Biological Control and Re-curing of Sweet Potato Roots as Alternatives for Managing Rhizopus Soft Rot

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 29^th, 2019 and grown according to standard practices at the Burden Center in Baton Rouge Figure 2. The sweetpotato roots were harvested on September 12^th, 2019 Figure 3, after which they were subjected to curing conditions (85^oF and >85% RH) for 5 days and then placed into storage (60^oF 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 9^th January – 21^st January, 2020. Rhizopus stolonifer isolate 92-Rs-2 was grown on Potato Dextrose Agar (PDA) for 6 days prior to inoculations at 28^oC 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 (60^oF) 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 3^rd February - 14^th 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 (60^o 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 (85^oF and >85% RH) and then placed in storage (60^oF 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 (22^nd January and 5^th February, 2021) and 150 days (14^th February – 26^th 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.

Literature cited

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.