Volatile Sulfur Compounds and Garlic Germination

Detection and Quantification of Bioactive Sulfur Compounds in Onion and Garlic for White Rot Control

Michael Qian Oregon State University, Corvallis, OR Feb 8, 2016 Background

• Can spread quickly and destroy the plant • Sclerotia can survive Sclerotium cepivorum more than 40 years in soil • Once infected, the field can no longer be used for garlic production

Sclerotia Current Challenges for White Rot Control

• Fumigation with methyl bromide – the most effective treatment – killing more than 95% of the sclerotia in the soil – phased out and uneconomical • Soil treatment with metam-sodium – results have been erratic – retreatment is necessary – risk to the environment – a general biocide. • Crop rotation – Ineffective – sclerotia are dormant in the absence of specific sulfides exuded from roots of Allium crops Sclerotia Germination

• The fungus will only colonize on Allium plants • sclerotia germinate only in response to exudation by Allium roots • These exudates contain alkyl and alkenyl-L-cysteine sulphoxides • Alkyl and alkenyl-L-cysteine sulphoxides are metabolized by the soil microflora to yield a range of volatile thiols and sulfides • Volatile thiols and sulfides activate the dormant sclerotia. Research Rationale

• Apply stimulants to the ground in the absence of an Allium crop • The sclerotia may be “tricked” into germinating. • In the absence of a host, the mycelium from germinating sclerotia will persist for a few days to several weeks depending on the soil temperature, then die after exhausting nutrient reserves. • Thus, the total population of sclerotia in a field is susceptible to control with this method (Crowe et al., 1980).

What do we know?

• Diallyl disulfide forced 90-99% of the sclerotia to germinate (Davis et al, 2009). – similar to methyl bromide fumigation – DADS is no longer commercially available • We know natural products will work (Davis et al, 2009)

(http://www.vgavic.org.au/pdf/r&d_VG00048_DADS_evaluation.pdf) Objectives

 Identify natural sulfur-organic compounds in onion and garlic  Develop analytical method(s) to quantitate the concentration of volatile sulfur compounds  Search for potential sources of biostimulates  High concentration of volatile sulfur compounds  Economical  develop a sustainable approach using garlic/onion oil or waste products as biostimulants to manage onion and garlic white rot (long term goal)

Headspace-GC-MS Method

Headspace (HS) Sample trays Volatile Sulfur Compounds Analysis

• Method development – Sample preparation • Fresh garlic/onion: dried with liquid nitrogen, blended into powder  2g of garlic/onion powder + 6 mL H2O + 2 mL methanol + 50 µl IS (518 ppm Ethyl Methyl Sulfide, 776 ppm Isopropyl Disulfide) + 2g salt – Instrument analysis • Headspace sampling: Samples placed into a 20 ml headspace vial; Equilibrated at 50ºC for 50 min; Syringe temp 50°C; Inject 500 µl, 1:5 split • GC-MS Parameters: HP-Wax Column, 30m length, 250 µm diameter, 0.25 µm film thickness; Oven program: 40 °C, 2 min

Calibration curve development

Dia llyl Dis ulfide

Response Ratio

0.4

0.2 Coef of Det (r˄2)= 0.995

0 5 10 Conce ntra tion Ra tio Calibration Curve Development

Average Actual Linear dynamic Compound ppm Stdev ppm %recovery range (mg/L) Equation R2 Allyl Methyl Sulfide 10.2 0.25 10.00 102% 1.25~40 Y=0.9435x 0.998 Dimethyl Disulfide 4.0 0.10 4.00 101% 0.5~16 Y=1.386x 0.998

Allyl Sulfide 4.0 0.14 4.00 100% 0.5~16 Y=0.5307x 0.997 Dimethyl Trisulfide 3.8 0.06 4.00 94% 0.5~16 Y=0.1485x 0.998 Diallyl Disulfide 11.3 0.72 10.00 113% 1.25~20 Y=0.1485x 0.996 Diallyl Y=0.003145x2 Trisulfide 2.88 0.27 4.00 72% 0.5~16 +0.01669x 0.992 Volatile sulfur compounds in Fresh Garlic 250000 Volatile compounds in fresh garlic (pH 7) *IS2 5 200000

150000

*IS1

Abundance 100000

4 50000 6 1 3 2 0 0 10 20 30 40 50 Retention time (min)

*IS1: Ethyl Methyl Sulfide 1: Allyl Methyl Sulfide 3: Allyl Sulfide 5: Diallyl Disulfide *IS2: Isopropyl Disulfide 2: Dimethyl Disulfide 4: Methyl Allyl Disulfide 6: Diallyl Trisulfide *IS represents for Internal standard, which is added manually for quantitative analysis purpose Fresh Garlic (3 samples)

0 Allyl Methyl Dimethyl Allyl Sulfide Dimethyl Diallyl Disulfide Diallyl Trisulfide Sulfide Disulfide Trisulfide CE CL FTR CE: California Early CL: California Late FTR: Forked Tree Ranch fresh onion

Concentration(ppm) Onion 4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0 Allyl Methyl Dimethyl Allyl Sulfide Dimethyl Diallyl Diallyl Sulfide Disulfide Trisulfide Disulfide Trisulfide Onion Onion Waste

Abundance 1 TIC: Garlic-HS-wax 2014-10-02-onion waste2.D\data.ms

190000

180000

170000

160000

150000

140000

130000

120000

110000

100000

90000

80000

70000

60000

50000

40000

30000 2 3 20000 5 4 10000

0 5.006.007.008.009.0010.0011.0012.0013.0014.0015.0016.0017.0018.0019.00 Time-->

1: Ethanol 2: Propanol 3: S-Methyl thioacetate 4: Isobutyl alcohol 5: Acetic acid Soil amendment

Concentration(ppm) soil amendment 0.30

0.25

0.20

0.15

0.10

0.05

0.00 Allyl Methyl Dimethyl Allyl Sulfide Dimethyl Diallyl Diallyl Sulfide Disulfide Trisulfide Disulfide Trisulfide soil amendment Garlic juice 1 (n=3)-2015

Concentration(ppm) GJ1(2+6+2) 30

0 Allyl Methyl Dimethyl Allyl Sulfide Dimethyl Diallyl Disulfide Diallyl Sulfide Disulfide Trisulfide Trisulfide GJ1(2+6+2)

GJ1: LOT # 4555-315 ( the Garlic Company, CA) Garlic juice 2 (n=3)-2015

Concentration(ppm)

GJ2(2+6+2) 25

0 Allyl Methyl Dimethyl Allyl Sulfide Dimethyl Diallyl Diallyl Sulfide Disulfide Trisulfide Disulfide Trisulfide GJ2(2+6+2)

GJ 2: Garlic Juice Crop App, Formula #2, LOT # 4466-315 ( the Garlic Company, CA) Garlic Juice Samples-2016

Concentration of sulfur compounds (mg/ kg)

Compounds GC 1522903 K 032651

Allyl Methyl Disulfide 5.50 ± 0.26 0.57 ± 0.03

Dimethyl Disulfide 0.50 ± 0 0.15 ± 0

Allyl Disulfide 4.13 ± 0.23 0.35 ± 0

Dimethyl Trisulfide 0.32 ± 0.03 0.05 ± 0

Diallyl Disulfide 3.22 ± 0.20 0.12 ± 0.03

Diallyl Trisulfide 0.00 ± 0 0.00 ± 0 Commercial DADS sample

(%) Allyl sulfide (45.1%) 50 451,000ppm

40 DADS (24.8%) 30 247,000ppm

20 DATS (9.2%) 91,500 ppm 10

0 Allyl Dimethyl Allyl Dimethyl Diallyl Diallyl Methyl Disulfide Sulfide Trisulfide Disulfide Trisulfide Sulfide How to make more DADS?

S CH2 H2C S Formation of VOC in Garlic Formation of VOC in fresh garlic

Sample preparation

• Garlic cloves were dried with liquid nitrogen and blended into a powder. + 2.0 g of frozen garlic powder + 6.0 mL of 0.01 M phosphate buffer (pH 2,4,6,7, or 8) • Samples were incubated: -Room temperature, 35°C and 45°C degrees. -GC/MS analysis at 5 hours, 1 day, 3, 7, 14 days. • Preparation for analysis: + small stir bar + 2.0 mL methanol (MeOH) + 50 µL internal standard (538 ppm ethyl methyl sulfide, 760 ppm isopropyl disulfide. + 2.0 g salt

Effect of pH on the formation of DADS in garlic paste

400 Control RT-1day Conclusion:

300 35C-1day (1) DADS formation is 45C-1day more favorable at higher pH ( i.e. pH 7 mg/kg ) ( and pH 8) 200 (2) acidic condition will inhibit the alliinase activity, (3) DADS is also not 100 stable in acidic concentration conditions.

0 pH2 pH4 pH6 pH7 pH8 pH effect on DADS concentration Effect of incubation time on the formation of DADS at pH 7

RT 300 35C 45C Conclusion: (1) After 1 day incubation, the 200 DADS concentration will increase about 2.5 time compared to no incubation 100 treatment at pH 7 (2) Mild heating (35C)

concentration (mg/kg) will improve enzyme activity compared to room 0 temperature Control 1day 3day 7day 14day Time effect on DADS concentration Other compounds- Allyl Methyl Sulfide RT 80

60 Concentration (mg/kg) 40 highest concentration is 20 pH8 labeled in red pH7 0 pH6 14day pH4 7day 3day pH2 1day 45C Control 35C 400 160 300 140

120 200 100

80 100 60 pH8 40 pH7 0 pH6 20 pH8 14day pH4 pH7 7day 0 pH6 3day pH2 1day 14day pH4 7day control 3day pH2 1day control Other compounds- Dimethyl Disulfide

RT 4

1 pH8 pH7 0 pH6 14day pH4 7day 40 45C 3day pH2 1day Control 35C 30 20

10 10 pH8 pH7 0 pH6 14day pH4 7day pH8 3day pH2 pH7 1day 0 control pH6 14day pH4 7day 3day pH2 1day control Other compounds- Allyl Sulfide RT 100

20 pH8 pH7 0 45C pH6 14day pH4 7day 400 3day pH2 1day 350 35C Control 200 300 250

200

150

100 100 50 pH8 pH7 0 pH6 14day pH4 7day pH8 3day pH2 pH7 1day 0 pH6 control 14day pH4 7day 3day pH2 1day control Other compounds- Dimethyl Trisulfide RT 8

2 pH8 pH7 0 pH6 14day pH4 7day 3day pH2 1day 40 Control 45C 35C 30

20 20

10 pH8 pH7 0 pH8 pH6 pH7 14day pH4 0 7day pH6 3day pH2 1day 14day pH4 7day control 3day pH2 1day control Other compounds- Diallyl Trisulfide RT 50

40 30 20 10 pH8 pH7 0 pH6 14day pH4 7day 45C 3day pH2 120 1day Control 100 35C 80 60 80 40 60 20 pH8 40 pH7 0 pH6 20 14day pH4 pH8 7day pH7 3day pH2 0 1day pH6 control 14day pH4 7day 3day pH2 1day control Limits of the Sky

• Dynamic conversions of various forms of volatile sulfur compounds • Can achieve 250 ppm of DADS, 10-100x of the garlic juice products • All forms of volatile sulfur compounds were about 1000 ppm • 1000x less than the commercial DADS Homemade garlic juice (revised protocol)

Sample preparation

• Garlic cloves were dried with liquid nitrogen, blended into powder. -50 g of frozen garlic powder -100g of 0.05 M phosphate buffer (pH 7, 8, 9) • Samples were incubated: -35°C water bath, gentle shaking, 1day. -Centrifuge at 4°C, 7000rpm,10min • Save the supernatant in a plastic tube and keep in 4 °C refrigerator (Garlic Juice) • Instrument analysis - HS-GC-FID - 50 µL internal standard (538 ppm ethyl methyl sulfide, 760 ppm isopropyl disulfide). - 2ml Garlic Juice+ 6ml Salt Water+2ml MeOH + 2g Salt

Concentration of sulfur compounds in home made garlic juice during storage

40 pH 9 (fresh) pH 9 (1 month) (mg/kg 30

20 AMS: Allyl Methyl Sulfide, 10 DMDS: Dimethyl Disulfide, AS: Allyl Sulfide,

concentration DMTS: Dimethyl Trisulfide, 0 DATS: Diallyl Trisulfide, AMS DMDS AS DMTS DADS DATS DADS: Diallyl Disulfide 40

30 (mg/kg)

pH 7(1 month) 20 pH 8 (1 month) pH 9 (1 month) 10

concentration 0 AMS DMDS AS DMTS DADS DATS New Directions

• Waste from garlic processing plants – Waste water? – Effluent from drying? – Other materials Garlic powder?

• Study showed the efficacy of using garlic powder, effluent (garlic waste) at 500 liter/ha, DADS at 0.5 ml/m2, and Methyl bromide at 448 kg/ha

Numbers of sclerotia of Sclerotium cepivorum in 500 cm3 of soil sampled immediately before treatments were applied (the first sampling date in each trial) and periodically thereafter in two locations. (RM Davis et al. 2007)

Garlic powder?

• Soil-applied garlic powder/paste could be a better option compared to the garlic juice product.

• DADS at 0.5 ml/m2 , and methyl bromide at 448 kg/ha

• We do not know the minimum concentration of volatile sulfur compound required for sclerotia generation • Jeremiah has prepared some viable sclerotia ( ~150) for us • We want to develop a device to study sclerotia generation in lab • Screen various volatile sulfur compounds and products in lab before field trial