Final report for GS19-200
Rhizopus soft rot (RSR), caused by the wound-dependent fungus Rhizopus stolonifer is among the most devastating postharvest diseases of sweetpotato (Ipomoea batatas). Roots are normally cured following harvest to heal wounds, thereby minimizing the risk of RSR infection in storage. During packing however, sweetpotato roots are treated with the fungicide dicloran (Botran®). Increasing concerns about pesticide residue on the sweetpotato roots poses the need for alternative ways of managing RSR. Even though the potential of using biological control or re-curing roots has been demonstrated by a few authors, there is a need to research further to determine the optimal application and determine if combining these methods would improve the level of control. The objective of this two-year project was, therefore, to evaluate the effectiveness of integrating improved resistance to RSR with the biological control product, P. syringae strain ESC-10 (Bio-Save 10LP), applied either as an overhead spray or as a dip to sweetpotato roots; and re-curing sweetpotato roots on the incidence of RSR in comparison to the standard dicloran. These practices were evaluated individually and in combination. Five predominant commercial cultivars Beauregard, Bayou Belle, Bellevue, Orleans, and Covington were grown according to standard practices. At 120 and 145 days in year 1 or 130 and 150 days after harvest in year 2, sweetpotato roots were artificially wounded, inoculated, subjected to the treatments, and evaluated for RSR incidence up to 14 days. Analysis of the response variable percent RSR revealed significant interactions, as well as significant differences between treatments, cultivars, and sampling times in each year. The results suggested the need for cultivar and time-specific recommendations, and the need to consider all possible variables in the development of RSR management programs.
- To evaluate the effectiveness of the biological control product, Pseudomonas syringae strain ESC-10 (Bio-Save 10LP) on the roots of five sweetpotato cultivars with varying resistance at the recommended label rate and applied either as a spray or as a dip.
- Assess the effects of re-curing roots for 24 hours after wounding, five sweetpotato cultivars with varying resistance, inflicted with one wound type (bruise) on the incidence of RSR.
- Assess the combined effects of the biological control product Bio-Save 10LP, applied to sweetpotato roots of varying RSR resistance as a dip and spray, followed by re-curing 24 hours on the incidence of RSR.
Year 1 (2019/2020)
Five cultivars with different levels of resistance to RSR were selected, based on predominance among sweetpotato growers and/or packers. These included Beauregard, Bayou Belle, Bellevue, Covington, and Orleans Figure 1 . Sweetpotato slips of all 5 cultivars were transplanted into the field on May 29th, 2019 and grown according to standard practices at the Burden Center in Baton Rouge Figure 2. The sweetpotato roots were harvested on September 12th, 2019 Figure 3, after which they were subjected to curing conditions (85oF and >85% RH) for 5 days and then placed into storage (60oF and >85%RH). At 120 and 145 days after harvest, roots of each cultivar were sampled for inoculation and treatment application Table 1. The dicloran was already available in the lab, so was not purchased. The Bio-Save 10 LP was donated by Jet Harvest Solutions, Longwood, FL. The curing room utilized in the study was located at the Burden Center in Baton Rouge, where all experiments were also conducted. Because of low yields and because some roots had to be eliminated because they developed end rots during storage Figure 4, a few modifications were made to the methods to ensure there were at least enough storage roots for the two sampling times. Consequently, only one wound type (bruise) was used Figure 5 with no non-wounded control and at most 10 roots were used per treatment for each cultivar. Additionally, only one re-curing period (24 hours) was used, based on the most effective for managing RSR determined in Sweany et al (2020).
- 120-day inoculations
These were performed between 9th January – 21st January, 2020. Rhizopus stolonifer isolate 92-Rs-2 was grown on Potato Dextrose Agar (PDA) for 6 days prior to inoculations at 28oC to promote sporulation. A day prior to inoculations and treatment application, a total of 1050 roots (10 roots/cultivar * 7 treatments * 3 replications * 5 cultivars) were washed using tap water and air dried. The roots were left in the open in readiness for the next day, which was a mistake as rats fed on several roots which had to be replaced the next day reducing the numbers further for subsequent inoculations. On the day of inoculation, a 6-day-old culture of R. stolonifer Figure 6 was used to prepare a spore suspension Figure 6 for inoculations as described by Edmunds and Holmes (2009). Spores were dislodged from the surface of the culture plate by adding 0.01% Triton-X solution and scraping the surface using a flame-sterilized scalpel. The resulting spore suspension was poured through four layers of sterile cheesecloth in a funnel. The final spore concentration was adjusted to 106 sporangiospores/ml based on hematocytometer counts. A total volume of 877 ml was prepared to inoculate 1050 sweetpotato roots. The sweetpotato roots were wounded with a rubber band impelled wooden dowel also known as wackadero to inflict a bruise wound. The roots were then inoculated by brushing the spore suspension over the wounded surface using a foam paintbrush.
The sweetpotato roots were left to air dry before applying the treatments. While the spore suspension dried out, the mixtures of dicloran Figure 7 and Bio-Save Figure 8 were prepared, by mixing 18 ml in 15 L water and dissolving 67 g in 15 L water, respectively. When the spore suspension had dried out, roots were either placed directly in storage (non-treated controls), submerged for 30s in the dicloran (dicloran dip), submerged for 30s in Bio-Save (Bio-Save dip), placed immediately in the curing room for 24 hours (re-curing), submerged for 30s in Bio-Save and re-cured for 24 hours (Bio-Save dip + re-cure), sprayed with Bio-Save using a backpack sprayer (Bio-Save spray) or sprayed with Bio-Save then re-cured for 24 hours (Bio-Save spray + re-cure). For the spray treatments, roots were placed on baskets and a 2-gal capacity backpack sprayer was used, spraying twice over all the roots by turning the roots to ensure coverage of the entire root Figure 9. All roots except for those that received the re-curing treatment were placed under storage conditions (60oF) immediately following treatment application. All roots that were re-cured were moved to storage conditions after 24 hours in the curing room. All roots were then evaluated for RSR incidence 12 days after inoculation, by counting how many roots out of the total developed soft rot symptoms Figure 10.
- 145-day inoculations
These were performed between 3rd February - 14th February, 2020. This time, roots were washed with tap water three days prior to inoculations and treatment application, then air-dried. Due to limited root availability caused by increasing end rots, the number of roots per cultivar between treatments and replication was reduced, with 10 roots for Covington, Bayou Belle, and Orleans, 9 roots for Beauregard, and 8 roots for Orleans. The root size was also more variable since there were not many roots to choose from to obtain uniform roots. After washing, the roots were placed back in the storage room until inoculation day. A 6-day-old culture of R. stolonifer was used to prepare a spore suspension for inoculations as described above. The final spore concentration was adjusted to 106 sporangiospores/ml based on hematocytometer counts. A total volume of 1260 ml was prepared to inoculate 987 sweetpotato roots. Wounding, inoculation, and application of treatments were performed as described above, except that the Bio-Save spray treatment was modified to improve the coverage on roots. Four rounds of spray were applied, two on each side of the root, and all the mix in the 2-gal backpack sprayer was used. All roots were incubated in storage conditions (60o F) and evaluated for RSR incidence at 11 days after inoculation, by counting how many roots out of the total had soft rot symptoms.
Year 2 (2020/2021)
The same five cultivars used in year 1 were grown according to standard practices at the Sweet Potato Research Center in Chase, LA in 2020. The sweetpotato roots were harvested and subjected to recommended curing conditions (85oF and >85% RH) and then placed in storage (60oF and >85%RH). The roots were transported to the Burden Center in Baton Rouge on December 14, 2020 and placed in storage until ready for use. At 130 days (22nd January and 5th February, 2021) and 150 days (14th February – 26th February, 2021) after harvest, roots were taken out of storage for inoculation and treatment application. At each sampling time, roots of each cultivar were inoculated with an R. stolonifer spore suspension prepared from a 7-d old R. stolonifer culture and randomly assigned to the same treatments described for year 1. The source of dicloran and Bio-Save was the same as described for year 1. The only difference between the two years is that for the first inoculation at 130 days, two wound types (scrape and bruise) were used Figure 11, while at the second inoculation at 150 days only one wound type (bruise) was used due to reduced root numbers for all cultivars. As in the first year, at both sampling times, no non-wounded control was used due to insufficient root numbers, and only one re-curing period (24 hours) was used, based on the most effective for managing RSR determined in Sweany et al (2020).
Experimental design and statistical analysis
The roots were arranged on baskets in a completely randomized design with 3 replications. The total number of sweetpotato roots with RSR was recorded at 12 days and 11 days, for the 120- and 145-day sampling, respectively in year 1 and 14 days for both sampling times in year 2. The root counts were converted to percent, by dividing the roots with RSR by the total roots inoculated. The percent RSR was subjected to ANOVA using the PROC GLM procedure in SAS 9.4 (SAS Institute, Cary, NC). Mean separation was based on Fisher’s least significant differences (LSD) at a critical level of P < 0.05.
Edmunds, B.A. and Holmes, G.J., 2009. Evaluation of alternative decay control products for control of postharvest Rhizopus soft rot of sweetpotatoes. Plant Health Progress, 10, p.26.
Sweany, R.R., Picha, D.H. and Clark, C.A., 2020. Hot‐water baths, biologicals and re‐curing effects on Rhizopus soft rot during sweetpotato packing. Plant Pathology, 69, pp.284-293.
The analysis in both years was based on RSR observed on roots wounded with the wackadero (bruise), because that was the only wound type used in year 1 and for the second year no RSR developed on all roots wounded with the potato peeler (scrape) except for one root which was treated as an outlier. Significant differences in the overall RSR incidence were observed between years Figure 12, with the RSR incidence in year 2 being 2x more that in year 1. Differences up to 2.5x were also observed between sampling times within each year Figure 13, with significantly greater RSR incidence at the first sampling time compared to the second. Additionally, significant two-way and three-way interactions among all the variables (year, time, treatment, and cultivar) were observed demonstrating the complexity that can exist and why efforts to develop RSR occurrence predictor models have not been successful to date. Differences between years and locations have been also observed by previous researchers. Edmunds et al (2015) demonstrated that various preharvest factors (soil, weather) can vary by location, and consequently influence the susceptibility of sweetpotato roots to RSR. The differences between sampling times in each year could be attributed to the storage duration which has been shown to influence a roots susceptibility to RSR (Holmes and Stange, 2002).
Analysis was then performed by year and highly significant three-way (time*treatment*cultivar) and two-way (time*treatment, time*cultivar, and treatment*cultivar) interactions were observed in the 1st Table 2 and 2nd year Table 3, respectively. Further analysis by time within each year revealed significant differences among cultivars at each sampling time in both year 1 Figure 14 and year 2 Figure 15. In year 1 Figure 14, all cultivars had between 1.9-4x greater RSR incidence at the first sampling time (120 days) compared to the second sampling time (145 days). The cultivar Bellevue recorded the highest RSR incidence at both sampling times, while Bayou Belle had the least at both sampling times, with the other cultivars in between. In the second year Figure 15, the cultivars behaved differently than they did in the first year, with a significantly greater RSR disease pressure at the first sampling time (up to 74%). As in the first year however, the RSR incidence was lower at the second sampling time (up to 49%). The cultivar Covington consistently had the lowest RSR incidence at both sampling times, while the highest RSR incidence was recorded for Bellevue and Beauregard, at the first and second sampling time, respectively.
Lastly, analysis by time within each year also revealed significant differences among treatments in year 1 Figure 16 and year 2 Figure 17. In the first year Figure 16, the most effective treatments at both sampling times included the synthetic fungicide dicloran, the Bio-Save applied as a dip and the Bio-Save applied as a dip combined with recuring. These three treatments resulted in up to 80% and 98% decrease in RSR incidence, at the first and second sampling, respectively, compared to the non-treated control which was among the treatments with the highest disease pressure. The overall lower disease pressure in the first year may have contributed to the good control efficacy provided by the Bio-Save. The recuring unfortunately was among the treatments with higher RSR incidence, but it is not being ruled out as a management strategy given that the heating unit used in this project was not always consistent thus temperatures on several occasions went below 85oF (curing temperature). Bio-Save was therefore the only treatment comparable to dicloran in terms of control efficacy in the first year. In the second year Figure 17, the disease pressure was significantly higher (up to 89%), especially at the first sampling time and so the only effective treatment in reducing the RSR incidence was the synthetic fungicide dicloran (99% decrease compared to the non-treated control). Interestingly, all the treatments except for the dicloran had greater RSR incidence (74-89%) than the non-treated control (67%). At the second sampling, however, the decline in RSR incidence, contributed to the better control efficacy provided by the Bio-Save applied as a dip (68% decrease compared to non-treated) or a spray (43% decrease). The dicloran still consistently provided the most effective control resulting in an 83% reduction in RSR compared to the non-treated control. The treatments recuring, Bio-Save applied either as a spray or dip in combination with recuring had the highest RSR incidence, even greater than the non-treated control. The Bio-Save was therefore the only treatment that provided comparable RSR control to the dicloran at the second sampling time when the disease pressure was lower indicating its potential to be included in RSR management programs.
Edmunds, B.A., Clark, C.A., Villordon, A.Q. and Holmes, G.J., 2015. Relationships of preharvest weather conditions and soil factors to susceptibility of sweetpotato to postharvest decay caused by Rhizopus stolonifer and Dickeya dadantii. Plant Disease, 99(6), pp.848-857.
Holmes, G.J. and Stange, R.R., 2002. Influence of wound type and storage duration on susceptibility of sweetpotatoes to Rhizopus soft rot. Plant disease, 86, pp.345-348.
Educational & Outreach Activities
Even though there were limitations to conduct in-person outreach events due to COVID, a few efforts were successful. Firstly, the first-year results were shared during an oral presentation given during the department seminar series in Fall 2020, with the audience being graduate students, research associates and faculty in the department. Another oral presentation entitled ‘Evaluating Options for the Control of Rhizopus Soft Rot on Sweetpotato’ was then given at the 98th American Phytopathological Society Southern Division 2021 virtual meeting. Since this was a scientific meeting, the audience was primarily comprised of graduate students, faculty, and industry representatives. The results obtained from both years and other similar research was also shared as a video during the 2021 LSU AgCenter virtual sweetpotato field day in August (https://www.youtube.com/watch?v=gz-APqsxIWQ). The virtual sweetpotato field day targets various sweetpotato stakeholders including growers and packers who would benefit greatly from the research. Additionally, a recorded oral presentation entitled ‘Investigating the Use of Biologicals and Re-curing for the Management of Rhizopus Soft Rot on Sweetpotato Roots’ was given at the National Sweetpotato Collaborators group meeting that took place from February 10-12, 2022, in New Orleans, LA. Finally, the results will eventually be published in the Horticulture journal for the scientific community and will comprise a chapter of my dissertation.
Based on the results obtained from both years, the biological product Bio-Save provided good control when the disease pressure was the least and seemed to be influenced by how long the roots had been stored, the treatment combination, and the cultivars suggesting the need for cultivar and time-specific control options. The differences and interactions between years, cultivars, and sampling times within each year demonstrate the variability that can exist in susceptibility and also suggests the need to screen cultivars for RSR at various times in storage rather than one time point as is usually the case. Overall, applied as a dip, the Bio-Save was more effective and, in some cases, comparable to the dicloran thus could potentially be used by growers/packers to manage RSR. The re-curing treatment both alone and in combination with Bio-Save was not effective for all cultivars in both years, thus cannot be recommended at this point but needs further investigation. Having another alternative comparable to dicloran would result in reduced fungicide use, improved environmental stewardship, and better quality of produce to meet the needs of various markets.
It has both been a learning experience as well an eye-opener to some research aspects that could be looked at in the future. During the project, one thing that came out was the fact that many other variables (root source, storage duration, disease pressure) could be overlooked which can influence the control efficacy of the various treatments. One important factor is the disease pressure which should probably be considered when comparing conventional fungicides with biologicals in the future, as that determines how effective they are as seen in the project results. Overall, a holistic approach when developing management programs for RSR would be valuable if feasible.
One recommendation based on observation was that the susceptibility of sweetpotato roots to RSR can be quite sensitive to small temperature differences, thus it is worth exploring re-curing as a management strategy further. Based on the experiences during the entire project, another recommendation would be to develop RSR management recommendations specific to the time in storage and cultivar because different responses were observed. It would also be good to test the control products used in this study at an actual packing facility before making recommendations to growers and packers to see what the control efficacy would be like in routine packing operations. Additionally, it would be good to perform a cost-benefits analysis using Bio-Save which showed the most potential in comparison to the synthetic fungicide dicloran. Based on the variability observed related to root source it would be good to use roots from various locations representing various growing conditions so that recommendations would be specific to certain areas. Lastly, collaborating with sweetpotato growers in the future to evaluate the different control treatments would be a good strategy to have increased root numbers for evaluations since this was one limitation in both years.