Two greenhouses for strawberry cultivation and a packing area was located in Jinju, South Korea. These greenhouses were 10 × 88 meters and had 6 high-bed cultivation lines system. Tests were carried out in three lines and spores were collected from each line at three locations, one at the exit, one at the center and one at the farthest point from the exit. Also, in order to investigate the differences between spores’ density according to the micro-meteorological conditions, experiments were carried out in two greenhouses, named greenhouse A (GA) and greenhouse B (GB). GA and GB planted cultivar Maehyang with a high-bed cultivation system and the general management for cultivation was the same, except GA had installed customized machinery to control temperature and humidity. The export strawberry packing facility was located in the same area as the greenhouses. The packing facility was 30 × 40 m in size and had 6 screening and packing lanes.

Collection of spores and survey of the gray mold disease incidence were conducted from November 2014 to May 2015 and from November 2015 to May 2016 every two weeks. Three air spore samplers (UCK Bio-Culture^TMUPump, USA) were used to place in the strawberry greenhouses or the packing facility. B. cinerea spore selective media, Botrytis Spore Trap Medium (BSTM; glucose 2 g, NaNO₃ 0.1 g, K₂HPO₂ 0.1 g, MgSO₄7H₂O 0.2 g, chlorampenicol 0.2 g, pentachloronotrobenzene 0.02 g, Maneb 80 0.02 g, Rubigan 0.1 ml, tannic acid 5 g, agar 20 g per 1 L) (Kim et al., 2015), inside the sampler. Then the sampler was activated to suck air for 2 min. The media’s collection of airborne spores was done in triplicate. When this finished the inhaled air, the BSTM plates were sealed with parafilm and incubated at 28^oC for 7 days, and the number of colonies on the fungus was measured and the value was calculated in Colony Forming Units (CFU Log / L of air).

The strawberry greenhouse and the survey period were the same as described above and a study on disease incidence was conducted in both GA and GB (Fig. 1). Disease incidence was investigated by randomly selecting 100 strawberries from each greenhouse at the investigation date every year and the experiment was conducted in triplicate (Kim et al., 2015).

Fig. 1. 
Gray mold disease symptoms of strawberries.

(A: Crown, B: Fruit, C: Primary inoculum on strawberry fruit in packing place)

Strawberries that were packed at the distribution center in Sugok-myeon, Jinju-si, Gyeongsangnam-do were brought into the laboratory and stored at low temperature (4^oC) and room temperature (21^oC) for 10 days, respectively. After 10 days of storage, the number of strawberries in each package was counted. Then, the number of strawberries with gray mold was counted and the number of diseased fruits in the total number of fruits was converted into percentage. The treats at low temperature (4^oC) and at room temperature (21^oC) were packed with 12 fruits respectively and they were examined by a total of 8 repeats.

Multiple regression analysis was performed using forward and stepwise methods and the differences in the environmental factors leading to gray mold disease and spore scattering in the strawberry cultivation area were analyzed using total of 10 environmental factors [soil surface-humidity, electrical conductivity (EC) and temperature, inside the soil-humidity, EC and temperature, atmosphere-temperature, humidity, solar and CO₂ concentration]. In addition, the differences in environmental factors’ impact on spore scattering in the packing area were investigated using three environmental factors-temperature, humidity and CO₂. All environmental factors were automatically measured every 5 min by customized sensors (Elsys Co., Korea). Based on this, the micro-meteorological factors significantly influencing the incidence of gray mold disease and spore scattering were analyzed through principal component analysis.

Statistical analysis was done using ANOVA analysis (Tukey’s HSD) according to the complete randomization method for each treatment and graphs were generated using the Sigma Plot (Systat Software, Inc., Chicago, IL, USA) program.

The results of the first-year experiment showed that the density of B. cinerea spores in GA ranged from 105 to 106 (Log CFU/L) from December 3, 2014 to January 10, 2015 and it was investigated from 10⁴ to 10⁶ on the next date of collection. The density result of B. cinerea spores in GB showed that spore density was 10⁵ to 10⁶ from December 3, 2014 to January 15, 2014 and it was investigated from 10⁴ to 10⁵ on the next date of collection. While the spore scattering period in both greenhouses lasted from December to January, the increase in gray mold began in late February. The reason for this was due to time differences. The results of the second year experiment showed that the B. cinerea spore density of GA ranged from 102 to 103 from November 4, 2015 to February 11, 2016 and it was investigated from 10⁴ to 10⁶ from March 9, 2015 to May 6, 2016. The spore density of GB ranged from 10² to 10³ from November 4, 2015 to February 11, 2016 and it was investigated from 10⁴ to 10⁶ from March 9, 2015 to May 6, 2016. (Fig. 2). Spore scatterings in both greenhouses had increased since February 2016 and the incidence of gray mold disease rapidly increased from early March in both greenhouses.

Fig. 2. 
Floating spore densities of gray mold pathogen in strawberry cultivation fields on BSTM (A,B,C) media. BSTM media were incubated at 28^oC for 7 days. Average floating spore densities during survey periods (D, E, F, G). D: 2014~2015 Greenhouse A (no dehumidifying heater installed), E: 2014~2015 Greenhouse B (dehumidifying heater installed), F: 2015~2016 Greenhouse A (no dehumidifying heater installed), G: 2015~2016 Greenhouse B (dehumidifying heater installed). Statistical analysis With Tukey’s HSD (p=0.05), vertical bar indicates relative standard errors.

The first-year experiment showed that the density of B. cinerea spores in the sorting and packing place was investigated from 10⁵ to 10⁶ in spore density from December 3, 2014 to January 15, 2015 and it was investigated from 10⁴ to 10⁵ from January 28, 2015 to February 24, 2015. Then, from March 25 to May 6, it was investigated from 10⁴~10⁵. The results of the second-year experiment showed that the spore density in the packing place ranged from 10¹ to 10² from November 4, 2015 to February 24, 2016 and it was investigated from 10³ to 10⁴ from March 9, 2016 to May 6, 2016 (Fig. 3).

Fig. 3. 
Floating spore densities of gray mold pathogen in sorting and packing workplace on BSTM media. A: 2014~15 sorting and packing workplace, B: 2015~2016 at packing place. Statistical analysis With Tukey’s HSD (p=0.05), vertical bar indicates relative standard errors.

The results of the first-year experiment showed that the incidence of gray mold disease in GA ranged from 6% to 7% between December 3, 2014 and January 28, 2015. The incidence of disease rose to 25% per the result on February 11, 2015 but then fell again until late March. Since March 25, 2015, the rate of disease incidence increased sharply to 86%. It emerged that GB recorded disease incidence of less than 10% from December 3, 2014 to March 11, 2015, but it increased from March 25, 2015 and reached 70%. GB had 16% less disease than GA. In the second year of the experiment, gray mold disease in GA was 0 to 14% from November 4, 2015 to February 24, 2016 but results for March 9, 2016, reported that the incidence of disease increased sharply to 38%, finally reaching approximately 70%. The investigation’s result for the incidence of gray mold disease in GB reported 0 to 11% from November 4, 2015 to February 24, 2016, while the disease rapidly increased to 37% on March 9, 2016. Then the incidence of the disease increased by approximately 70%. The incidence of gray mold disease began to increase rapidly from early March 2016 in both greenhouses. Although from early February to early March in 2016, the control of gray mold was actively carried out using a chemical strategy, gray mold disease continued to increase from then on (Fig. 4).

Fig. 4. 
Gray mold disease incidence on strawberries. A: 2014~2015 Greenhouse A (no dehumidifying heater installed), B :2014~2015 Greenhouse B (dehumidifying heater installed), C: 2015~2016 Greenhouse A (no dehumidifying heater installed), D: 2015~2016 Greenhouse B (dehumidifying heater installed). Statistical analysis with Tukey’s HSD (p=0.05), vertical bar indicates relative standard errors.

Referring to the first-year experiment’s results, the storage temperature experiment was conducted at low temperature and room temperature for packaged strawberries in the laboratory. The percentage of disease incidence of fruit stored at room temperature was 69-100%, and 0-17% at low temperature (Fig. 5A). The result of the second-year experiment indicated that the disease occurred 100% at room temperature after January 2016 and the incidence of disease was less than 10% at low temperature (Fig. 5B). Thus, the disease incidence rate of gray mold was remarkably small at low temperature storage but higher at room temperature storage.

Fig. 5. 
Gray mold disease incidence based on storage temperature conditions. Packed strawberries were stored at low temperature (4^oC) for 10 days (A, B, C). The packages were stored at room temperature for 10 days (D, E, F). Decay ratio of gray mold disease in stored strawberries for both temperature conditions (G: 2014~15, H: 2015~16).

In GA, temperature and humidity were identified as independent variables that have a significant influence on the amount of B. cinerea spores. Furthermore, the independent variables affecting disease incidence also appear to be humidity and temperature (Table 1). In GB, temperature and humidity were identified as being independent variables that wield a significant influence on the amount of B. cinerea spores. Here the independent variables affecting disease incidence also appear to be humidity and temperature (Table 2). Then, since the multiple regression analysis showed that the significance is below 0.05, this result was considered to be important. Relative humidity in the packing facility was the most affecting environmental factor in the spore disperse. However, the result was not consistent with statistical analysis in both years.

While the two greenhouses had different moisture conditions, temperature and relative humidity showed a similar correlation with spore scattering and disease incidence. If dehumidification is not available, the correlation between quantity of light and CO₂ will be reversed, but it was confirmed that no complete correlation was established when dehumidification was not available. If the micro-meteorological environment was not well controlled by a dehumidifier, only temperature was grouped into the same location among nine independent and it was confirmed that the incidence of gray mold and spore scattering were affected by temperature in GA. If the micro-meteorological environment was well controlled by a dehumidifier, only temperature was grouped into the same location among nine independent variables and it was confirmed that the incidence of gray mold and spore scattering was affected by temperature in GB. It was confirmed that interference by temperature occurred in both greenhouses (Fig. 6). In the case of the packing workplace, there was some overlap between temperature and humidity. Overall, the fact that temperature and humidity were highly involved in the microenvironment and greatly influenced the incidence of gray mold disease and spore scattering was confirmed by the PCA results where multiple regression analysis and micro-meteorological data were used to evaluate the impact of humidity.

Fig. 6. 
Principle analysis between diseases and environmental factors in strawberry cultivation. A: Greenhouse A (no dehumidifying heater installed), B: Greenhouse B (dehumidifying heater installed), C: Sorting and packing workplace.