Accepted Manuscript. Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato

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1 Accepted Manuscript Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato Phillip S. Wharton, William W. Kirk PII: S (14) DOI: Reference: YBCON 3091 To appear in: Biological Control Received Date: 13 September 2013 Accepted Date: 3 March 2014 Please cite this article as: Wharton, P.S., Kirk, W.W., Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato, Biological Control (2014), doi: /j.biocontrol This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Wharton and Kirk 1 Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato. Phillip S. Wharton 1, William W. Kirk 2, 1 University of Idaho, Aberdeen R&E Center, 1693 S W., Aberdeen, ID, Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, 48824, USA Corresponding author; tel: (517) , fax: (517) , kirkw@msu.edu

3 ABSTRACT Wharton and Kirk 2 Seed-borne diseases of potato represent a significant constraint to potato production in the US. The use of an effective fungicide in combination with good management practices during cutting and storage, prior to planting, is essential to reducing disease. The efficacy of two biocontrol agents (Bacillus subtilis and Trichoderma harzianum), and a commercially formulated mixture of the chemicals fludioxonil plus mancozeb, applied as seed treatments in combination with different management practices, were evaluated over two years for the control of seed piece decay and sprout rot caused by Fusarium sambucinum. Treatments were made 10 days prior to planting and at planting, and tubers were re-stored at either 18 C and 95% RH with forced air ventilation at 5950 l min -1 (optimal conditions), at 25 C in the dark without ventilation (suboptimal), or not stored at all prior to planting. Seed piece and sprout health were evaluated in vitro and agronomic impacts evaluated in field experiments. Results showed that the biological control agents B. subtilis and T. harzianum provided good control of sprout rot and seed piece decay caused by F. sambucinum, when seed was re-stored under optimal conditions or not restored at all. Under optimal conditions, treatment with B. subtilis reduced sprout rot and seed piece decay on average by 66 and 84% respectively. Treatment with T. harzianum reduced sprout rot and seed piece decay on average by 70 and 81% respectively. Treatment with fludioxonil + mancozeb reduced sprout rot and seed piece decay under both re-storage regimes. Under optimal conditions, disease incidence and severity was reduced on average by 81 and 97% respectively. Neither biological control agent reduced seed piece decay incidence under either re-storage regime compared to the untreated controls. Key words: Solanum tuberosum, Fusarium sambucinum, Bacillus subtilis, Trichoderma harzianum, seed-piece decay, sprout rot, plant establishment.

4 Wharton and Kirk 3 1. Introduction Seed-borne diseases of potato (Solanum tuberosum L.) represent a significant constraint to potato production in the US (Secor and Salas, 2001) and in a recent study fungal seed-borne diseases were identified as a major constraint to healthy seed production (Frost et al., 2013). Fusarium dry rot of potato is a major postharvest disease worldwide and is caused by several Fusarium species with Fusarium sambucinum Fuckel. being the most aggressive species in Michigan (Boyd, 1972; Secor and Salas, 2001; Wharton et al., 2007a; Gachango et al., 2011a, b). Fusarium sambucinum is readily transmitted by seed-borne inoculum (Secor and Salas, 2001; Carnegie et al., 1998). Dry rot affects tubers in storage and seed tuber pieces in the field (Wharton et al., 2007a; Gachango et al., 2012a,b; Kirk et al., 2013). Losses associated with dry rot have been estimated to range from 6 to 25%, and occasionally losses as great as 60% have been reported during long-term storage (Estrada Jr. et al 2010; Secor and Salas, 2001). In the spring, F. sambucinum typically relies on seed infection to establish a moderate level of sprout infection, thereby initiating seasonal epidemics on stems either above or below ground (Hanson et al., 1996; Gachango et al 2012a). In severe cases, the pathogen can kill developing sprouts outright, resulting in delayed or non-emergence, which is usually, expressed as poor and uneven stands with weakened plants (Wharton et al., 2006). Fusarium sambucinum may also rot seed pieces completely (Ray and Hammerschmidt, 1998; Wharton et al., 2007). Furthermore, bacterial pathogens such as Pectobacterium spp. are known to frequently invade through Fusarium dry rot lesions causing soft rot decay of seed pieces and blackleg. Blackleg is infection of the stem which begins with bacteria moving up from an infected seed piece. Dry rot infected seed pieces are known to have a high incidence of black leg (Secor and Salas, 2001). In late summer/ autumn, infection of potato tubers by dry rot pathogens occurs through wounds inflicted during harvesting, grading, cutting, and handling of tubers (Wharton et al., 2007a;

5 Powelson et al., 2008). Wharton and Kirk 4 In the past ten years in the potato growing regions of the mid-western states of the US, three factors have enhanced seed-borne disease problems, a lack of information on effective fungicides for both post-harvest and pre-planting use against seed-borne pathogens, an increase in the acreage of potatoes grown by fewer growers leading to management issues such as timing of pre-cutting of seed prior to planting (MPIC Pesticide Survey, ), and thirdly climatic factors such as increased rainfall during the planting phase of the season (Andresen et al., 2001; Baker et al., 2005). In combination, these factors can delay planting and increase the impact of fungal and secondary bacterial seed piece decay and sprout rot during the early part of the growing season, subsequently affecting yield and quality of the crop. Dry rot has traditionally been managed by implementing practices that reduce tuber bruising and provide conditions for rapid wound healing (Secor and Salas, 2001; Secor and Johnson, 2008) and by application of the benzimidazole fungicide thiabendazole (TBZ) either as tubers are going into storage or before planting (Hide et al., 1992). Isolates of F. sambucinum resistant to TBZ and other benzimidazoles were first discovered in Europe in 1973 (Hide et al., 1992) and in the United States in 1992 (Desjardins, 1995). Gachango et al. (2012b) reported that all samples of F. sambucinum isolated from Michigan potato seed were resistant to TBZ. Other fungicides used to control Fusarium dry rot in US include fludioxonil alone (Maxim Seed Potato, Syngenta Inc. Greensboro, NC, USA) or in combination with mancozeb (Maxim MZ, Syngenta; Zitter, 2010). Fludioxonil has been shown to reduce seed piece decay and sprout rot resulting in increased plant stands and the production of healthy progeny tubers (Wharton et al., 2007b). In the past five years, fludioxonil-resistant isolates of Fusarium spp. have been reported in Canada and the US and included isolates of F. sambucinum, F. coeruleum, and F. oxysporum (Peters et al., 2008a,b; Gachango et al., 2011a,b; 2012a). This has resulted in fewer alternatives for controlling potato seed piece decay and sprout rot caused by these pathogens (Gachango et

6 Wharton and Kirk 5 al., 2012b). Thus, the use of an effective seed treatment in combination with good management practices during the cutting process and storage of cut seed prior to planting are essential to reducing these diseases in cut seed prior to planting. In the past five years several new biological control agents based on the biocontrol bacteria Bacillus subtilis (Serenade Max ; AgraQuest Inc.) and the biocontrol fungus Trichoderma harzianum (T-22 Planter Box ; Bioworks Inc.) have been registered for use on potato. These have shown promise in the control of seed and soil borne diseases such as late blight, black scurf and dry rot and silver scurf (McBeath and Kirk, 2000; Sadfi et al., 2002; Brewer and Larkin, 2005; Johnson, 2007; Wharton et al., 2012). None of these products has been evaluated as seed treatment for the control of Fusarium seed piece decay and sprout rot on potato. However, in greenhouse studies B. subtilis and T. harzianum were shown to have some efficacy in controlling Rhizoctonia stem canker and black scurf (Brewer and Larkin, 2005). Preliminary trials using a related Trichoderma species T. atroviride, as a seed treatment showed that the fungus was as effective in controlling seed-borne late blight as the conventional fungicide mancozeb (McBeath and Kirk, 2000). These biological control agents have the additional benefit of being endophytic and persisting on the host surface and thus may protect emerging sprouts from disease. Research with the biological control agent B. cereus showed that colonization of the potato surface increased until 61 days after planting (Sadfi et al. 2002). Previous studies showed that cutting and treating potato seed with fludioxonil + mancozeb up to ten days before planting could significantly reduce Fusarium seed piece decay, improve plant establishment and subsequent crop vigor and early development (Wharton et al. 2007c). However, the presence of fungal inoculum and poor seed storage conditions after seed cutting and prior to planting can negatively impact plant establishment, crop vigor and development (Wharton et al. 2007c). Fusarium insensitivity in laboratory studies may not translate directly to commercial production. This disparity may result from interactions not

7 Wharton and Kirk 6 experienced in mixed populations or within a living host. There has been no compelling evidence to suggest that fludioxonil has failed to perform because of insensitivity to Fusarium (Gachango et al., 2012a). However, in light of the discoveries of fungicide resistant Fusarium isolates, the objectives of this investigation were two-fold. First, to evaluate in vitro the effectiveness of the biological control agents as seed treatments for controlling seed piece decay and sprout rot caused by F. sambucinum prior to planting. Secondly, to determine the best conditions to re-store seed after cutting and treating with these biological seed treatments in order to minimize seed piece decay and sprout rot and maximize early plant development and vigor. 2. Materials and methods 2.1 Fungal cultures A mixture of 10 virulent single spore isolates of Fusarium sambucinum, which produces dry rot symptoms in potato tuber tissue and was determined to be resistant to thiabendazole but sensitive to fludioxonil, was used. Conidia of the isolates were maintained at 4 C in the dark on filter paper and axenic cultures of the isolates were produced by placing a 1 mm 2 section of filter paper containing conidia of the stored cultures on PDA. For inoculum production, cultures of F. sambucinum were grown on PDA in the dark at 8 C for 14 days prior to the date of inoculation. Conidia were harvested by flooding the surface of the Petri dish with sterile distilled-deionized water (5 ml) and gently scraping the surface of the media with an L-shaped glass rod to dislodge the conidia. The conidial suspension was stirred with a magnetic stirrer for 1 h and strained through 4 layers of cheesecloth to remove mycelial fragments. The concentration was then adjusted to about 1 x 10 3 conidia ml -1 using a hemacytometer. Conidial suspensions of each of the 10 isolates were then mixed together to make the inoculation mixture.

8 2.2 Inoculation and seed treatment Wharton and Kirk 7 Potato tubers of the commonly used chip processing potato cultivar Snowden which is susceptible to F. sambucinum (Wharton et al., 2007) was used in all experiments. Whole tubers were harvested in October from certified seed crops grown in northern Michigan in 2005 and Tubers free from symptoms of dry rot and other diseases were selected for the experiments. The tubers were stored in the dark at 3 C and 95% RH until the spring (when conditions became suitable for planting; usually the beginning of May in Michigan) of the following year. Tubers were removed from storage, warmed from 3 C in 2 C increments, every 2 days, up to 12 C over a period of 10 days in the dark in a controlled environment chamber with forced air ventilation at 5950 l min -1. Tubers were cut in half longitudinally with a sterilized knife ensuring that viable sprouts were present on both halves. Treatments applied to seed pieces were 1) not inoculated, 2) inoculated with F. sambucinum, 3) inoculated with F. sambucinum as in (2) and treated with either the commercial seed protectant Maxim MZ (active ingredients: fludioxonil 5 g kg -1 + mancozeb 96 g kg -1 ; Syngenta Crop Protection Inc., Greensboro, NC, USA) at the manufacturers recommended rate (500g per 100 kg potato seed), Bacillus subtilis (Serenade Max, strain QST 713; AgraQuest Inc. CA) at 250g per 100 kg potato seed, or Trichoderma harzianum (T-22 Planter Box, Rifai strain KRL-AG2; Bioworks Inc.) at 125g per 100 kg potato seed. The manufacturers recommended the rates of the biological control agents. The non-inoculated treatment consisted of cut seed pieces sprayed with sterile distilled water only. Inoculated seed pieces were produced by spraying 200 ml of conidial suspension (1 x 10 3 conidia ml -1 ) over the entire cut surface to give a final dosage of about 1 ml per seed piece. Care was taken to limit inoculum spray to the cut surface only. All seed treatments were applied to the cut seed pieces 30 minutes after inoculation using a Gustafson revolving drum seed treater. Sufficient tubers were treated to give a total of 160 seed pieces per treatment with 4 replicates per treatment.

9 Wharton and Kirk Re-storage regimes for the cut and treated seed After seed had been cut and treated as described above they were subjected to three different re-storage regimes. The first two regimes were actual storage regimes and the third was no re-storage. The first pre-planting re-storage regime (A) was considered to be the optimal restorage regime and followed current guidelines for recommended pre-planting practices (Michigan State University extension bulletin E-2995; Wharton et al 2007b). Cut and treated tubers (not inoculated, inoculated but not treated or inoculated and treated as described above in section 2.2) were returned to controlled environment chambers, held at 8 C for 2 days and over the next 10 days the temperature was increased every 2 days by 2 C intervals up to 18 C in a 14/10 h photoperiod and 95%RH with forced air ventilation at 5950 l min -1 and held at 18 C until planting. The second regime (B) was developed to simulate non-recommended preplanting practices. In this regime, the cut and treated seed pieces were placed in large paper bags (20 x 40 cm) and stored in the dark at 25 C without added humidity or ventilation. The total storage time for both storage regimes (A and B) was 14 d. The third storage regime (C) was a no storage treatment. In this regime, tubers were stored under optimal storage conditions (in the dark at 12 C and 95% RH) until the day of planting. On the day of planting, tubers were cut, and either not inoculated, inoculated but not treated or inoculated and treated with a fungicide (as described above in section 2.2), and planted all on the same day. The different storage regimes are listed as A, B or C, respectively in Tables 2, 3, 5, Evaluation of seed treatment and re-storage conditions on seed piece decay and sprout rot To evaluate the effect of seed treatment and re-storage conditions on the development of Fusarium seed piece decay and its effect on sprout health, samples from each treatment (n = 40) were removed prior to planting and incubated at 18 C (95% RH with forced air ventilation at

10 Wharton and Kirk l min -1 ) in controlled environment chambers for 14 d. The total number of healthy and diseased sprouts was counted and the percentage of diseased sprouts calculated to determine sprout rot incidence and severity caused by F. sambucinum. The incidence of seed piece decay caused by F. sambucinum was calculated by counting the number of seed pieces with symptoms of seed piece dry rot. Severity of seed piece decay was calculated as the percentage volume of decay per tuber. The percentage of decay per seed piece was scored by estimating subjectively the percentage surface area covered by darkening after cutting four transverse sections through the seed piece as follows. The maximum length, width, and depth of the seed piece was measured and multiplied to give an approximate volume of the seed piece. The same measurements were then taken on the lesion to calculate the approximate volume of the dry rot lesion. To determine the percentage volume of decay, the volume of the lesion was divided by the volume of the tuber and the result expressed as a percentage to give the percentage seed piece decay per tuber. The experiment was carried out in 2006 and repeated in Field experiments To evaluate the agronomic effects (e.g. effects disease on emergence and stand count) of the seed treatments and re-storage conditions before planting on crop health, the treated seedpieces were planted at the Michigan State University Muck Soils Research Farm, Bath, MI into 24 two-row by 3 m plots (ca. 20 cm between plants to give a target population of 30 plants per two-row plot). Each treatment was replicated four times in a randomized complete block design. Stand count and rate of emergence were measured and after canopy closure the treatments were sampled for the presence of disease. For Fusarium wilt symptoms, stems from 16 plants per treatment (4 per replicate) were cut at an angle and rated for the presence/absence of vascular browning. In both years all the treatments were planted on the same day; June 12 in 2006 and June 4 in 2007.

11 Wharton and Kirk Collection and analyses of field data Analysis of variance showed that there were significant differences between results from each year, so data from each year were analyzed separately. Data were analyzed by analysis of variance using the statistical analysis software package JMP (SAS Institute Inc., SAS Campus Drive, Cary, North Carolina 27513, USA). Emergence was rated as the cumulative number of plants breaking the soil surface. The number of emerged plants was recorded over a 22 d period after planting, at which time emergence was complete in non-inoculated tubers from the optimal pre-planting/storage treatment. The final plant stand was expressed as the percentage of emerged plants divided by the expected number based on the planting rate. The rate of emergence was calculated initially as the area under the plant progress curve (AUEPC; max=100). From this, the relative area under the emergence progress curve (RAUEPC) was calculated by modification of the method used to calculate the relative area under the disease progress curve [RAUDPC, (Kirk et al., 2001)], using the following equation: Σ( ti RAUEPC = + 1 Ei Ei ti) * 2 ; Ttotal *100 where t was the time in days after planting and E was the percentage of plant emergence. As plant emergence was assessed at various time intervals, the area under emergence progress curve (AUEPC) was calculated by adding the area under the linear progression of the number of emerged plants between consecutive estimations of emergence from planting to full emergence. The RAUEPC was calculated by dividing the sum of individual AUEPC values by the maximum AUEPC (100 duration of emergence period, from planting to full emergence). Treatment means for all experiments were analyzed by analysis of variance (least squares method) using the JMP program version

12 3. Results Wharton and Kirk Evaluation of seed treatment and storage conditions on seed piece decay and sprout rot Statistical analysis showed that there were significant differences between results from 2006 and 2007 for seed piece and sprout health so results were analyzed separately (Table 1). However, the results from both years showed similar trends, with re-storage of seed pieces under suboptimal conditions (at 25 C in the dark without ventilation) significantly increasing the levels of seed piece decay and sprout rot caused by the pathogen (Table 2). In experiments where seed pieces were inoculated with F. sambucinum, treatments re-stored under optimal conditions (between 12 and 18 C at 95% RH and with forced air ventilation), or not stored had significantly fewer diseased sprouts (sprout rot) and a lower severity of seed piece decay than the inoculated/non-treated controls (Table 2, Treatment x Storage section). In 2006 the levels of dry rot were higher than in The levels of disease in the non-inoculated treatments illustrated this (Table 3). However, seed treatment with the biological control agents Bacillus subtilis (strain QST 713), and Trichoderma harzianum (Rifai strain KRL-AG2) and re-storage under optimal conditions provided good control of sprout rot and seed piece decay caused by F. sambucinum (Tables 2 and 3). In both years, non-inoculated treatments with all three seed treatments were not significantly different from the non-inoculated/non-treated controls (Table 2, Treatment x inoculation section). However, in 2007 inoculated treatments with the biological control agents and fungicide were also not significantly different from the non-inoculated/nontreated controls (Table 2, Treatment x inoculation section). Results from both years also showed that the presence of fungal growth on the seed piece surface (rated as disease incidence) was not correlated with the severity of seed piece decay as treatments with high incidence of disease often had low levels of seed piece decay (Table 3).

13 3.2 Field experiments Wharton and Kirk 12 Results of the field experiments to evaluate the agronomical effects of fungicidal seed treatments and re-storage conditions before planting on crop health are shown in Tables 4-6. Statistical analysis showed that there were significant differences between results from 2006 and 2007 so results were analyzed separately (Table 4). In both years, treatment with the biological control agents did not increase RAUEPC or final plant stands in treatments that were inoculated with F. sambucinum (Table 5, Treatment x inoculation section). Furthermore, there were no significant differences between treatment with the biological control agents and the fludioxonil + mancozeb seed treatment. However, in 2006, in experiments that were not inoculated with F. sambucinum, B. subtilis did increase RAUEPC (Table 5, Treatment x inoculation section). In both years there were no significant differences between the two types of storage regime (Table 5, inoculation x storage; Table 6). However, there was a significant difference between the storage treatments and treatments that were cut, treated and planted on the same day (storage type C). Cutting, treating and planting on the same day significantly reduced RAUEPC and final plant stand regardless of whether the treatment was inoculated or not (Table 5, inoculation x storage; Table 6). In trials where seed pieces were inoculated with F. sambucinum, very few plants were found with vascular browning and the results were not significantly different between treatments in 2006 or 2007 (results not shown). 4. Discussion This systematic study to investigate the use of biological control agents as seed treatments in combination with good management practices during the cutting and re-storage of potato seed prior to planting on Fusarium dry rot complements the study on late blight by Wharton et al. (2012). Potato seed piece treatments have been shown to be useful for the control of seed-borne diseases and have been used to control diseases such as black scurf and stem

14 Wharton and Kirk 13 canker (Rhizoctonia solani Kuhn), silver scurf (Helminthosporium solani Durieu & Mont) and dry rot [Fusarium spp. (Hide and Lapwood, 1982; Frazier et al., 1998; Nolte et al 2003); Wharton et al., 2007c)]. Previous studies have shown that applying a fungicide seed treatment to cut seed up to 10 days prior to planting can provide effective control of both seed piece decay and sprout rot caused by F. sambucinum (Wharton et al., 2007c). However, these studies relied on the use of good re-storage conditions (12 18 C at 95% RH with forced air ventilation at 5950 l min -1 ) prior to planting. In recent years, factors such as increased rain events have lead to delays in planting of up to 21 days, during which time seed may be stored under sub-optimal conditions. In our study, results showed that B. subtilis and T. harzianum could provide good control of sprout rot and seed piece decay caused by F. sambucinum, when seed was re-stored under optimal conditions. In inoculated treatments, under optimal re-storage conditions B. subtilis was able to reduce seed piece decay by 56.5% in 2006 and 94.3% in Trichoderma harzianum was able to reduce seed piece decay by 59.2 and 83.5 percent in 2006 and 2007, respectively. In 2007, this was statistically comparable to the control achieved through the use of fludioxonil + mancozeb. In 2006, it was not statistically comparable to the conventional fungicide treatment but was still significantly better than the non-treated, inoculated controls. These results are in agreement with previous studies that have shown that biological control of sprout rot and seed piece decay can be obtained with B. subtilis and T. harzianum (McBeath and Kirk, 2000; Sadfi et al., 2002; Wharton et al., 2012). Although the biocontrol agents were effective under optimal restorage conditions, they were unable to overcome the effects of poor storage conditions and were not effective against disease under these conditions. Results showed that re-storage of seed pieces under sub-optimal conditions (at 25 C in the dark without ventilation) significantly increased the levels of seed piece decay and sprout rot caused by F. sambucinum. However, there were no significant differences in the rate of emergence and final plant stand between

15 Wharton and Kirk 14 optimal and sub-optimal re-storage regimes. Even in the experiments with the standard commercial seed treatment fludioxonil + mancozeb, levels of sprout rot and seed piece decay were elevated in treatments stored under sub-optimal conditions. Unexpectedly, in this study there were the lower rates of emergence and final plant stands in all treatments where seed was cut, treated and inoculated on the day of planting. This effect did not appear to be due to the application of a seed treatment as it also occurred in the noninoculated control. One explanation may be that the cut seed surface which was not given time to heal was more susceptible to seed piece decay that occurred naturally in the field after planting. Biological control agents are an attractive alternative to conventional seed treatments as previous studies have shown that these organisms will colonize the potato surface and persist for considerable periods of time. Trichoderma harzianum has been shown to be able to grow and colonize new stems, stolons and roots of the developing potato plant throughout the growing season, thus maintaining its controlling efficacy (Harman, 2000; Howell, 2003). In contrast, the chemical seed treatment will be effective for a much shorter period, as it will eventually be washed away from the seed piece during the growing season. This may be the case when these biological control organisms are applied in-furrow. However, it does not appear to be the case when they are applied as seed treatments to cut seed pieces surfaces. Although both biological control organisms provided good control of disease under optimal storage conditions and elevated inoculum pressure, they were not as effective as the chemical seed treatment, which was able to provide disease control even under sub-optimal re-storage conditions and elevated inoculum pressure. In our field studies there were no significant differences between the treatments in terms of the rate of emergence, final plant stand or yield. Differences in the agronomic variables may have been observed if the biological control organisms were persisting on the potato surface as

16 Wharton and Kirk 15 described in previous studies which have reported that T. harzianum reduced Rhizoctonia stem canker in the field and black scurf on progeny tubers (Wilson et al., 2008). The results obtained in this study suggest that biological control agents may be an alternative to conventional seed treatments for the control of sprout rot and seed piece decay if they are applied to healthy seed, which is then re-stored under optimal storage conditions. With the advent of fludioxonil-resistant isolates of Fusarium spp. (Peters, R. et al., 2008; Peters, J. et al., 2008; Gachango et al., 2011ab; Gachango et al., 2012ab), the use of biocontrol agents may become an important tool in the control of seed piece decay and sprout rot caused by these pathogens. ACKNOWLEDGEMENTS This work was funded by Project GREEEN (Generating Research and Extension to meet Economic and Environmental Needs) at Michigan State University, the Michigan Potato Industry Commission. The authors are grateful to AgraQuest Inc., BioWorks Inc, and Syngenta Crop Protection Inc. for providing the fungicide products used in this study. REFERENCES Andresen, J.A., Alagarswamy, G., Rotz, C., Ritchie, J., LeBaron, A., Weather impacts on maize, soybean, and alfalfa production in the Great Lakes region, Agron. J. 93, Baker, K.M., Kirk, W.W., Stein, J.M., Andresen, J.A., Climatic trends and potato late blight risk in the Upper Great Lakes region. HortTechnology 15, Boyd, A.E.W., Potato storage diseases. Rev. Plant Pathol. 51, 297e321.

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18 Wharton and Kirk 17 Gachango, E., Kirk,W., Wharton, P.S., Schafer, R., 2012b. Evaluation and comparison of biocontrol and conventional fungicides for control of postharvest potato tuber diseases. Biol. Control. 63, Hanson, L.E., Schwager, S.J., Loria, R.,1996. Sensitivity to Thiabendazole in Fusarium species associated with dry rot of potato. Phytopathology 86, Harman G.E., Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, Hide, G.A., and Lapwood, D.H., Disease aspects of potato production. In: Harris, P.M. (Ed.), The potato crop: the scientific basis for improvement. Chapman and Hall, London, UK, pp Howell, C.R., Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, Johnson, S.B., Evaluation of a biological agent for control of Helminthosporium solani. Plant Pathology J. 6, Kirk, W.W., Felcher, K.J., Douches, D.S., Coombs, J., Stein, J.M., Baker, K.M., Hammerschmidt, R., Effect of host plant resistance and reduced rates and frequencies of fungicide application to control potato late blight. Plant Dis. 85, Kirk,W., Gachango, E., Schafer, R., Wharton. S., Effects of in-season crop-protection combined with postharvest applied fungicide on suppression of potato storage diseases caused by Fusarium pathogens. Crop Prot. 51:

19 Wharton and Kirk 18 McBeath, J.H., Kirk, W.W., Control of seed-borne late blight on pre-cut potato seed with Trichoderma atroviride. In: Huber, D.M. (Ed.), Biocontrol in a new millennium: Building for the Future on Past Experience, Estes Park Center, Colorado, pp Michigan Potato Industry Commission Pesticide Survey. MPIC, Schavey Rd., Suite #7, DeWitt, MI Nolte, P., Bertram, M., Bateman, M., McIntosh, C.S., Comparative effects of cut and treated seed tubers vs. untreated whole seed tubers on seed decay, rhizoctonia stem canker, growth, and yield of Russet Burbank potatoes. Am. J. Potato Res. 80, 1-8. Peters, J.C., Lees, A.K., Cullen, D.W., Sullivan, L., Stroud, G.P., Cunnington, A.C., Characterization of Fusarium spp. responsible for causing dry rot of potato in Great Britain. Plant Pathol. 57, Peters, R.D., Platt, H.W., Drake, K.A., Coffin, R.H., Moorehead, S., Clark, M.M., Al-Mughrabi, K.I., Howard, R.J First report of fludioxonil-resistant isolates of Fusarium spp. causing potato seed-piece decay. Plant Dis. 92, 172. Powelson, M. L., Rowe, H. C., Managing diseases caused by seedborne and soilborne fungi and fungus-like pathogens. Pages in: Potato Health Management, D. A. Johnson, ed. APS Press, St. Paul MN. Ray, H., Hammerschmidt, R., Responses of potato tuber to infection by Fusarium sambucinum. Physiol. Mol. Plant P. 53, Sadfi, N., Cherif, M., Hajlaoui, M.R., Boudabbous, A., Biological control of the potato tubers dry rot caused by Fusarium roseum var. sambucinum under greenhouse, field and storage conditions using Bacillus spp. isolates. J. Phytopathol. 150, Secor, G.A., Salas, B., Fusarium Dry Rot and Fusarium Wilt. In: Stevenson, W.R., Loria, R., Franc., G.D., Weingartner, D.P. (Eds.), Compendium of Potato Diseases, APS Press, St. Paul, USA, pp

20 Wharton and Kirk 19 Wharton, P.S., Tumbalam, P., Kirk, W.W., First report of potato tuber sprout rot caused by Fusarium sambucinum in Michigan. Plant Dis. 90, Wharton, P., Hammerschmidt, R., Kirk, W. 2007a. Fusarium dry rot. Michigan State University. Extension Bulletin E East Lansing, Michigan. 4 pages. Available at Wharton, P., Kirk, W. 2007b. Potato seed piece health management. Michigan State University. Extension Bulletin E East Lansing, Michigan. 4 pages. Available at Wharton, P.S., Kirk., W.W., Berry, D., Tumbalam, P., 2007c. Seed treatment application-timing options for control of Fusarium decay and sprout rot of cut seed pieces. Am. J. Potato Res. 84, Wharton, P.S., Kirk, W.W., Schafer, R.L., and Tumbalam, P., Evaluation of biological seed treatments in combination with management practices for the control of seed-borne late blight in potato. Biol. Control 63, Wilson, P.S., Ahvenniemi, P.M., Lehtonen, M.J., Kukkonen, M., Rita, H., and Valkonen, J.P.T., Biological and chemical control and their combined use to control different stages of the Rhizoctonia disease complex on potato through the growing season. Ann. Appl. Biol. 153,

21 Wharton and Kirk 20 Table 1. Summary of the analysis of variance of the main effects of seed treatment application and inoculation with Fusarium sambucinum on sprout rot, and incidence and severity of seed piece decay in 2006 and Table 2. Least square means differences table showing the effects of seed treatment application and inoculation with Fusarium sambucinum, on sprout rot, incidence and severity of seed piece decay in 2006 and Table 3. Effect of storage method, seed treatment application and inoculation with Fusarium sambucinum on sprout rot, and seed piece incidence and severity in 2006 and Table 4. Summary of the analysis of variance of the main effects of seed treatment application and inoculation with Fusarium sambucinum, on emergence and final plant stand of potato in 2006 and Table 5. Least square means differences table showing the effects of seed treatment application and inoculation with Fusarium sambucinum on emergence and final plant stand in 2006 and Table 6. Effect of seed treatment, inoculation with Fusarium sambucinum, and storage type on emergence and final plant stand of potato in 2006 and 2007.

22 Table 1 Wharton and Kirk 21 Source P value a Diseased sprouts (%) Incidence of seed piece decay (%) Seed piece decay (%) Treatment < < < < < Inoculation < < < < < < Storage < < Treatment Inoculation < < < < Treatment storage Inoculation storage a Significance indicated by P 0.05.

23 Table 2 Wharton and Kirk 22 Source Sprout rot (%) Seed Piece Incidence (%) a Seed Piece Decay (%) b Treatment Non-treated control (UTC) 62.6 a c 44.9 a 68.8 a 62.5 a 39.7 a 25.9 a Bacillus subtilis 48.3 b 39.5 ab 45.8 b 60.4 a 27.7 b 24.3 a Trichoderma harzianum 52.7 b 28.7 bc 50.0 b 42.9 b 29.1 b 12.4 ab Fludioxonil + mancozeb 31.7 c 26.4 c 27.9 c 22.1 c 8.1 c 4.8 b Inoculation no 35.7 a 16.4 a 34.8 a 27.9 a 8.8 a 5.5 a yes 62.0 b 53.3 b 61.5 b 66.0 b 43.5 b 28.2 b Storage Type d A 45.8 b 21.3 b 44.4 b 37.2 b 25.8 ab 11.1 b B 54.5 a 57.1 a 52.2 a 67.5 a 29.2 a 26.7 a C 46.2 b 26.3 b 47.8 ab 36.3 b 23.5 b 12.8 b Treatment x inoculation UTC,no 32.7 d 7.5 e 39.2 cd 35.8 cd 7.9 c 2.3 c UTC,yes 92.5 a 82.3 a 98.3 a 89.2 a 71.5 a 49.5 a Bacillus subtilis,no 41.0 d 25.8 de 42.5 c 43.3 bc 9.0 c 16.1 bc Bacillus subtilis,yes 55.6 c 53.1 b 57.5 b 77.5 a 46.3 b 32.6 ab Trichoderma harzianum,no 37.5 d 7.6 e 30.0 cd 20.0 cd 12.6 c 3.0 c Trichoderma harzianum,yes 67.9 b 45.3 bc 61.7 b 65.8 ab 45.7 b 21.8 bc Fludioxonil + mancozeb,no 31.5 d 24.9 de 27.5 d 12.5 d 5.7 c 0.5 c Fludioxonil + mancozeb,yes 32.0 d 32.5 cd 28.3 cd 31.7 cd 10.6 c 9.0 bc Treatment x Storage UTC,A 61.1 a 40.7 bcde 66.3 ab 50.0 abcd 45.1 a 28.3 ab UTC,B 67.2 a 51.3 abc 62.5 abc 82.5 a 36.8 ab 35.1 a UTC,C 59.5 ab 42.8 bcd 77.5 a 55.0 abc 37.2 ab 14.3 ab Bacillus subtilis,a 46.1 bcd 22.1 def 45 cde 50.0 abcd 26.0 bc 5.8 ab Bacillus subtilis,b 54.3 abc 69.1 a 61.3 abcd 83.8 a 33.6 ab 34.6 a Bacillus subtilis,c 44.5 cd 27.2 cdef 43.8 cdef 47.5 bcd 23.4 bc 32.6 ab Trichoderma harzianum,a 43.8 cd 16.6 ef 42.5 defg 30.0 cde 27.9 bc 9.2 ab Trichoderma harzianum,b 60.8 a 46.9 abc 50 bcde 65.0 ab 31.8 ab 24.0 ab Trichoderma harzianum,c 53.5 abc 15.8 f 45 cde 33.8 bcde 27.8 bc 4.0 ab Fludioxonil + mancozeb,a 32.3 de 5.8 f 23.8 g 18.8 de 4.1 d 1.0 b Fludioxonil + mancozeb,b 35.6 de 61.1 ab 35 efg 38.8 bcde 14.8 cd 13.0 ab Fludioxonil + mancozeb,c 27.2 e 19.2 def 25 fg 8.8 e 5.5 d 0.2 b Inoculation x storage no,a 35.6 c 5.3 c 29.4 c 18.1 c 8.0 c 2.5 c no,b 37.6 c 36.2 b 35.6 c 50.6 b 8.5 c 12.7 bc

24 Wharton and Kirk 23 no,c 33.8 c 7.9 c 39.4 c 15.0 c 9.9 c 1.1 c yes,a 56.0 b 37.3 b 59.4 ab 56.3 b 43.6 ab 19.6 bc yes,b 71.3 a 78.0 a 68.8 a 84.4 a 49.9 a 40.7 a yes,c 58.6 b 44.7 b 56.3 b 57.5 b 37.1 b 24.4 ab a Incidence of seed piece decay was calculated as the mean number of seed pieces showing symptoms of Fusarium dry rot. b Seed piece decay was calculated as the percentage volume of decay per tuber. c Numbers followed by the same letter within a column are not significantly different at P = 0.05 (Tukey multiple comparison method). d Storage types were: A, stored for 10 DBP under optimal conditions (12 18 C at 95% RH with forced air ventilation at 5950 l min -1 ); B, stored for 10 DBP under poor conditions (25 C in dark with no ventilation); C, treated and inoculated on the day of planting.

25 Table 3 Wharton and Kirk 24 Fungicide treatment and Inocul Storage Sprout rot (%) d Seed Piece Incidence (%) b Seed Piece Decay (%) c rate/100kg seed a ation b type c None A 32.8 d-g f 3.4 g f 35.0 d-g 12.5 ef 5.1 e 1.0 c B 37.5 d-g 14.3 fg 25.0 fg 75.0 abc 4.6 e 4.9 c C 27.7 fg 5.0 g 57.5 b-e 20.0 def 14.0 e 0.9 c + A 89.4 a 78.0 ab 97.5 a 87.5 ab 68.9 ab 55.6 ab + B 96.9 a 88.3 a 100 a 90.0 ab 85.1 a 65.4 a + C 91.2 a 80.7 ab 97.5 a 90.0 ab 60.4 bc 27.6 ab Bacillus subtilis A 45.0 c-g 9.5 g 40.0 c-g 25.0 def 8.5 e 6.0 c (250g) B 42.4 d-g 61.9 a-d 45.0 c-f 82.5 ab 11.6 e 40.1 ab C 35.6 d-g 6.0 g 42.5 c-g 22.5 def 6.9 e 2.2 c + A 47.2 c-f 34.6 c-g 50.0 b-f 75.0 abc 43.5 cd 5.7 c + B 66.1 bc 76.3 ab 77.5 ab 85.0 ab 55.5 bc 29.1 ab + C 53.4 cde 48.5 b-f 45.0 c-f 72.5 abc 40.0 cd 63.0 a Trichoderma harzianum A 33.5 d-g 4.7 g 27.5 fg 15.0 ef 15.0 e 2.0 c (125g) B 37.5 d-g 12.1 g 32.5 d-g 32.5 c-f 11.0 e 5.9 c C 41.6 d-g 5.8 g 30.0 efg 12.5 ef 11.9 e 1.3 c + A 54.0 cd 28.4 d-g 57.5 b-e 45.0 b-f 40.8 cd 16.5 bc + B 84.2 ab 81.7 ab 67.5 bc 97.5 a 52.5 bc 42.2 ab + C 65.4 bc 25.8 efg 60.0 bcd 55.0 b-e 43.8 cd 6.8 c Fludioxonil + mancozeb A 31.2 efg 3.4 g 15.0 g 20.0 def 3.4 e 1.3 c (500g) B 33.0 d-g 56.5 a-e 40.0 c-g 12.5 ef 6.9 e 0.1 c C 30.1 fg 14.8 fg 27.5 fg 5.0 f 6.9 e 0.2 c + A 33.4 d-g 8.2 g 32.5 d-g 17.5 ef 4.9 e 0.8 c + B 38.2 d-g 65.7 abc 30.0 efg 65.0 a-d 22.8 de 26.0 ab + C 24.4 g 23.7 efg 22.5 fg 12.5 ef 4.1 e 0.3 c Tukey s HSD (P=0.05) Replicate Prob(F) Treatment Prob(F) a Seed pieces were treated with the seed treatment 30 min after inoculation. All rates are manufacturers recommended rate. b Seed pieces were inoculated with Fusarium sambucinum = + ; seed pieces were not inoculated = ; seed pieces were inoculated immediately after cutting 14 days prior to planting. c Storage types were: A, stored for 10 DBP under optimal conditions (12 18 C at 95% RH with forced air ventilation at 5950 l min -1 ); B, stored for 10 DBP under poor conditions (25 C in dark with no ventilation); C, treated and inoculated on the day of planting. d Incidence of seed piece decay was calculated as the mean number of seed pieces showing symptoms of Fusarium dry rot. e Seed piece decay was calculated as the percentage volume of decay per tuber. f Numbers followed by the same letter within a column are not significantly different at P = 0.05 (Tukey multiple comparison method).

26 Table 4 Wharton and Kirk 25 Source P value a RAUEPC (%) Final Plant Stand (%) Treatment < < Inoculation Storage < < < < Treatment Inoculation Treatment storage Inoculation storage a Significance indicated by P 0.05.

27 Table 5 Wharton and Kirk 26 Source RAUEPC (%) a Final Plant Stand (%) b Treatment Non-treated control (UTC) 11.8 b c 5.8 b 71.0 b 63.9 b Bacillus subtilis 11.6 b 6.9 a 70.1 b 76.0 a Trichoderma harzianum 13.2 ab 6.8 a 79.7 ab 73.6 a Fludioxonil + mancozeb 14.8 a 7.5 a 88.1 a 78.6 a Inoculation no 13.8 a 6.8 a 82.0 a 74.0 a yes 11.9 b 6.6 a 72.4 b 72.0 a Storage Type d A 15.2 a 7.4 a 89.1 a 79.7 a B 13.5 b 7.7 a 79.4 b 80.5 a C 9.8 c 5.0 b 63.2 c 58.9 b Treatment x inoculation UTC,no 12.2 bcd 5.8 b 73.1 abc 65.0 ab UTC,yes 11.3 cd 5.7 b 68.9 bc 62.8 b Bacillus subtilis,no 14.6 ab 7.0 ab 76.7 abc 78.1 ab Bacillus subtilis,yes 11.8 bcd 6.7 ab 63.6 c 73.9 ab Trichoderma harzianum,no 12.7 abcd 7.0 ab 86.4 ab 76.4 ab Trichoderma harzianum,yes 10.4 d 6.6 ab 73.1 abc 70.8 ab Fludioxonil + mancozeb,no 15.5 a 7.4 a 91.9 a 76.7 ab Fludioxonil + mancozeb,yes 14.1 abc 7.5 a 84.2 ab 80.6 a Treatment x Storage UTC,A 14.9 abc 6.8 bcd 86.7 ab 75.0 abc UTC,B 13.1 abc 7.1 abcd 77.9 abc 76.3 abc UTC,C 7.3 e 3.4 f 48.3 d 40.4 d Bacillus subtilis,a 14.0 abc 7.3 abcd 83.3 abc 82.1 ab Bacillus subtilis,b 11.9 bc 7.6 abc 68.8 bcd 79.2 ab Bacillus subtilis,c 8.7 de 5.6 de 58.3 cd 66.7 bc Trichoderma harzianum,a 15.2 ab 8.2 ab 90.0 ab 85.0 ab Trichoderma harzianum,b 13.5 abc 7.4 abcd 80.4 abc 77.9 abc Trichoderma harzianum,c 10.9 cde 4.7 ef 68.8 bcd 57.9 cd Fludioxonil + mancozeb,a 16.6 a 7.4 abcd 96.3 a 76.7 abc Fludioxonil + mancozeb,b 15.4 ab 8.9 a 90.4 ab 88.8 a Fludioxonil + mancozeb,c 12.4 abcd 6.1 cde 77.5 abc 70.4 abc Inoculation x storage no,a 15.6 a 7.2 a 90.6 a 78.3 a no,b 14.9 a 8.1 a 87.5 a 84.6 a

28 Wharton and Kirk 27 no,c 10.8 bc 5.1 b 67.9 b 59.2 b yes,a 14.8 a 7.6 a 87.5 a 81.0 a yes,b 12.1 b 7.4 a 71.3 b 76.5 a yes,c 8.9 c 4.8 b 58.5 b 58.5 b a Incidence of seed piece decay was calculated as the mean number of seed pieces showing symptoms of Fusarium dry rot. b Seed piece decay was calculated as the percentage volume of decay per tuber. c Numbers followed by the same letter within a column are not significantly different at P = 0.05 (Tukey multiple comparison method). d Storage types were: A, stored for 10 DBP under optimal conditions (12 18 C at 95% RH with forced air ventilation at 5950 l min -1 ); B, stored for 10 DBP under poor conditions (25 C in dark with no ventilation); C, treated and inoculated on the day of planting.

29 Table 6 Wharton and Kirk 28 Fungicide treatment Storage RAUEPC (%) d Final plant stand (%) e and rate/100kg seed a Inoculation b type c None A 15.5 a f 7.1 a-d f 89.2 abc 78.3 abc B 14.7 a-d 7.4 abc 85.8 abc 82.5 ab C 6.5 g 2.9 f 44.2 d 34.2 d + A 14.4 a-d 6.4 a-e 84.2 abc 71.7 abc + B 11.5 a-g 6.8 a-d 70.0 a-d 70.0 abc + C 8.2 efg 3.9 ef 52.5 cd 46.7 cd Bacillus subtilis A 14.7 a-d 7.0 a-d 86.7 a-d 82.5 ab (250g) B 14.8 abc 7.7 abc 85.0 a-d 80.0 ab C 8.6 d-g 6.2 a-e 58.3 b-e 71.7 abc + A 13.4 a-f 7.6 abc 80.0 a-e 81.7 ab + B 9.1 b-g 7.5 abc 52.5 cde 78.3 abc + C 8.8 c-g 5.0 c-f 58.3 b-e 61.7 a-d Trichoderma harzianum A 15.1 ab 7.7 abc 88.3 a-d 81.7 ab (125g) B 14.4 a-d 8.1 ab 85.0 a-d 85.8 ab C 14.3 a-e 5.1 c-f 85.8 a-d 61.7 a-d + A 15.4 a 8.7 ab 91.7 ab 88.3 a + B 12.6 a-f 6.7 a-e 75.8 a-e 70.0 abc + C 7.5 fg 4.4 def 51.7 de 54.2 bcd Fludioxonil + mancozeb A 17.0 a 6.9 a-d 98.3 a 70.8 abc (500g) B 15.8 a 9.0 a 94.2 ab 90.0 a C 13.7 a-e 6.4 a-e 83.3 a-d 69.2 abc + A 16.1 a 7.9 abc 94.2 ab 82.5 ab + B 15.0 ab 8.7 ab 86.7 a-d 87.5 a + C 11.2 a-g 5.9 b-e 71.7 a-e 71.7 abc Tukey s HSD (P=0.05) Replicate Prob(F) Treatment Prob(F) a Seed pieces were treated with the seed treatment 30 min after inoculation. All rates are manufacturers recommended rate. b Seed pieces were inoculated with Fusarium sambucinum = + ; seed pieces were not inoculated = ; seed pieces were inoculated immediately after cutting 14 days prior to planting. c Storage types were: A, stored for 10 DBP under optimal conditions (12-18 C at 95% RH with forced air ventilation at 5950 l min -1 ); B, stored for 10 DBP under poor conditions (25 C in dark with no ventilation); C, treated and inoculated on the day of planting. d RAUEPC, relative area under the plant emergence progress curve, calculated from the day of planting to full emergence 28 days after planting (max = 100). e Final plant stand, was expressed as the percentage of emerged plants divided by the expected number based on the planting rate. f Numbers followed by the same letter within a column are not significantly different at P = 0.05 (Tukey multiple comparison method).

30 Wharton and Kirk 29

31 Wharton and Kirk 30 Biocontrol agents provide good control of seed piece decay under optimal conditions. Biocontrol agents provide good control of sprout rot under optimal conditions. Biocontrol agents provided no disease control under poor storage conditions.