Evaluation and comparison of biofungicides and fungicides for the control of post harvest potato tuber diseases ( ).

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1 Evaluation and comparison of biofungicides and fungicides for the control of post harvest potato tuber diseases ( ). E. Gachango, W. W. Kirk, R. Schafer and P. Tumbalam. Department of Plant Pathology, Michigan State University, East Lansing, MI Summary In 2005, approximately 20 million metric tons of potatoes with a total value of approximately $2.8 billion were produced in the United States and nearly 58% were stored for 2 months or longer (1). Control of storage pathogens has become increasingly important because of the value, and potential for economic loss,. Potatoes are susceptible to a variety of storage pathogens, including late blight (Phytophthora infestans), Fusarium dry rot (Fusarium sambucinum), pink rot (Phytophthora erythroseptica), Pythium leak (Pythium ultimum), tuber soft rot (Erwinia spp), silver scurf (Helminthosporium solani). Among these pathogens Pythium ultimum, P. infestans and P. erythroseptica are of major concern to potato producers due to the great losses they cause in stored potatoes (6). Few disinfectants and no effective fungicides are registered for direct application to tubers for control of these pathogens. Current recommendations for potato storage diseases include sanitation and exclusion as the primary controls for these pathogens in storage facilities (6). Few alternative compounds are available for potato tuber treatment in storage; these include chlorine-based disinfectants such as chlorine dioxide (5, 7). However, the use of chlorine-based products such as chlorine dioxide is complicated because of the potentially corrosive nature of the material (3) and the need to activate the product in order to generate the chlorine dioxide gas (5). Furthermore, limited information is available as to the effectiveness of chlorine dioxide on potato storage pathogens and results of some studies have suggested that chlorine dioxide does not provide effective tuber protection against Fusarium dry rot (4, 7). At present, management of late blight relies exclusively on the elimination of above ground sources of inoculum by frequent applications of foliar fungicides, rather than protecting tubers directly. In recent years, preliminary trials at MSU have investigated the use of several new biofungicides (Bacillus subtilis, Serenade and B. pumilus, Sonata; AgraQuest) and reduced risk foliar fungicides (azoxystrobin; Syngenta) for postharvest use on potato. These products have shown promise in the control of seed- and soil-borne diseases such as late blight, black scurf and pink rot. However, none of them has been registered for postharvest use on potatoes and further studies are needed to evaluate their postharvest use on potatoes including as mixtures. Results from the 2007/08 projects indicate that the efficacy of the biofungicides described above was more limited than previously thought. As this project develops it is clear that a strategy involving co-applications of biofungicides and conventional chemicals is warranted. New products are being developed for use in potato storage protection, including new biofungicides (e.g. Streptomyces strains, Wanner included in 2008/09), and new active ingredients previously not tested in storage, including difenoconazole (triazole for Fusarium dry rot management) and mandipropamid (for oomycete control). The objective of this study was to examine the effect of post-harvest crop protection programs on harvested tubers to various diseases.

2 The trials in 2008/09 were partially successful in that late blight did not develop sufficiently to effectively compare treatments. Overall in this study, a mixture of azoxystrobin, fludioxinil, and difenoconazole was the most effective in controlling the storage diseases. The biofungicides and the Streptomyces spp. were very promising and suppressed many of the diseases in these tests. In particular the BuPot materials gave excellent control of Pythium leak, and suppressed Fusarium dry rot and Pink rot development. Bacillus subtilis and Bacillus pumulis (Agraquest strains) suppressed Pythium leak, Fusarium dry rot and Pink rot. The Streptomycetes from ARS had efficacy against some of the pathogens in particular W103-5B 100D was effective against Fusarium dry rot in this study, Materials and Methods Experiments were carried out in November 2008 with potato cultivars FL1879. The tests were carried out at 10 o C (49 o F), the storage temperature used in the potato chip processing industry. The cultivar used in the 10 o C test was FL1879 a chip processing cultivar. Potatoes free from visible diseases were selected for the trials from tubers harvested in October Tubers were prepared for inoculation with Phytophthora infestans (Pi), P. erythroseptica (Pe), Pythium ultimum (Py), and Fusarium sambucinum (Fs) by grazing with a single light stroke with a wire brush, sufficient to abrade the skin of the tubers to a depth of 0.01 mm. Solutions (1 x 10 3 /ml) of sporangia/zoosporangia of Pi (late blight), oospores/sporangia of Pe (pink rot), oospores of Py (Pythium leak) and macroconidia of Fs (dry rot) were prepared from cultures of the pathogens previously isolated from potato tubers in Michigan. All pathogens were grown on PDA for 10 days prior to preparation of inoculum solutions. Two non-treated controls, either inoculated with one of the pathogens or non-inoculated were included in the trial. Damaged tubers, (50/replicate/treatment; total 200 tubers/treatment) were sprayed with 10 ml of pathogen suspension, for a final dosage of about 0.25 ml per tuber. Tubers were stored for 2-d after inoculation at 20 C before treatment. Fungicides and biofungicides were applied as liquid treatments in a water suspension with a single R&D XR11003VS spray nozzle at a rate of 1L/ton at 50 psi onto the tuber surfaces, with an entire seed surface being coated. After inoculation, tubers were incubated in the dark in plastic boxes at 10 C, 7 o C or 4 o C (depending on cultivar and disease combination) for 120 to 127 days. Tubers were cut open and the number of tubers with symptoms or signs of the individual pathogens were counted to determine incidence of disease. Disease severity was assessed using a disease severity index. Disease severity classes were determined as class 0 = 0%; 1 = 1-10%; 2 = 11-20%; 3 = 21-50; 4 > % internal area of tuber tissue with disease. The disease severity index was then calculated as the number in each class multiplied by the class number and summed. The sum was then multiplied by a constant to express severity on a scale. Data were analyzed using analysis of variance and the Tukey s HSD test in JMP (SAS Institute, NC).

3 Table 1. Details of Products evaluated in the study. Product name/code/ Active ingredient (%) Formulation Manufacturer FRAC Group Serenade QST 713 (44) Bacillus subtilis (10) 1.34 SC Agraquest Sonata QST 2808 (44) Bacillus pumulis (1.38) 1.38 SC Agraquest A12705 (11) Azoxystrobin (22.9) 250 SC Syngenta A9859A (12) Fludioxinil (0.5) 250 SC Syngenta A8574 (3) Difenoconazole (23.2) 360 SC Syngenta Revus (40) Mandipropamid (23.3) 250 SC Syngenta Mertect (1) Thiabendazole (42.3) 340 F Syngenta Phostrol (33) Phosphonic acid (53.6) 4.32 SC NuFarm Americas Oxidate Hydrogen dioxide (27) 27% SC BioSafe BuPot-1 Not Known 100 L Becker Underwood BuPot-2 Not Known 100 L Becker Underwood BuPot-2 Not Known 100 L Becker Underwood ME D Streptomyces scabies 100 D ARS (Wanner) W103-4C Streptomyces scabies 100 D ARS (Wanner) 1D01-15D Streptomyces scabies 100 D ARS (Wanner) M102-7B Streptomyces scabies 100 D ARS (Wanner) W103-5B Streptomyces scabies 100 D ARS (Wanner) Results and Conclusion In pythium leak (Pythium ultimum) studies, the inoculated non-treated control had significantly higher incidence and severity of disease in comparison with all other treatments (Table 2). Treatments with azoxysrobin and mandipropamid at both rates were the least effective in controlling pythium leak and were not significantly different from one another. Hydrogen peroxide, Bupot at the three rates and a mixture of fludioxonil, difenoconazale and azoxystrobin were the most effective in controlling pythium leak. The biofungicides; Bacillus subtilis and Bacillus pumilus and phosphonic acid had limited control of pythium leak, and were not significantly different from one another. In the Fusarium dry rot (Fusarium sambucinum) studies the inoculated non-treated control had the highest disease incidence and severity (Table 3). However, this was not significantly different from treatment with Bupot-1, Vermiculite, ME D, thiobendazole, W103-4C, 1D 01-15D, Hydrogen peroxide, M102-7B, and Bacillus pumilus. All the other treatments were effective in controlling Fusarium dry rot, and they were not significantly different from each other. In the pink rot (Phytophthora erythrospetica) studies, the inoculated non-treated control had the highest disease incidence (Table 4). Treatments with azoxystrobin, BuPot (all products), mandipropamid, Bacillus pumilus and the mixture of azoxystrobin, fludioxonil and difenoconazale were effective in controlling pink rot since their mean incidence was significantly lower compared to the inoculated non-treated control. All the other treatments (Hydrogen peroxide, phosphonic acid, and Bacillus subtilis were not different from the inoculated non-treated control.

4 In the late blight (Phytophthora infestans) studies, there was very low disease development for all the treatments (Table 4). All the treatments were not significant different from one another. Overall in this study, a mixture of azoxystrobin, fludioxonil, and difenoconazale was the most effective in controlling the storage diseases. The biofungicides and the streptomyces spp. provided excellent to moderate control of the diseases but some of the products were ineffective against some diseases. Supplementary Trials (Syngenta 2009) All treatments gave excellent control of Fusarium dry rot (Table 5). There were no problems in mixing the three-way mixtures at any of the rates. Revus gave excellent control of pink rot and the Maxim + Quadris treatment had significantly les pink rot than the inoculated check (Table 6).

5 Table 2. Incidence and severity of Pythium leak on potato tubers (FL 1879) stored at 10 C for 120 days after treatment with fungicides/biofungicides. Treatments and rate of application per Pythium leak cwt of tubers (4 pt/h 2 O per 20 cwt) Incidence (%) Severity Index z Azoxystrobin 250SC 0.06 fl oz 51.0 b y 43.6 a BuPot-1 100L oz fl oz 0.0 e 0.0 c BuPot-2 100L fl oz 0.0 e 0.0 c BuPot-3 100L fl oz 1.0 de 0.8 c Azoxystrobin 250SC 0.06 fl oz + Fludioxinil 250SC 0.18 fl oz + Difenoconazole 250SC 0.03 fl oz 0.0 e 0.0 c Hydrogen peroxide 27SC fl oz 0.0 e 0.0 c Phosphonic acid 53.6SC 1.28 fl oz 23.0 c 22.2 b Mandipropamid 249FS fl oz 49.0 b 46.4 a Mandipropamid 249FS fl oz 42.0 b 37.4 a Bacillus subtilis ASO 1.34SC 0.32 fl oz 18.0 c 13.2 bc Bacillus pumulis 1.38SC 0.32 fl oz 17.0 cd 13.0 bc Non-treated inoculated check 70.0 a 49.2 a LSD F-ratio p-value < < z Severity classes were determined as class 0 = 0%; 1 = 1-10%; 2 = 11-20%; 3 = 21-50; 4 > % internal area of tuber tissue with disease and incidence is percentage of tubers in class 1-4. y Mean values of diseased tubers within a column followed by the same letter are not significantly different

6 Table 3. Incidence and severity of Fusarium dry rot on potato tubers (FL 1879) stored at 10 C for 120 days after treatment with fungicides/biofungicides. Treatments and rate of application per Fusarium dry rot cwt of tubers (4 pt/h 2 O per 20 cwt) Incidence (%) Severity index z BuPot-1100L oz fl oz 12.0 ab y 5.0 a Vermiculite 100D 16 oz 11.0 ab 2.2 ab ME D 100D 16 oz 10.0 abc 3.4 ab Thiabendazole 43F fl oz 10.0 abc 2.6 ab W103-4C 100D 16 oz 7.0 abc 1.4 ab 1D01-15D 100D 16 oz 7.0 abc 1.4 ab Hydrogen peroxide 27SC fl oz 7.0 abc 1.8 ab M102-7B 100D 16 oz wt 6.0 abc 1.4 ab Bacillus pumilus 1.38SC 0.32 fl oz 5.0 abc 1.0 ab BuPot-2 100L fl oz 4.0 bc 1.2 ab BuPot-3 100L fl oz 4.0 bc 0.8 b Bacillus subtilis 1.34SC 0.32 fl oz 3.0 bc 1.4 ab W103-5B 100D 16 oz 3.0 bc 0.6 b Phosphonic acid 53.6SC 1.28 fl oz 3.0 bc 1.6 ab Fludioxinil 250SC 0.18 fl oz 2.0 bc 0.4 b Azoxystrobin 250SC 0.06 fl oz 2.0 bc 1.2 ab Azoxystrobin 250SC 0.06 fl oz Fludioxinil 250SC 0.18 fl oz Difenoconazole 360FS 0.03 fl oz 2.0 bc 1.2 ab Difenoconazole 360FS 0.03 fl oz 0.0 c 0.0 b Non-treated inoculated check 15.0 a 3.6 ab LSD F-ratio p-value < z Severity classes were determined as class 0 = 0%; 1 = 1-10%; 2 = 11-20%; 3 = 21-50; 4 > % internal area of tuber tissue with disease and incidence is percentage of tubers in class 1-4. y Mean values of diseased tubers within a column followed by the same letter are not significantly different

7 Table 4. Incidence of Pink rot on potato tubers (FL 1879) stored at 10 C for 120 days after treatment with fungicides/biofungicides. Treatments and rate of application per cwt of tubers (4 pt/h 2 O per 20 cwt) Incidence of tuber with pink rot symptoms (%) Azoxystrobin 250SC 0.06 fl oz 7.0 bc z BuPot-1100L fl oz 7.0 bc BuPot L fl oz 7.0 bc BuPot L fl oz 3.0 c Azoxystrobin 250SC 0.06 fl oz Fludioxinil 250SC 0.18 fl oz Difenoconazole 360FS 0.03 fl oz 4.0 bc Hydrogen peroxide 27SC fl oz 9.0 ab Phosphonic acid 53.6SC 1.28 fl oz 8.0 abc Mandipropamid 249 FS fl oz 7.0 bc Mandipropamid 249 FS fl oz 5.0 bc Bacillus subtilis 1.34SC 0.32 fl oz 8.0 abc Bacillus pumilus 1.38SC 0.32 fl oz 5.0 bc Non-treated inoculated check 13.0 a LDS F-ratio p-value <0.001 z Mean values of diseased tubers within a column followed by the same letter are not significantly different

8 Table 5. Supplementary Trial: Incidence and severity of Fusarium dry rot on potato tubers (FL 1879) stored at 10 C for 120 days after treatment with fungicides. Treatments and rate of application per cwt Fusarium dry rot of tubers (4 pt/h 2 O per 20 cwt) Incidence (%) Severity index z A FS fl oz 2.5 b y 1.0 b A FS 0.15 fl oz 2.0 b 0.8 b A FS fl oz + A SC 0.03 fl oz 0.5 b 0.3 b A FS fl oz + A SC fl oz 0.5 b 0.3 b A FS 0.15 fl oz + A SC 0.03 fl oz 1.5 b 0.5 b A FS 0.15 fl oz + A SC fl oz 2.5 b 0.5 b Non-inoculated Check 0.0 b 0.0 b Inoculated Check 60.0 a 26.3 a LDS F-ratio p-value < z Severity classes were determined as class 0 = 0%; 1 = 1-10%; 2 = 11-20%; 3 = 21-50; 4 > % internal area of tuber tissue with disease and incidence is percentage of tubers in class 1-4. y Mean values of diseased tubers within a column followed by the same letter are not significantly different

9 Table 6. Supplementary Trial: Incidence of Pink rot on potato tubers (Goldrush) stored at 10 C for 120 days after treatment with fungicides. Treatments and rate of application per cwt of tubers (4 pt/h 2 O per 20 cwt) Pink rot Incidence (%) Revus 249FS 0.03 fl oz 2.5 bc z Revus 249FS 0.03 fl oz 3.5 bc Revus 249FS 0.03 fl oz 2.5 bc Quadris 250SC 0.03 fl oz + Maxim 4FS fl oz 6.5 b Non-inoculated Check 0.0 c Inoculated Check 33.0 a LDS F-ratio p-value z Mean values of diseased tubers within a column followed by the same letter are not significantly different