collected from infected trees Witches broom disease of lime (WBDL)
• Not present in the U.S. • Field observations suggest that extensive natural spread of WBDL has occurred in Oman and UAE • Presumably, WBDL poses greater risks in arid areas such as California with a climate similar to that in Oman and UAE • Unlikely to pose a severe economic threat to Florida because our major scion cultivars are not susceptible Types of vector-borne plant pathogens • Viruses • Bacteria • Mollicutes: Spiroplasmas and phytoplasmas • Proteobacteria Case study: Leafhoppers, vectors of Xylella fastidiosa X. Fastidiosa host plants
• Broad host range includes over 30 families of monocotyledonous and dicotyledonous plants. • “Xylella fastidiosa, a bacterium in the class Gammaproteobacteria, is an important plant pathogen that causes phony peach disease in the southern United States, Bacterial Leaf Scorch, oleander leaf scorch, Pierce's disease, and citrus variegated chlorosis disease (CVC) in Brazil.” • 153 host species are known to harbor the bacterium • Infects most citrus cultivars • All cultivars in Florida are susceptible Xylella fastidiosa
• A gram negative bacterium • citrus variegated chlorosis • Pierce’s disease in grape • glassy-winged sharpshooter CVC: Causal agent
• Xylella fastidiosa the most important bacterial pathogen transmitted by Auchenorrhyncha • The xylem vessels are ultimately blocked by bacterial aggregates and by tyloses and gums formed by the plant. X. Fastidiosa host plants
• Broad host range includes over 30 families of monocotyledonous and dicotyledonous plants • 153 host species are known to harbor the bacterium • Infects most citrus cultivars • All cultivars in Florida are susceptible Citrus variegated chlorosis (CVC) • First described in 1987 • Found in Brazil, Argentina, Paraguay and recently identified in Costa Rica (not outside of South America) • Thrives in warm climates • If introduced, CVC has the potential to become a significant problem for Florida citrus production. Citrus variegated chlorosis (CVC) • In young trees, CVC causes severe leaf chlorosis between veins resembling nutritional deficiencies • CVC does not kill trees, but trees become less productive within a few years following infection • Fruits not suitable for processing or fresh market • No cure!
Pierce’s disease in grape
• Southern California • Blue green and glassy winged sharpshooter X. fastidiosa in Florida grapes
• Pierce’s disease has been the limiting factor in bunch grape production in Florida. • Cultivars of Vitis vinifera will not survive in Florida. • Many muscadine varieties have partial to near complete resistance • X. fastidiosa resistance important in the selection of both grapes and rootstocks. • Muscadine grapes do not require rootstocks. Why isn’t CVC in Florida?
• CVC strain not in Florida • Climatic conditions ideal for spread • Presence of alternate hosts • Variety of bacterial strains, and only selected strains induce diseases in specific hosts CVC: Vectors
• CVC can be graft-transmitted or vectored by sharpshooter leafhoppers (Family: Cicadellidae) • At least 11 species of sharpshooters vector CVC in Brazil • Despite the fact that sharpshooters can feed on various plants, the role of non-citrus hosts as a pathogen reservoir remains unknown CVC vectors
• Two Florida sharpshooter species transmit CVC under experimental conditions: • Sharpshooter leafhopper, Oncometopia nigricans Walker, native to Florida • Glassy-winged sharpshooter Homalodisca coagulata Say native to Florida, introduced in CA around 1990, vectors Pierce’s disease of grape Glassy-winged sharpshooter common name: glassy-winged sharpshooter scientific name: Homalodisca vitripennis (=coagulata) (Germar) (Insecta: Hemiptera: Cicadellidae: Cicadellinae)
http://entnemdept.ufl.edu/creatures/fruit/glassywinged_sharpshooter.htm CVC Vectors
Oncometopia nigricans Populations of Sharpshooters in Two Citrus Groves in East-central Florida as Indicated by Yellow Sticky Card Traps
David G. Hall and Wayne B. Hunter. 2008. Florida Entomologist 91(3):488-490. CVC: Transmission
• Foregut-borne (non-circulative) and propagative (multiply) inside the vector’s foregut • No latent period • Sharpshooter adults are infective for life • Bacterial cells acquired by sharpshooters from infected plants adhere to the foregut cuticle, particularly in the anterior portion of the esophagus, cibarium (suction pump), and precibarium • Nymphs lose infectivity after molting, suggesting that transmissible bacterial cells are limited to the vector’s foregut which is shed during molting Retention of X. fastidiosa in sharpshooters
(A) Dorsal view of the cibarium chamber of Acrogonia citrine (B) magnified view showing a bacterial biofilm (Bf) (C) bacterial cells forming a microcolony (Mc) on the precibarium
Alves et al. (2008) Retention of X. fastidiosa in sharpshooters
(D) bacterial aggregates on the precibarium wall and on the cibarium’s ventral surface, near the food meatus Challenges for CVC management
• If established, spread of the CVC pathogen would be rapid and extensive • In South America spread over thousands of miles occurred in 10 years • Long latent period (1 yr) between infection and symptom appearance Challenges for CVC management
• Asymptomatic infections in many host plant species makes identification of host species difficult without molecular diagnosis • Inoculum removal, chemical control • Eradication of alternate hosts • Cultural control CVC Cultural control
• Maintenance of weed-free tree rows with an herbicide program (feeding/breeding sites) • Avoidance of summer pruning (new leaf growth attractive to vectors) • Avoid planting near woods (overwintering/alternative feeding sites) Challenges for CVC management
• Asymptomatic infections in many host plant species makes identification of host species difficult without molecular diagnosis • Long latent period (1 yr) between infection and symptom appearance • Inoculum removal, chemical control • Eradication of alternate hosts • Cultural control • Planting resistant cultivars- this worked in grapes… Case Study: Psyllids and citrus greening
http://news.softpedia.com/news/How-Can-a-Parasite- Influence-the-Evolution-of-a-Host-Species-When-it- Does-Not-Even-Infect-it-37797.shtml Huanglongbing (HLB)
• Also called Citrus greening disease or Yellow Shoot/Dragon Disease • Pathogen: Candidatus Liberibacter asiacticus (Las) • Fastidious (uncultured) • Phloem-limited alpha-proteobacteria • Discovered in Florida August 2005, Brazil 2004 • Currently throughout all of Florida’s citrus production
Photo: Huanglongbing: A Destructive, Newly-Emerging, Century-Old Disease of Citrus, J.M. Bové Huanglongbing (HLB)
DPI (2008) http://www.doacs.state.fl.us/pi/chrp/greening/citrusgreening.html HLB: Symptoms • Yellow shoots • Leaf symptoms: blotchy mottle, yellow veins, vein corking • Blotchy mottle: a random pattern of yellowing (chlorosis) on leaves that is not the same on the right and left sides of the leaf • Fruit symptoms: misshaped, bitter-tasting, small green • Decline and eventual death of trees HLB: Leaf Symptoms Fruit Symptoms • Fruit symptoms: misshaped, bitter-tasting, small green HLB: Fruit Symptoms HLB: Fruit Symptoms HLB: Fruit Symptoms
HLB: Symptoms
Twig dieback
Yellow shoots HLB: Other Symptoms • Fruit drop • Decline and eventual death of trees Transmission: Inoculation
• ACP transferred to healthy citrus for 1-15 d inoculation access periods (IAPs)
ACP reared Las- on Las+ (1 ACP/plant) citrus
IAP (days) % Positive psyllids (N) Infected plants (%)
1 45.7 (35) 6.3 a 4 72.4 (58) 7.1 a 7 81.3 (48) 7.7 a 15 69.2 (39) 3.7 a Do psyllids retain the pathogen throughout their lifetime? • ACP reared from eggs on Las+ citrus • Held for 1-25 d, then tested for Las with real-time PCR • N= 10-40 ACP per day
ACP reared Las- on Las+ (1 ACP/plant) citrus 1.2 1 R² = 0.7229 0.8 Retention of Las by ACP 0.6 decreases over time 0.4 0.2 0
Proportion Infected ACP Infected Proportion 0 10 20 30 Days on healthy plant Pathogen-vector interaction: Transovarial Transmission
Developmental % Infected
stage n offspring
Eggs 49 2.0
Nymphs 48 6.3
Adults 42 2.4
• Low rate of vertical transmission (parent-offspring) Many hosts Huanglongbing (HLB)
DPI (2008) http://www.doacs.state.fl.us/pi/chrp/greening/citrusgreening.html Huanglongbing (HLB)
• Pathogen: Candidatus Liberibacter asiacticus (Las) • Fastidious (uncultured) • Phloem-limited alpha-proteobacteria • What is Koch’s postulates? • https://www.medicinenet.com/kochs_ postulates/definition.htm • Discovered in Florida August 2005, Brazil 2004 • Currently throughout all of Florida’s citrus production http://news.softpedia.com/news/How-Can-a-Parasite- Influence-the-Evolution-of-a-Host-Species-When-it- Does-Not-Even-Infect-it-37797.shtml
Photo: Huanglongbing: A Destructive, Newly-Emerging, Century-Old Disease of Citrus, J.M. Bové Diaphorina citri
• Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) (Ca. L. asiaticus and Ca. L. americanus) • Discovered in Florida June 1998 • Trioza erytrea (Ca. L. africanus) Diaphorina citri
• Sternorrhyncha: Liviidae • Found in most citrus growing regions globally • Primarily feed on Rutaceous plants • Vector of Candidatus Liberibacter asiaticus (CLas)
Hall et al.,2008 Diaphorina citri
• Persistent, propagative transmission • Insects become infective after a latent period of 5-7 days following acquisition of Las • Infective throughout lifetime • Acquisition most effective at nymphal stage First report Persistent transmission
Asian citrus psyllid Midgut
Salivary glands
Candidatus Liberibacter Cuticle asiaticus Epidermal cells
Parenchyma cells
Spongy mesophyll Citrus Citrus plant Xylem cells
Phloem
Bundle sheath cells Persistent transmission
• Bacteria replicate in the psyllid • There is evidence
Asian citrus psyllid that transovarial Midgut
Salivary glands transmission of Las
Candidatus Liberibacter Cuticle asiaticus Epidermal cells occurs in D. citri
Parenchyma cells
Spongy mesophyll Citrus Citrus plant Xylem cells
Phloem
Bundle sheath cells Review: Persistent transmission
Barriers to transmission: 1. midgut infection barrier 2. dissemination Asian citrus psyllid (including midgut Midgut
Salivary escape and glands salivary gland Candidatus Liberibacter Cuticle asiaticus Epidermal cells infection) barriers Parenchyma cells 3. salivary gland Spongy mesophyll Citrus Citrus plant Xylem cells escape barrier
Phloem Bundle sheath cells 4. transovarial transmission barriers Does Las infection alter psyllid fitness?
• Infection with pathogens may have important effects on the metabolism and fitness of vector species • Longer evolutionary relationships may translate to lower pathogenicity of pathogen to the vector (e.g. Purcell 1982) • Well-adapted pathogen-host associations may benefit the vector (pathway to mutualism?) (Beanland et al. 2000) Transmission: Acquisition
70 p < 0.0001 c 60 50 bc SEM) 40 infected ACP ACP infected - b 30 ab ab ab (mean ± (mean 20 a Eggs or 50 Las- citrus adult ACP on 10
For 1 week Percent LAS Las+ citrus 0 1-6 7-13 14-20 21-27 28-34 >35 nymph
Acquisition access period (days)
• Acquisition of Las by ACP greatest when reared on infected plant Vector fitness: survival decreases
100 p < 0.0001
80 SEM) ±
60
HLB- 40 HLB+
20 Percent adult survival (mean (mean adult survival Percent 0 0 5 10 15 20 25 30 35 Day Vector fitness: More egg production
Cumulative egg production: 250
± p < 0.0001 200 150 Egg production: SEM 100 HLB-Las - Eggs from single female counted 50 HLB+Las + every 5 days for 25 days 0 Cumulative No. Eggs Cumulative 5 10 15 20 25 Eggs per female per 5 days: Days 50 b p < 0.0001 40 SEM)
± 30 a 20 10 No. Eggs ( Eggs No. 0 LasHLB- - HLB+Las + Psyllid condition Vector fitness
• Las alters fitness of adult psyllids: increased egg laying, faster development time, shorter lifespan • Vertical transmission of Las and increased fecundity of infected psyllids suggest that Las is well-adapted to psyllids • Possible fitness trade-off between reproduction and longevity when infected with Las • Greater egg production in Las-infected D. citri may increase disease spread Global distribution of the Asian Citrus Psyllid (Source: EPPO global database, www.eppo.int) Complicating factors
• Uneven distribution of pathogen in trees • Asymptomatic trees Complicating factors
• Reinfestation • Abandoned and backyard trees. Disease management
• Chemical control with pesticides • Biological control with parasitoids • Problems with insecticide resistance in the psyllid populations to most of the commonly used insecticides. • Need for repeated applications with the rapid growth of new shoots and the negative impact on populations of natural enemies • Long-term: Develop psyllid populations incapable of transmitting Las Disease management
• Pathogen-free nursery stock • Supplemental nutrition Disease management
• Cultural control • Quarantines, eliminate ornamental hosts Impact Common sight in FL ca. 2008
Common sight in 2016
Richard Perry/The New York Times Economic Impact of HLB
Florida citrus lost an estimated • 6600+ Jobs lost $7.80 billion between 2006-2014 • 30% loss in orange production Control of vector-borne diseases
• Prevention : - Used certified disease-free bud/graftwood - Quarantine - Prevent mechanical spread - Mild strain protection (e.g. CTV) • Vector control? Difficult to get 100% • Control of alternate hosts Questions?