HUANGLONGBING - A RESEARCH AND EXTENSION UPDATE

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1 HUANGLONGBING - A RESEARCH AND EXTENSION UPDATE An overview Dr. Megan Dewdney Assistant Professor and Extension Specialist Citrus Research and Education Center, Lake Alfred

2 HLB Management Psyllid (vector) control Scouting for HLB infected trees Removal of HLB infected trees (inoculum source)

3 Scouting Groves should be scouted 4 or more times/year More often if HLB has been found nearby Most difficult during spring flush and early summer A combination of methods is generally bests Elevated platforms Walking ATV or small vehicle Careful marking of row and tree is important Name and date EDIS document with details coming soon!

4 Three Species of Bacteria Cause HLB Ca. Liberibacter asiaticus only known species in Florida Vectored by 2 psyllid species Diaphorina citri and Trioza erytreae (experimentally) Heat tolerant Found on the Indian Subcontinent, Southeast Asia, Arabian peninsula, Brazil, Louisiana and Florida

5 Ca. Liberibacter africanus Has not been found in Florida Vectored by 2 psyllid species Trioza erytreae and Diaphorina citri (experimentally) Bacterium and vector heat sensitive Found in many African countries, Madagascar and Yemen at higher altitudes

6 Ca. Liberibacter americanus Newest species of bacteria that cause HLB Not found in Florida Vectored by Diaphorina citri Disease is not as severe as when caused by Asian species Heat tolerant Only found in Brazil currently Is not found in citrus as often as in the past

7 New Disease of Potato Zebra Chip Caused by a newly identified Ca. Liberibacter Identified in New Zealand Vectored by the potato/tomato psyllid Bactericera cockerelli Also damages tomatoes and peppers From South Texas to Colorado as well as Mexico, Guatemala

8 Approaches to Address HLB What factors can be altered to attempt to manage the disease? Host The insect The pathogen The tree Insect The interaction between the tree and the pathogen The interaction between the plant and the insect The interaction between the pathogen and the insect Pathogen Some approaches have to wait until more is known

9 Huanglongbing Assays are Difficult Uneven distribution of pathogen High concentrations of bacteria in some areas, low in others Uneven concentrations over time Cycles from high to undetectable with time Plants difficult to inoculate Many buds on infected trees have no bacteria Symptoms take from 3 to 9 months Need to repeat experiments many times months for a single experiment

10 HLB Populations Vary Among Tissues Ca. Liberibacter is found in most tissues Greenhouse plants Number of bacteria drastically different Population determined with real-time PCR Data taken at a single time point

11 Ca.. Liberibacter often is unevenly distributed in infected trees Madam Vinus sweet orange Cleopatra mandarin Ct = 22 Ct = 22 Ct = 38 Ct = 33 Ct = undet Ct = 21 Population estimates from real- time PCR Ct = 29 Ct = undet Population with low Ct

12 Distribution of Bacteria with EM Bacteria in low numbers in citrus Phloem necrosis found in many samples Both elongate and round forms found also reported by Garnier and Bové Necrosis Ca.. Liberibacter cells Elongate Round

13 Periwinckle tool to study Ca. Liberibacter asiaticus Bacteria transmitted to periwinkle via dodder Periwinkle symptoms of yellowing, wilting and death Bacteria more prevalent in periwinkle than in citrus Sweet orange infected with Dodder HLB symptoms in periwinkle

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15 HOST RANGE EXPERIMENTS Questionable whether resistance occurs in Citrus species and relatives Poncirus may be resistant, but not hybrids Lots of tolerance in greenhouse, but plants may eventually die

16 Citrus varieties and relatives that have been inoculated 1 Citrus macrophylla (Alemow) 17 Sour orange 2 Valencia Sweet Orange 18 Meiwa kumquat 3 Ruby Red Grapefruit 19 Calamondin 4 Nules clementine (mandarin) 20 Citrus hystrix 5 Minneola Tangelo 21 Swingle citrumelo 6 Eureka lemon 22 Sun Chu Sha (mandarin) 7 Volkamer Lemon 23 Palestine Sweet lime 8 Persian lime 24 Murraya paniculata 9 Mexican lime 25 Citrus latipes 10 Citron 11 Carrizo 12 Poncirus trifoliata 13 Citrus halimii 14 Citrus indica 15 Citrus micanthra var. microcarpa 16 Citrus ambyllocarpa 26 Cleopatra mandarin 27 Madam Vinous Sweet Orange 28 Duncan grapefruit 29 Ling Ping Yau pummelo 30 Hirado Buntan Pink pummelo 31 Siamese Sweet pummelo 32 Severinia buxifolia

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18 Research Results No citrus cultivar or relative found to be resistant to HLB Some cultivars more tolerant to HLB Symptoms less severe Cleopatra mandarin Citrus macrophylla Persian lime Healthy Infected Healthy Infected Healthy Infected

19 Disease Symptoms Are Correlated Abnormal Sugar Distribution Phloem appears to be blocked phloem necrosis callous deposits Large amounts of starch accumulation starch granules accumulate and disrupt chloroplasts starch accumulation is diagnostic for greening

20 Iodine staining of starch Cut leaf edges after 1 min of incubation in iodine solution Ed Etxeberria Asymptomatic greening Symptomatic greening Field control Field yellowing leaf

21 Anatomical Changes are Observed Infected Healthy Starch Phloem collapse Healthy phloem

22 Healthy Infected

23 In the Vascular System Also Infected Healthy Callose deposits Phloem Healthy phloem

24 Phloem Up Close Infected Healthy Blocked sieve plate and pores Blocked sieve plate Companion cell and pores Sieve plate and pores

25 Light Intensity and Duration Affects HLB Symptoms Symptoms more severe with 24 hrs light than in the greenhouse. 24 hour light Greenhouse 24 hour light Healthy Infected Infected Healthy Infected Healthy

26 Trees Resistant to Ca.. Liberibacter (important to have complete immunity) Find naturally resistant plant (needs to be edible/marketable) Transgenic antibacterial genes/peptides approval and acceptance issues Transgenic with natural resistance genes Transgenic with inducible genes for increased resistance downside time to use transgenics in the field (~15 years)

27 Problem!! No resistance to HLB exists in commercial citrus germplasm Even if tolerant citrus species are found, tolerance cannot be moved into commercial scions by conventional breeding

28 Related Questions Inhibit bacterial function in plants What are bacterial functions? Which are critical? Sequence of the bacterium Bacterial microarray How does the bacterium cause disease? What genes are important? What does the bacterium need from the plant? Why does the bacterium cause disease in some plants and not others? Inhibit bacterial movement?

29 Genetic Engineering Approaches Agrobacterium mediated transformation Protoplasts with GFP with regeneration via somatic embryogenesis Antimicrobial genes Target expression to phloem tissue Hope to minimize foreign gene products in fruit

30 Antimicrobial Gene Constructs 2 promoters: constitutive (d35s) or phloem specific Constructs in testing: AttacinE - Lytic peptide gene from Hyalophora cecropia CEAD - Codon optimized cecropin A-cecropin D lytic peptide gene CEMA - Codon optimized cecropin A-melittin lytic peptide gene CEME - Codon optimized cecropin A-melittin lytic peptide gene LIMA - Lytic peptide gene - works against Xylella in grape! PTA - Codon optimized N terminally modified Temporin A gene

31 GUS Expression in the Phloem To ensure that promoters work in Citrus GUS is a marker gene that is placed in a construct to show that a promoter is working where anticipated A B Phloem specific GUS expression by the Arabidopsis (A) and Rice (B) sucrose synthase promoters

32 Greening and Canker Education Activities Education and Outreach Displays eg. Citrus EXPO, Indian River Seminar, Florida Ag Expo (> 6,000 attendees collectively) > 50 trainings conducted and meetings assisted > 2300 attendees > 19,000 educational items distributed CDs, DVDs, laminated ID sheets, ID pocket guides > 7,000 EDIS brochures, magazine articles etc.

33 Greening and Canker Education Activities cont. Master Gardener canker and greening training notebooks created and distributed to every county extension office Educational brochures developed to specifically target homeowners and other non-industry clientele

34 Web Sites

35 Interactive HLB Training Internet based History Psyllids Symptoms Confounding symptoms Quizzes Supervisor provided with results To be β-tested by growers

36 Citrus Research and Education Center Dr. Ron Brlansky Dr. William Dawson Dr. Ed Etxeberria Dr. Jude Grosser Dr. Nian Wang Ms. Jamie Yates