Protect your trees in the ground: What s new on the antimicrobial front?

Transcription

1 June 12-14, 2013 Ninth Annual Florida Citrus Industry Annual Conference Protect your trees in the ground: What s new on the antimicrobial front? Hyatt Regency Coconut Point, Bonita Springs June 12-14, 2013 A Researcher s Perspective

2 Combined Projects on Discovery of Most Effective Antimicrobials and best Application Strategies USDA UNIVERSITY OF FLORIDA AND CRDF Robert Shatters David Hall Ed Stover Yong-Ping Duan Gary Luzio Chuck Powell Mu-Qing Zhang Tom Turpen Harold Browning Calvin Arnold Research Supported by the Florida Specialty Crops Research Initiative and Florida Research and Development Foundation

3 TREATMENT ID ANTIMICROBIAL HISTORY New Products Antibiotics DELIVERY HISTORY Use of Penetrants Classical Injection FIELD DELIVERABLE SOLUTION

4 COMPONENTS OF A SUCCESSFUL ANTIMICROBIAL TREATMENT Citrus Canker Successful Treatment Citrus Greening (HLB) Extended Exposure

5 ID ANTIMICROBIAL HISTORY Antibiotics TREATMENT New Products FIELD DELIVERABLE SOLUTION

6 1. Injections of tetracycline and penicillin improve overall plant health and fruit quality Studies since the early 1970 s show this. 2. Antibiotic residues are short lived (~15 days or less). 3. Only injections were used 4. Improvement lasts for 1 year (often with two injections) 5. Only method that was successful was injection: 1. Costly 2. Problems with phytotoxicity using this method.

7 6-year old trees 3 injections 2 months apart

8 Finding the Antimicrobial CLASSICAL ANTIBIOTICS NEW COMPOUNDS 1. Biocides: 2. Peptides: 3. Fungicide: 4. Systemic Acquired Resistance (SAR Copyright 2009 substances 8

9 Crowd Sourcing approach: 1. Internet-based incentive-based strategy 2. International posting of alternative compounds to test 3. Expert review panel reviews entries selected 80 to test 4. Testing conducted in Dr. Chuck Powell s lab through collaboration with Drs M. Q. Zhang and Y. P. Duan.

10 Using the above two optimized screening system, more than 80 molecules were evaluated against HLB bacterium 1. Antibiotics: Biocides: Peptides:5 4. Fungicide: 6 5. SAR substances: 4 6. Others: 10

11 HLB-Symptomatic citrus HLB-Symptomatic shoots Shoots treated with Candidate Antimicrobial overnight Graft-based protocol For Testing Candidate Antimicrobials: Monitor Liberibacter in treated scion Monitor ability of antimicrobial to effect migration of Liberibacter into seedling Grafted onto the Las-free citrus seedlings Treated Scions

12 Noneffective, 39 Highly effective, 9 Effective, 23 Partly effective, 11 Tested compounds were divided into 4 groups based on the Q- PCR Ct value in the inoculated plants Highly effective: Ct 36.0; Effective: 36.0>Ct 32.0; Partly effective: 32.0>Ct 28.0; Noneffective: Ct<28.0.

13 Eleven Effective Compounds Are Currently Being Advanced and Include: 1) Antimicrobial Peptides 2) Natural antimicrobial compounds 3) Agricultural antibiotics 4) Sulfonamide compounds

14 Infected rates (%) Ct value Scion-infection (%) Las transmission (%) A B C CK Class 1 compounds Pre-Tr Post-Tr A B C CK Class 1 compounds

15 TREATMENT DELIVERY HISTORY Use of Penetrants Classical Injection FIELD DELIVERABLE SOLUTION

16 TREATMENT DELIVERY Basal Bark Foliar Root Application Assume application strategy will have to be optimized to the antimicrobial being used

17 Developing New Application Strategies for HLB Control: Antimicrobials and How to Apply Them Research Supported by the Florida Specialty Crops Research Initiative Ashley Voss OUTSTANDING TECHNICAL SUPPORT Research Supported by the Florida Specialty Crops Research Initiative and Florida Research and Development Foundation Dr. Ric Stange Dr. Rocco Allesandro Belkis Diego and Carol Malone Dr. John Ramos

18 Basal Bark Application Concept CANOPY TRUNK Trunk Applications: Allow Minimum Surface Area (reduced chemical cost) Potential for reduced environmental exposure ROOT MASS

19 FIRST QUESTION: Can We Get Antimicrobials to Move Systemically?

20 MEASURING ANTIMICROBIAL MOVEMENT IN CITRUS (We have done this on up to 3-year old trees) Monitoring Systemic Movement Leaf Sampling Trunk Application About 2 g of tissue is required for 100 µl of clarified leaf extract.

21 Systemic Movement of Penicillin- G Bark Applications Penicillin moves systemically without any penetrants WITH PENETRANT AID WITHOUT PENETRANT

22 Systemic Movement of Difference Tetracycline Derivatives-Bark Applications Tetracycline requires combination with penetrant WITHOUT PENETRANTS Tet-d1 Tet-d2 WITH PENETRANT AID Tet-d1 Tet-d2

23 Log2 of avg. radius (mm) Duration of Penicillin in Plant Days after treatment A avg B avg C avg

24 Comparison of Time-Release Matrices Used with Basal Bark Applications

25 PCR Ct Value for Clas Detection SECOND QUESTION: Can Basal Bark Application Treatment Reduce Clas Abundance in Infected Citrus? (Monthly Applications over ~ 1 year of symptomatic potted trees ~ 1 meter) * * * * * ½ of trees had no detectable CLas Water A5P1 15 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Month of Sampling

26 120 healthy trees in ten blocks with 12 treatments in each block. Trees are grafted sweet orange from certified nursery. Treated Monthly and Quantitate CLas Monthly

27 July '10 Aug '10 Sept '10 Oct '10 Nov '10 Dec '10 Jan '11 Feb '11 Mar '11 Apr '11 May '11 Jun '11 July '11 Aug '11 Sept '11 Oct '11 Nov '11 Avg. CT July '10 Aug '10 Sept '10 Oct '10 Nov '10 Dec '10 Jan '11 Feb '11 Mar '11 Apr '11 May '11 Jun '11 July '11 Aug '11 Sept '11 Oct '11 Nov '11 Dec' 11 Avg. CT T1 - WATER Avg. CT low Temp. Linear (Avg. CT) Bark Treatment Avg. CT low Temp.

28 Thermal treatments eliminate or suppress the bacterial pathogen in Huanglongbing-affected citrus Melissa S. Doud 1, Michele T. Hoffman 1, Mu-Qing Zhang 2, Ed Stover 1, David Hall 1, Shouan Zhang 3, and Yong Ping Duan 1 1 USDA-ARS-USHRL, Fort Pierce, FL, 2 IFAS-IRREC, University of Florida, Fort Pierce, FL, 3 IFAS-TREC, University of Florida, Homestead, FL

29 Doud et al., 2012 In Dr. Duan s Lab Before Heat Treatment After Heat Treatment Thermal exposure at C for >/= 48 h reduced titer or eliminated Las bacteria entirely in potted HLBaffected citrus seedlings.

30 Ct value Pre-tr Old Post-Tr New Post-Tr Old EPL SDX STZ CK Thermo- and chemo-therapy Notes: Combinations of chemical compounds (EPL, SDX, STZ) and heat treatments (42 o C and 45 o C) Old is the old leaf, New is the new flush leaf. The qpcr test was done in two months after treatment.

31 The Next Two Years Verify results in production citrus systems Optimize combination of antimicrobial and application method. Ideally, several alternatives will be optimized that can move through approval process. Identify best method for extending exposure (time release) Determine if combination of antimicrobial and thermal therapy is advantageous. Determine if combination of chemical (antimicrobial and /or nutritional?) and heat