Bent J. Nielsen

Anders Borgen, Lars Kristensen

In an organic barley production, seed borne diseases like leaf stripe (Pyrenophora graminea) and loose smut (Ustilago nuda) can relatively quickly give rise to serious problems with reduction in yield. Chemical seed treatment is not possible in the last generations of organic seed production and the current strategy in production of healthy organic seed in Denmark is to discard seed lots with infections above defined threshold values (Nielsen et al., 1998). In this way large amounts of seeds can be discarded and increasing the area for production of organic seeds is at present the only way to ensure enough seeds in quantities and qualities of different varieties.

Today, the number of acceptable control measures in organic seed production is rather limited. Use of resistant varieties is an obvious possibility, but we know only little about the distribution of resistance against seed borne diseases in modern varieties. Other methods could be to reintroduce old techniques like hot water treatment or to use chemicals different from the conventional seed treatments that are acceptable to the organic production.

The heat treatment in water to control seed pathogens in cereals were invented by J.L. Jensen in the 1870-80's (Jensen 1888). Heat treatment in water is usually divided in hot-water (high temperatures (>50° C) and short duration (<10 minutes)) and warm-water (low temperatures (<50° C) and long duration (1-3 hours)). From about 1890's to about 1930 several papers were published on the effect, especially the warm-water method, recommending the method for a wide range of pathogens, including leaf stripe and loose smut (Lind & Ravn, 1918). More recently studies were carried out on hot-water treatment aiming at control of leaf stripe and warm-water treatments to control loose smut of barley (Winter et al., 1996, 1998).

Treatments based on pH-effects, i.e. manipulation the pH-value on the seed surface have been known for a long time (Buttress & Dennis 1947). These methods have primarily been used to control common bunt in wheat, and they have been based on increasing pH with alkaline treatments using chalk and wood-ash. More recently Spie & Dutschke (1991) tested different alkaline treatments showing good effects against common bunt (Tilletia caries). Treatments based on decreasing pH by use of acids to control barley leaf stribe and loose smut has not been tried before to our knowledge.

The objective of the present study was to investigate combinations of hot- and warm-water treatments on control efficacy and side effects on germination properties on barley leaf stripe and loose smut. Further, the objective was to study the effect of treatments based on pH-effects, using acetic acid as an example.

All experiments were carried out at Research Centre Flakkebjerg, Slagelse, Denmark

The seeds used in the experiments were natural infected with either leaf stripe (Pyrenophora graminea) or loose smut (Ustilago nuda). Field emergence was counted at the 1-leaf stages and later on, the number of plants with leaf stripe or loose smut was counted. Plot size in all experiments was 9 m rows with 200 plants in 4 replicates.

Chemical seed treatment was included as standard in the trials with leaf stripe (Fungazil A or Fungazil bejdse, imazalil, 50g/l) or loose smut (Fungazil C, imazalil, 25g/l + carboxin 400 g/l)

Seeds of spring barley were treated with 5% acetic acid, pH 2,5 (same as vinegar) and then dried on filter paper for 24 hours. The seed was sown in a field trial with complete randomised blocks and 4 replicates.

The first experiment with hot water was performed as a three factorial trial in spring barley. At first, the seeds were soaked in water at 20⁰C for 10, 20 and 30 minutes (factor 1) and then treated with hot water at 45^°C, 50°C and 55°C (factor 2) for 2, 4 or 6 minutes (factor 3).

The second experiment with hot water was performed with randomised, complete blocks (4 rep.) in spring barley. The seeds were first, as a pre treatment, soaked in water (20°C or 45°C in 60 or 120 minutes) and then treated with hot water at 50°C or 55°C in 2 or 4 minutes.

There was a clear dose response effect of acetic acid on leaf stripe (table 1) but at the same time also reduction in seed germination at the high dose. Increasing the dose to 50 ml/kg gave full control in 1999 but only 81% control in 2000. The disease pressure was very high in 2000 but the number of plants was low. At the dose level 50 ml/kg a significant reduction in plants to 77-83% compared to untreated were observed.

Table 1. Control of leaf stripe (Pyrenophora graminea) in spring barley

by seed treatment with 5% acetic acid. 1999 and 2000.

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1. 2000

Dose rate Rel. plant Pct. Rel. plant Pct.

per kg seed emergence leaf stripe emergence leaf stripe

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Control 100a 7,8a 100a 15,0a

imazalil 0,05g/ kg 99a 0c 126a 0,2c

5 % Acetic acid 10 ml/kg 102a 8,3a 117a 9,8b

5 % Acetic acid 20 ml/kg 101a 6,5a 104a 10,1b

5 % Acetic acid 30 ml/kg 97a 4,5b 120a 5,7bc

5 % Acetic acid 40 ml/kg 94a 3,4b 128a 4,8bc

5 % Acetic acid 50 ml/kg 83b 0c 77a 3,1c

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Figures with the same letter are not significantly different

1. Pre-treatment (soaking) at 20^°C and then hot-water treatment at 45°C - 55°C.

Soaking the seeds first at 20°C as a pre treatment and then treating them with hot water at 45°C - 55^°C had no effect on loose smut (results not shown).

There was a considerable reduction in leaf stripe after hot water treatment at 50°C and especially at 55°C (table 2). The effect of hot water was increased if a pre-treatment for 10, 20 or 30 minutes was included. There seems to be no difference between the duration of the pre treatment.

Table 2 Relative plant emergence and attack of leaf stripe (Pyrenophora graminea) in

spring barley after pre treatment in water (20°C ) and then hot water at

45°C, 50°C and 55°C in 2, 4 or 6 minutes.

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Hot water Pre-treatment in minutes

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0 10 20 30 0 10 20 30

temp. min. Pct. leaf stripe Relative plant emergence

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45^°C 0 9,0abc 9,9a 7,8b-g 7,1b-h 100a-g 105abc 105a-d 104a-f

2 8,5a-e 6,9c-h 7,8a-g 6,2fgh 98e-i 103a-f 101a-f 104a-f

4 9,1ab 7,4b-h 6,5e-h 7,1b-h 100a-g 99b-h 98c-i 102a-f

6 6,5e-h 8,1a-f 6,9d-h 7,3b-h 103a-f 101a-f 100a-h 102a-f

50°C 0 6,7d-h 7,6b-g 6,2fgh 5,4hi 102a-f 104a-e 100a-h 100a-h

2 6,0fgh 5,9gh 3,3ij 2,8jkl 103a-f 99b-h 102a-f 107a

4 3,0jk 1,8j-n 2,2j-n 1,0k-n 103a-f 103a-f 102a-f 106ab

6 2,6jm 0,2n 0,7lmn 0,5lmn 99b-h 100a-h 104a-f 93g-j

55°C 0 5,3hi 8,8ad 6,8d-h 6,1fgh 98d-i 100a-g 105a-e 100a-h

2 2,2jn 0,4lmn 0,2n 0,3n 100a-g 98b-h 103a-f 98b-h

4 1,5jn 0,6mn 1,0k-n 0,6mn 101a-g 91ij 100a-h 97f-j

6 0,3n 1,5j-n 0,3n 0,6mn 93hij 72k 61l 47m

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Only the high water temperature (55°C) for 6 minutes had a negative effect on the emergence of plants in the field. Combination of pre treatment and high water temperature decreased the emerged plants significantly (table 2).

2. Pre-treatment at 20°C (soaking) or 45°C (warm water) and then hot-water treatment with 50°C or 55°C.

Loose smut could only be controlled by first soaking the kernels at 45°C and then the hot water treatment at 50°C or 55°C (table 3). However, only hot-water at 50°C in 2 minutes showed a high treatment efficacy without side effects on germination.

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20⁰C 60 min. 16,4a-d 15,5a-e 16,0a-d 16,5a-d 15,5a-e 106a-g 118a 109a-d 111abc 105a-g

120 min 14,3a-f 16,8ab 17,4a 12,5ef 15,6a-e 109a-d 114ab 110a-d 104b-g 105a-g

45⁰C 60 min. 14,2a-f 13,3c-f 11,1fg 0,6h 0h 109a-d 108a-e 109a-d 80j 56k

120 min 3,4h 0,2h 0,3h 0h 0h 106a-g 97d-i 94f-i 88ij 56k

carb.+ iamz. ¹⁾ 8,4g 98d-i

Seed treatment in conventional agriculture normally gives a control level of 99-100% of seed borne diseases like common bunt, leaf stripe and loose smut. This high and consistent level is difficult to achieve with alternative methods but the results with acetic acid and hot water are promising.

Acetic acid in the same concentration as vinegar had a good effect on leaf stripe but the volume used was high (50 ml/kg) and had a negative effect on the number of emerging plants in the field. New trials where concentration and volume is changed will show if acetic acid can be used in practice for controlling leaf stripe.

Our results show that pre treatment (soaking) in water at 20⁰C could enhance the effect of hot-water treatment on leaf stripe. Water temperature at 50⁰C for 6 minutes and 55⁰C for 2, 4 or 6 minutes had a high effect on leaf stripe comparable to chemical standard. However, combination of pre treatment and then water temperature at 55⁰C seems to be harmful to the seeds. Winter et al. (1996, 1998) also found that leaf stripe could be controlled at levels comparable to chemical standards by both hot-water (52° C, 10 min.) and warm-water (45° C, 2 h), but hot-water had negative effects on germination. Our results indicate that effects comparable to chemical standard can be obtained without negative side effects on germination.

The infection of loose smut is deep in the seed and long pre treatment in water is necessary for controlling the disease. Combination of high water temperature for pre treatment and high temperature in the succeeding hot water treatment can, however, be harmful to the seeds. Our results indicate that the best combination was pre treatment at 45C for 120 minutes succeeded by hot water treatment at 50° C in 2 minutes. This combination seems to control loose smut (98- 99% control) without side effects on germination. Winter et al. (1996, 1998) used 3 hours at 45° C treatment to obtain same efficacy. Based on our results, longer duration than 2 hours should not be necessary. Compared to the chemical standard in the trial the effect was very high. Seed treatment with the chemical standard gave in our trial an unexpected low control level. Normally a control level at 95-98% would be expected using this product.

Generally, to minimise problems of re-drying and to avoid side effects on germination, treatments with shortest duration and lowest possible temperatures should be chosen. Optimisation, however, is necessary if these methods are going to be implemented on a larger scale.

To avoid losses of large quantities of seeds in organic agriculture due to infections of seed borne diseases, it is necessary to have some kind of control measures. The results presented here indicate that there are alternatives to the conventional seed treatments, but the methods have to be optimised further. Also the practical feasibility and the economy have to be considered carefully, especially concerning re-drying procedures and costs.

It is also necessary to revise if the existing threshold values for seed pathogens are applicable for organic seed production and to look more at specific threshold values, dependent on whether the seeds represent basic, pre basic or later stages of certified seeds. Furthermore, it is necessary to evaluate how the acceptable efficacy level for the individual diseases has to be.

Buttress, F.A. og R.W. Dennis, 1947: The early history of cereal seed treatment in England. Agricultural History 21:93-103.

Nielsen, B. J., Borgen, A., Nielsen, G. C. & Scheel, C. 1998. Strategies for controlling seed borne diseases in cereals and possibilities for fungicide seed treatments. The 1998 Brighton Conference - Pest and Diseases, vol. 3: 893-900.

Jensen, J.L. 1888. Om kornsorternes brand (Anden meddelelse). Triers Bogtrykkeri, Kjøbenhavn. 72 pp.

Lind, J. & F. K. Ravn 1918. Forsøg med midler mod byggets stribesyge. Tidsskrift for Planteavl 25: 56-116

Spie og Dutschke 1991: Bekämpfung des Weizensteinbrandes (Tilletia caries) im biologisch-dynamischen Landbau unter experimentellen und praktischen Bedingungen. Gesunde Pflanzen, 43(8):264-270.

Winter, W., I Bänziger, H. Krebs & A. Ruegger 1996. Warm- und Heisswasserbehandlung von Gerstensaatgut. Agrarforschung 3(1): 25-28

Winter, W., I Bänziger, H. Krebs & A. Ruegger 1998. Getreidebrände und Gerste-Stereifenkrankheit sanft behandeln. Agrarforschung 5(1): 29-32