How effective is hydrochloric acid (HCl) treatment to control Clavibacter michiganensis subsp. michiganensis contamination in tomato Seed?

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1 How effective is hydrochloric acid (HCl) treatment to control Clavibacter michiganensis subsp. michiganensis contamination in tomato Seed? Prakash Pradhanang and George Collier HeinzSeed Company

2 Acid Treatment Seed is submerged in hydrochloric acid (HCl) solution (1%) for 3 min. Removed from acid solution and rinsed in clean water. Tumble dried under ambient temperature or at 35 o C for 1 hr.

3 Effect of Acid Treatment (UC, Davis) 4 reps, 15 plants/rep Year Canker incidence (%) in Highly (1 4 CFU/g) contaminated seed lot Acid treated Untreated Canker incidence (%) in Lowly (1 2 CFU/g) contaminated seed lot Acid treated Untreated dead 96. affected. dead 4. affected 24.3 (2 plants)

4 Top Pruning Greenhouse at UC, Davis, 23

5 Small Plot Trial: Untreated seed, 23

6 18, top pruned plants were transplanted in 2.5 Ac land to verify the effect of acid treatment

7 Large Grow-out Trial: Monitoring for Canker (1,5 of 18, plants examined)

8 Results: Grow-Out Trial Heavily Contaminated Seeds Treated With Hydrochloric Acid 23 Small Plot Experiment 24 Small Plot Experiment Transplants pruned Transplants not pruned Complete canker control Complete canker control Large Plot Experiment Large Plot Experiment Transplants pruned Transplants not pruned 1 out 18, plants tested positive 1 plant with primary symptoms

9 Key Results No canker epidemic from acidtreated seeds No economic yield loss

10 Why few among thousands of plants developed primary or secondary type canker symptoms both years? Could there be a barrier to acid activity on some seed? Can Cmm survive acid treated seeds? Was there a natural infection in the field? What could those barriers be? Gelatinous coat of pulp on some seed surfaces Seed that float on acid solution or stuck on walls of a container and escape treatment Pathogen inside seed coat

11 Can Cmm survive 1% HCl solution? Method 1μl of 4 x 1 8 to 4 x 1 2 Cmm CFU/ml added to 9μl of 1% HCl solution Undiluted suspension was plated on D2ANX medium Results No viable Cmm recovered from any treatment Conclusion Cmm cannot survive in 1% HCl solution Cmm CFU/ml Viable colonies 4 x x x x x x x 1 1

12 Can Cmm Survive Acid Seed Treatment? Methods: 1g each of untreated and acid-treated seeds were inoculated with Cmm density ranging from 3.1 x 1 8 to 3.1 x 1 4 CFU/ml Cmm Log CFU per g seed Untreated Cmm Log CFU per g seed Acid treated. Month.5 Month 3 Months 6 Months 12 Months. Month.5 Month 3 Months 6 Months 12 Months Cm m survival ( Months after inoculation) Cmm survival (Months after inoculation) 7.7x17 7.7x16 7.7x15 7.7x14 7.7x x17 7.7x16 7.7x15 7.7x14 7.7x13 Results: From inoculated untreated raw seeds, viable Cmm were recovered even from the lowest density tested Viable Cmm populations dropped sharply on acid treated seeds, declining from 3.1 x 1 8 CFU/ml to 6 CFU/ml after six months. No viable Cmm recovered from rest of the treatments. Conclusions: Result is convincing that Cmm can not survive for long on seed dipped in 1% HCl solution

13 Barrier to HCl activity: Dried up pulp on some seed surface?!! Seed extraction treatment 1. Overnight fermentation: (No pulp on seed surface) 2. Forced extraction: washed 2 hrs. after pulping: (Pulp on seed surface) 3. Enzyme extraction: washed 2 hrs. after enzyme treatment: (No pulp on seed surface) 4. Acid extraction (No pulp on seed surface)

14 Barrier to HCl activity Seed extraction treatment 1. Overnight fermentation Raw seed Cmm CFU/g seed. 7.6 x 1 3 After acid treatment Cmm CFU/g seed ELISA Forced extraction: washed 2 hrs. after pulping 8.5 x x Enzyme extraction: washed 2 hrs. after enzyme treatment 6.5 x Acid extraction 2.4 x 1 4 +

15 Monitoring of Cmm in Contaminated Raw Seeds Seed extraction treatment Raw seed: Cmm CFU/g seed (months after extraction) 1. Overnight fermentation 2. Forced extraction: washed 2 hrs. after pulping Immediate. 7.6 x x x months Not tested Test Discontinued Not tested 2.8 x Enzyme extraction: washed 2 hrs. after enzyme treatment 6.5 x 1 5 Test Discontinued 4. Acid extraction 2.4 x x 1 4

16 Why did a few among thousands of plants develop primary or secondary type canker symptoms both years? Forced extracted seed had dried up gel around them. They yielded relatively large Cmm populations (6.4 x 1 6 ) but subsequent HCl treatment eliminated Cmm on or in seed. So this cannot be the reason. Acid-treated seed eliminated Cmm seed contamination to a non-detectable level. Seed transmission on acid-treated seed will require a large grow-out test with carefully planned controls to check field as natural source of contamination Improper seed treatment can not be ruled out Checking internal presence of Cmm in acid-treated seed would be like trying to find a needle in a haystack

17 Conclusions 1% HCL solution can eliminate Cmm Seed treatment with 1% HCL solution eliminated Cmm from artificially inoculated or naturally infested tomato seeds Gelatinous coat of pulp was not a barrier to acid activity Cmm was not detected from acid-treated ground seeds Cmm survived on acid-extracted seeds but eliminated when dry seeds were treated again with 1% HCl Seed washing after overnight natural fermentation eliminated or reduced contamination Enzyme-extracted seed carried a large population of Cmm

18 Advantages of Acid Seed Treatment Inexpensive technology No adverse effect on seed germination Broad spectrum effect on pathogens