# The Colonization of Grape Bunch Trash by Microorganisms for the Biocontrol of Botrytis cinerea as Influenced by Temperature and Humidity

^{1}

^{2}

^{*}

## Abstract

**:**

^{2}> 0.87, correctly predict BCA growth under field conditions, they would help farmers select the BCA to be used for a specific application based on weather conditions at the time of treatment and in the following days. The equations would also help predict how long an early season BCA application remains effective and thereby help farmers decide whether and when a second BCA application may be needed.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Plant Material

#### 2.2. Treatment of Bunch Trash with BCAs

#### 2.3. Assessment of Colony Forming Units (CFUs)

#### 2.4. Data Analysis

^{8}, which occurred at 25 °C, 100% RH, and with 3 days of incubation; the average number of CFUs per day was 1.12 × 10

^{8}(i.e., 3.35 × 10

^{8}/3). At 15 °C and 100% RH, in contrast, the number of CFUs was the highest (2.75 × 10

^{7}) with 13 days of incubation, and the average number of CFUs per day was 2.81 × 10

^{6}(i.e., 2.75 × 10

^{7}/13). Rescaled values for the two cases were 1.000 (1.12 × 10

^{8}/1.12 × 10

^{8}) and 0.025 (2.81 × 10

^{6}/1.12 × 10

^{8}), respectively.

_{eq}= -(VPD - VPDmin) * 10, where VPD is as previously described and VPDmin is equal to 1.584; and E’ is calculated as follows:

^{b}(1-Teq)]

^{c}, accounts for the effect of temperature according to the bell-shaped curve of Analytis [35], with parameters a, b, and c defining the top, symmetry, and size of the curve, respectively. The second term of Equation (1), [1-d(1-e

^{VPD})], accounts for the combined effect of T and RH (as VPD) on Y (the rescaled number of CFUs) according to an asymptotic equation, where 1 is the maximum attainable value for Y, d is the value for Y at VPD = 0, and e is proportional to the relative rate of decrease for Y when VPD increases. The second term of Equation (3), {E {exp[(VPD

_{eq}-f)g/(h+1)}/{1 + exp[(VPD-f)g]}}, accounts for the effect of T and RH (as VPD) on Y according to a Weibull equation [36]. The equation defines a unimodal curve in which the response declines from 1, which is the maximum attainable value for Y, and approaches a lower limit of 0 as VPD increases or decreases from the optimum. The intrinsic rate of decline and degree of asymmetry in VPD response are described by the parameters g and h, respectively.

_{0}is the initial population (i.e., the population at time 0), t

_{cg}and t

_{cd}represent the characteristic times of the growth and the decay, respectively, had they been unimpeded; m

_{1}and m

_{2}are coefficients that account for the steepness of the curve, respectively.

_{cg}, t

_{cd}, m

_{1}, and m

_{2}in Equation (6). The parameterized equations were evaluated for goodness-of-fit based on the adjusted R

^{2}, the root mean square error (RMSE), the coefficient of residual mass (CRM), and the concordance correlation coefficient (CCC) [41,42]. The adjusted R

^{2}was estimated by conducting a linear regression between the observed values and the model predicted values; the linear regression was conducted with the lm function of the R “stats” package [40]. The RMSE was obtained using the rmse function of the R “modelr” package [43]. The CCC was obtained using the CCC function of the R “DescTools” package [44]. In brief, RMSE represents the average distance of real data from the fitted line, and CRM is a measure of the tendency of the equation to overestimate or underestimate the observed values (a negative CRM indicates a tendency of the model toward overestimation) [42]. CCC is the product of two terms: the Pearson correlation coefficient and the coefficient Cb, which indicates the difference between the best fitting line and the perfect agreement line (CCC = 1 means perfect agreement) [45].

## 3. Results

^{2}> 0.89, RMSE ≤ 0.105, CRM ≤ 0.069, and CCC > 0.945 (Table 2). Similarly, Equation (3) provided a good fit for the relative colonization data for AMY, NOL, and VIN, with R

^{2}> 0.92, RMSE ≤ 0.09, CRM ≤ 0.127, and CCC > 0.902 (Table 2). This indicated that solving Equation (1) or Equation (3) for any combination of T (between 15 and 35 °C) and RH (between 60 and 100%) provided a reliable prediction of the relative colonization of bunch trash by the BCAs. The contour plots of the relative colonization of bunch trash by the six BCAs are shown in Figure 4; the relative colonization values were obtained by solving Equation (1) or Equation (3) with the parameters of Table 2. The relative colonization values used for Figure 4 were grouped into five categories to clarify the different responses of the BCAs to T/RH, from low to high colonization, as follows: low, 0 to 0.2 relative colonization; medium-low, 0.2 to 0.4; medium, 0.4 to 0.6; medium-high, 0.6 to 0.8; and high: 0.8 to 1. For TAE, high relative colonization required a high level of relative humidity (Figure 4E). BOT (Figure 4B) and NOL (Figure 4C) were able to colonize the bunch trash at lower levels of RH than the other three BCAs. Relative colonization was highest at 90 < RH < 95% for AMY (Figure 4A) and VIN (Figure 4F), and at 80 < RH < 95% for NOL (Figure 4C). Both SER (Figure 4D) and VIN (Figure 4F) were able to colonize bunch trash at a wider range of temperature than the other BCAs.

^{2}> 0.87, RMSE < 0.84, CRM < 0.07, and CCC > 0.86 (Table 3). This indicates that solving Equation (5) for any temperature (between 15 and 35 °C) and RH = 100% provides a reliable prediction of the time required for the maximal colonization of each BCA on bunch trash. In Figure 5, Equation (5) and the parameters listed in Table 3 were used to calculate the number of days required by each BCA to reach the maximal colonization at the different temperatures, with RH = 100%. Overall, the time required for the maximal colonization was shorter at optimal temperatures, meaning that the length of the colonization period was temperature-dependent. Some BCAs, like AMY and TAE (Figure 5A,E), required fewer than 5 days at 35 °C and more than 13 days at temperatures <20 °C. NOL (Figure 5C) required fewer than 5 days to reach the maximal colonization across the whole temperature range (15–35 °C), indicating that the length of the colonization period was less temperature-dependent for this BCA.

^{2}> 0.79, RMSE < 0.154, CRM < 0.277, and CCC > 0.87 (Table 4). This indicated that solving Equation (6) for optimal temperature and RH = 100% provides a reliable prediction of the time required for the growth and the decay of each BCA on bunch trash. In Figure 6, Equation (6) and parameters listed in Table 4 were used to calculate the relative colonization dynamics of bunch trash over time. As was the case for RH and temperature data, these data also indicate that the duration of colonization differed among the BCAs (Figure 6).

## 4. Discussion

## Author Contributions

## Funding

## Conflicts of Interest

## References

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**Figure 1.**Bunch-trash aliquots were arranged in Petri plates (one aliquot per plate) over a wet filter paper and inoculated with the BCA suspension using a micropipette (

**A**). Petri plates were then incubated at different temperature/relative humidity regimes. After 1, 3, 6, 9, and 13 days, the bunch trash was placed in a 15-mL flask containing 10 mL of sterile double-distilled water, shaken by hand and serially diluted from 1:1 to 1:n, where 1:n is the dilution making the colony enumeration possible (

**B**). The number of colony forming units (CFUs) was finally determined by plating 100 µL of each dilution on potato dextrose agar (PDA) plates (

**C**), and expressed as CFUs per g of bunch trash.

**Figure 2.**Coefficient of variation (CV, %) for the numbers of colony forming units (CFUs) of the six BCAs that developed on bunch trash. After it was treated with one of six BCAs, the bunch trash was subjected to different temperature and relative humidity regimes for different BCA colonization periods. Full BCA names and background information are provided in Table 1.

**Figure 3.**Relative colonization on bunch trash by the six BCAs as affected by (

**A**) the length of the colonization period (in days), and by (

**B**) the temperature and (

**C**) relative humidity level that the bunch trash was subjected to after the BCA treatment. Bars are overall means of different T/RH regimes in (

**A**) and of different numbers of days after the BCA treatment in (

**B**) and (

**C**); whiskers show standard error.

**Figure 4.**Relative colonization of bunch trash that was treated with one of six BCAs (

**A**: AMY;

**B**: BOT;

**C**: NOL;

**D**: SER;

**E**: TAE;

**F**: VIN, see Table 1) and that was then incubated at different temperatures (T) and with different relative humidity (RH) levels. The contour plots, which were generated with Equations (1) and (3), and the parameters listed in Table 2, identify five areas of relative colonization of bunch trash by the six BCAs: L (low, 0 to 0.2 relative colonization; the white area); ML (medium-low, 0.2 to 0.4; the light grey area); M (medium, 0.4 to 0.6; the medium grey area); MH (medium-high, 0.6 to 0.8; the dark grey area); and H (high: 0.8 to 1; the black area).

**Figure 5.**Effect of temperature on the length of the BCA colonization period required by the six BCAs (

**A**: AMY;

**B**: BOT;

**C**: NOL;

**D**: SER;

**E**: TAE;

**F**: VIN, see Table 1) to attain their maximal colonization of bunch trash. Bunch trash was treated with the BCAs listed in Table 1 and was then incubated in plates at different temperatures and with different RH levels (values in this figure are for RH = 100%); after 1, 3, 6, 9, and 13 days, the number of CFUs was assessed. Lines were drawn using Equation (5) and the parameters listed in Table 3.

**Figure 6.**Progress of the relative colonization of bunch trash that was treated with one of six BCAs (

**A**: AMY;

**B**: BOT;

**C**: NOL;

**D**: SER;

**E**: TAE;

**F**: VIN, see Table 1) and then incubated at the optimal temperature (AMY: 30 °C, BOT: 25 °C, NOL: 15 °C, SER: 25 °C, TAE: 25 °C, and VIN: 30 °C) and 100% RH; after 1, 3, 6, 9, and 13 days, the number of CFUs was assessed. Lines were drawn using Equation (6) and the parameters listed in Table 4.

Active Ingredient | Commercial Product (Acronym) | Producer | Label Dose (g/ha) |
---|---|---|---|

Bacillus amyloliquefaciens D747 | Amylo-X (AMY) | CBC S.r.l. | 2000 |

Aureobasidium pullulans DMS 14941-14940 | Botector (BOT) | Manica S.p.A. | 400 |

Metschnikowia fructicola | Noli (NOL) | Koppert Italia | 2000 |

Bacillus subtilis QST 713 | Serenade max (SER) | Bayer S.p.A. | 3000 |

Bacillus amyloliquefaciens FZB24 | Taegro (TAE) | Syngenta | 370 |

Trichoderma atroviride SC1 | Vintec (VIN) | Belchim S.p.A. | 1000 |

**Table 2.**Parameters of Equations (1) and (3) for the six BCAs, and statistics for goodness-of-fit to real data.

BCA | Tmin/Tmax ^{1} | Equation Parameters ^{2} | Statistics ^{3} | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|

a | b | c | d | e | f | g | h | R^{2} | RMSE | CRM | CCC | ||

AMY | 5/40 | 2.102 (0.118) | 3.000 (0.348) | 3.000 (0.207) | - | - | 15.40 (0.759) | 1.538 (1.174) | 8.00 (10.513) | 0.918 | 0.090 | −0.037 | 0.961 |

BOT | 5/35 | 2.313 (0.076) | 2.511 (0.176) | 5.000 (1.279) | 0.686 (0.066) | 0.063 (0.043) | - | - | - | 0.892 | 0.105 | 0.069 | 0.945 |

NOL | 0/37 | 3.641 (0.475) | 1.148 (0.217) | 5.643 (5.135) | - | - | 12.00 (4.356) | 1.00 (0.564) | 0.389 (1.206) | 0.956 | 0.071 | 0.127 | 0.902 |

SER | 0/35 | 2.207 (0.070) | 2.712 (0.160) | 1.985 (0.361) | 0.991 (0.042) | 0.096 (0.022) | - | - | - | 0.990 | 0.032 | 0.007 | 0.995 |

TAE | 0/35 | 2.091 (0.069) | 3.126 (0.243) | 6.350 (1.997) | 0.837 (0.084) | 0.0001 (0.0004) | - | - | - | 0.933 | 0.085 | 0.060 | 0.967 |

VIN | 0/35 | 1.750 (0.238) | 54.762 (1.815) | 0.920 (0.704) | - | - | 12.00 (1.293) | 1.00 (0.234) | 0.269 (0.283) | 0.984 | 0.048 | 0.043 | 0.991 |

^{1}Estimates of Tmin and Tmax used for the calculation of equivalents of temperature, Teq, used in Equations (1) and (3);

^{2}estimates of parameters of Equation (1): Y = [a Teq

^{b}(1-Teq)]

^{c}[1 -d(1-e

^{VPD})] and Equation (3): Y = [a Teq

^{b}(1-Teq)]

^{c}{E’{exp[(VPD

_{eq}-f)g/(h+1)}/{1+exp[(VPD-f)g]}};

^{3}adjusted R

^{2}, root mean square error (RMSE), coefficient of residual mass (CRM), concordance correlation coefficient (CCC).

**Table 3.**Parameters of Equation (5) for the six BCAs, and statistics for goodness-of-fit to real data.

BCA | Equation Parameters ^{2} | Statistics ^{3} | ||||||
---|---|---|---|---|---|---|---|---|

m^{1} | Tmin | Topt | Tmax | R^{2} | RMSE | CRM | CCC | |

AMY | 3 | 17.24 (2.95) | 35.00 (24.41) | 40.00 (89.08) | 0.976 | 0.514 | 0.018 | 0.992 |

BOT | 3.8 | 11.80 (1.52) | 27.00 (2.40) | 30.14 (0.56) | 0.973 | 0.468 | −0.018 | 0.989 |

NOL | 2.7 | 11.21 (1.83) | 27.25 (2.21) | 60.00 (61.90) | 0.959 | 0.212 | 0.005 | 0.984 |

SER | 7 | 10.00 (7.99) | 24.75 (6.20) | 33.29 (18.68) | 0.874 | 0.695 | 0.069 | 0.862 |

TAE | 4.5 | 16.09 (9.28) | 35.485 (41.61) | 50.00 (389.91) | 0.907 | 0.844 | −0.033 | 0.963 |

VIN | 9 | 13.83 (2.41) | 27.57 (1.36) | 40.65 (6.98) | 0.999 | 0.012 | −0.014 | 0.993 |

^{1}Estimates of the minimum length of colonization period (in days) required for the maximal colonization of BCA at the optimum temperature (Topt, °C);

^{2}estimates of parameters of Equation (5);

^{3}adjusted R

^{2}, root mean square error (RMSE), coefficient of residual mass (CRM), concordance correlation coefficient (CCC).

**Table 4.**Parameters of Equation (6) for the six BCAs, and statistics for goodness-of-fit to real data.

BCA | Equation Parameters ^{1} | Statistics ^{2} | ||||||
---|---|---|---|---|---|---|---|---|

t_{cg} | t_{cd} | m_{1} | m_{2} | R^{2} | RMSE | CRM | CCC | |

AMY | 0.33 | 3.00 (14.758) | 0.621 (0.832) | 1.436 (2.391) | 0.919 | 0.091 | 0.031 | 0.949 |

BOT | 0.50 | 3.573 (630.049) | 1.196 (31.143) | 5.00 (1482.421) | 0.942 | 0.078 | 0.207 | 0.966 |

NOL | 0.30 | 3.602 (24.803) | 0.50 (0.891) | 1.116 (1.874) | 0.872 | 0.111 | 0.027 | 0.946 |

SER | 1.00 | 4.00 (6.459) | 0.770 (0.534) | 1.418 (0.374) | 0.889 | 0.095 | −0.071 | 0.941 |

TAE | 0.50 | 2.999 (3.041) | 0.994 (0.253) | 2.299 (1.182) | 0.989 | 0.033 | 0.058 | 0.994 |

VIN | 0.70 | 2.557 (305.636) | 1.204 (41.006) | 2.173 (89.386) | 0.792 | 0.154 | 0.277 | 0.871 |

^{1}Estimates of parameters of Equation (6);

^{2}adjusted R

^{2}, root mean square error (RMSE), coefficient of residual mass (CRM), concordance correlation coefficient (CCC).

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## Share and Cite

**MDPI and ACS Style**

Fedele, G.; Brischetto, C.; González-Domínguez, E.; Rossi, V.
The Colonization of Grape Bunch Trash by Microorganisms for the Biocontrol of *Botrytis cinerea* as Influenced by Temperature and Humidity. *Agronomy* **2020**, *10*, 1829.
https://doi.org/10.3390/agronomy10111829

**AMA Style**

Fedele G, Brischetto C, González-Domínguez E, Rossi V.
The Colonization of Grape Bunch Trash by Microorganisms for the Biocontrol of *Botrytis cinerea* as Influenced by Temperature and Humidity. *Agronomy*. 2020; 10(11):1829.
https://doi.org/10.3390/agronomy10111829

**Chicago/Turabian Style**

Fedele, Giorgia, Chiara Brischetto, Elisa González-Domínguez, and Vittorio Rossi.
2020. "The Colonization of Grape Bunch Trash by Microorganisms for the Biocontrol of *Botrytis cinerea* as Influenced by Temperature and Humidity" *Agronomy* 10, no. 11: 1829.
https://doi.org/10.3390/agronomy10111829