3.1. The effect of different O3 treatment conditions on the TNC in freshly-peeled garlic
As shown in Fig. 1(A), during storage, the TNC on the surface of fresh garlic inoculated with G. intermedia and A. ochraceus at different O3 concentrations was significantly lower than that of the blank group (P < 0.05). Cunkun Chen et al. (2020) reported similar findings that O3 treatment can inhibit the bacteria and fungi that lead to the corruption of Hami melon by postharvest pathogens at appropriate doses. With an increase in storage time, the TNC on the surface of fresh garlic in each experimental group increased. For the fresh garlic inoculated with G. intermedia, the TNC increased with an increase in the O3 concentration, while 6 ppm was the optimal O3 concentration level. For freshly-peeled garlic inoculated with A. ochraceus, the TNC displayed a lower level of decline when the O3 concentration increased from 1 ppm to 3 ppm, while the decrease was higher when the O3 concentration changed from 4 ppm to 6 ppm. When the garlic was stored for 10 d, no significant difference was evident between the freshly-peeled garlic treated with 1 ppm O3 and the blank group (P > 0.05). Therefore, it can be concluded that an O3 treatment concentration of 6 ppm was optimal in the freshly-peeled garlic inoculated with A. ochraceus.
Fig. 1(B) shows that an extended storage time increased the TNC at the same O3 treatment time. After inoculation with G. intermedia, the TNC on the fresh garlic's surface was low after O3 treatment for 15 min, while the germicidal effect had improved. When fresh garlic inoculated with A. ochraceus was stored for 10 d, the difference between O3 treatment and blank groups was significant (P < 0.05), while the optimal O3 treatment time was 15 min. These results are similar to those revealed by Showkat et al. (2019), who studied the effect of O3 treatment on bacteria in fruit. After a certain period of O3 treatment, the number of kiwifruit colonies decreased and prevented bacterial growth.
Fig. 1(C) shows that under the same O3 treatment humidity, the TNC on the surface increased with an extension in storage time. A significant difference was evident between the O3 treatment group with 85% humidity and the treatment group with 90% humidity (P < 0.05). During the 10 d of storage, the TNC on the fresh garlic's surface inoculated with the types of bacteria exhibited no significant change after treatment with O3 gas at 90% and 95% humidity, respectively. After storage for 10 d, the TNC on the surface of freshly-peeled garlic inoculated with G. intermedia and A. ochraceus was 3.37 lg CFU/g and 3.92 lg CFU/g, respectively. Therefore, when considering lower costs, the optimal lg CFU/g treatment humidity was 90%.
3.2. The effect of different O3 treatment conditions on the TNMY in freshly-peeled garlic
As shown in Fig. 2(A), the TNMY in freshly-peeled garlic inoculated with G. intermedia and A. ochraceus at different O3 concentrations changed with storage time. At the same storage time, the TNMY decreased with an increase in the O3 concentration. After the same concentration of O3 treatment, with the increase of storage time, the TNMY increased. A significant difference (P < 0.05) was evident between the O3 treatment groups and the blank group in the fresh garlic inoculated with G. intermedia. A similar phenomenon was apparent between the groups subjected to different O3 concentrations. The results showed that 6 ppm was the better O3 concentration. No significant mold and yeast levels were detected in the 1 ppm, 2 ppm, and blank groups of the fresh garlic inoculated with A. ochraceus. When the O3 concentration increased to 3 ppm, the TNMY decreased as the treatment concentration continued to increase. Moreover, the TNMY in the fresh garlic was the lowest after treatment with 6 ppm O3.
As shown in Fig. 2(B), the TNMY on the surface of the fresh garlic decreased with an increase in the treatment time and storage period at the same storage time. When the O3 treatment time increased from 15 min to 18 min, no significant difference was evident in the TNMY (P > 0.05) levels, but it was significantly lower than in the blank group and the previous experimental group (P < 0.05), while the better O3 treatment time was 15 min. P. Sarig et al. (1996) found that when berries were exposed to O3 for 20 min, the number of CFU of the fungi, yeast, and bacteria that naturally exist on the surface of the fruit was significantly reduced. O3 treatment substantially reduced the level of decay caused by fungi after cold storage and prolonged the quality assurance period of the berries. This is consistent with the results indicating that the quantity of mold and yeast decreased after the fresh garlic was treated with O3 for a particular time at a specific concentration.
Fig. 2(C) shows that after O3 treatment at 90% and 95% humidity, respectively, the TNMY in the freshly-peeled garlic inoculated with G. intermedia and A. ochraceus was always lower than in the treatment group subjected to 85% humidity. After 10 d of storage, the TNMY in the O3 treatment group at 90% humidity was 3.55 lg CFU / g and 3.48 lg CFU / g, respectively, which was considerably lower than in the treatment group exposed to 85% humidity.
3.3. The effect of different O3 treatment conditions on the incidence of freshly-peeled garlic
As shown in Fig. 3(A), O3 treatment significantly inhibited the incidence of freshly-peeled garlic inoculated with G. intermedia and A. ochraceus. After inoculation with G. intermedia, the garlic incidence decreased in conjunction with an increase in the O3 concentration at the same storage time. After 10 d of storage, the fresh garlic incidence in the 6 ppm treatment group was the lowest at only 44.85%, which was substantially lower than in the blank group (98.68%). Furthermore, when the fresh garlic was inoculated with A. ochraceus, the differences between the blank, the 1 ppm, and the 2 ppm O3 treatment groups were small, and the total incidence was high at the same storage time. Additionally, when the O3 concentration increased to 6 ppm, the fresh garlic incidence was the lowest of all the storage times, while the germicidal effect was the best.
After O3 treatment at different times, the incidence of the freshly-peeled garlic inoculated with G. intermedia and A. ochraceus during storage is shown in Fig. 3 (B(a)) and Fig. 3 (B(b)), respectively. With an extended storage time, the incidence of the garlic increased gradually. After 10 d of storage, the incidence of the blank group reached 100%. With an increase in the treatment time, the garlic incidence decreased slowly at the same storage time. After 15 min of O3 treatment, the incidence of the fresh garlic inoculated with the two types of mold remained low. When the treatment time continued to increase, no significant reduction was evident. After 10 d of storage, the incidence was 49.75% and 44.55%, respectively.
Fig. 3(C) indicated that during the entire storage period, the curve of the O3 treatment group at 85% humidity exceeded the curve of the O3 treatment groups at 90% and 95% humidity, and the incidence of fresh garlic treated with 90% and 95% O3 was significantly lower than that of the O3 treatment group at 85% humidity. At the end of the storage period, the freshly-peeled garlic incidence exposed to 90% humidity was 49.75% and 44.55%, respectively. From the perspective of saving resources, 90% O3 humidity was selected as appropriate, which was consistent with the results suggesting that O3 could reduce the incidence rate of papaya and red peppers, according to Marcin (2016).
3.4. The effect of different O3 treatment conditions on the lesion diameter of freshly-peeled garlic
Fig. 4(A) shows that extended storage time increased the lesion diameter of freshly-peeled garlic as the O3 concentration became higher, while the lesion diameter of the blank group always remained at the maximum level. After O3 treatment at different concentrations, the 6 ppm group displayed the best bacteriostatic effect and the smallest lesion diameter during storage. After 10 d of storage, the lesion diameter of the freshly-peeled garlic inoculated with G. intermedia and A. ochraceus was 3.07 mm and 2.67 mm, respectively, which was substantially lower than in the blank group (5.53 mm and 5.90 mm respectively).
Fig. 4(B) shows that at the same storage time, the lesion diameter of the freshly-peeled garlic inoculated with G. intermedia and A. ochraceus gradually decreased with an increase in treatment time. After 10 d of storage, little change occurred in the lesion diameter after 15 min of O3 treatment (3.37 mm and 3.27 mm, respectively), and 18 min of O3 treatment (3.28 mm and 3.14 mm respectively). Therefore, O3 treatment for 15 min achieved better control effect on the disease spot diameter.
Fig. 4(C) shows the changes in the lesion diameter of freshly-peeled garlic inoculated with Pythium after O3 treatment at different humidity levels. The lesion diameter increased with the storage time. From the beginning to the second day of storage, the lesion diameter increased sharply, and then rose gradually over time. At the same storage time point, the change in the lesion diameter with O3 humidity was small, but the lesion diameter of the fresh garlic treated with O3 at 90% and 95% humidity was still slightly lower than that of the O3 treatment group at 85% humidity. Based on these indicators' experimental results, 90% humidity was selected as the appropriate O3 treatment level.
3.5. The effect of different O3 treatment conditions on the lesion depth of freshly-peeled garlic
Fig. 5(A) indicates that for the freshly-peeled garlic inoculated with G. intermedia, at the same storage time point, the change of the lesion depth changes slowly with the increase of concentration, but in general, the total treatment value of lesion depth of freshly-peeled garlic treated with 6 ppm O3 was smaller. For fresh garlic inoculated with A. ochraceus, when the O3 concentration was lower than 4 ppm, the lesion depth change with the concentration was smaller under the same storage time. When the O3 concentration increased from 3 ppm to 4 ppm, the fresh garlic's lesion depth exhibited a visible change. When the O3 concentration increased to 6 ppm, the lesion depth was the smallest.
The change in lesion depth in conjunction with storage time is shown in Fig. 5(B) after treatment with the same O3 concentration at different times. It indicated that the lesion depth of the freshly-peeled garlic increased with extended storage time. Furthermore, it demonstrated that the lesion depth exposed to O3 treatment for 3 min coincided with that of the blank group, with no significant difference. A further increase in treatment time significantly affected the inhibition of lesion depth. Regarding the freshly-peeled garlic inoculated with A. ochraceus, when the O3 treatment time increased from 15 min to 18 min, the depth of the garlic spots decreased slightly. Therefore, an O3 treatment time of 15 min improved the inhibition. Regarding the fresh garlic inoculated with G. intermedia, no significant difference was evident between the three treatment groups (12 min, 15 min, and 18 min) in terms of the degree of curve coincidence. The curve basically coincided, and the lesion depth always remained low. To save costs and protect the environment, the 12 min O3 treatment time was chosen.
Fig. 5(C) shows that from the beginning of storage and at the same storage time point, when the humidity increased from 85% to 90%, the disease spot depth of the freshly-peeled garlic inoculated with G. intermedia decreased significantly. When the humidity continued to increase to 95%, the disease spot depth remained unchanged. Regarding the freshly-peeled garlic inoculated with A. ochraceus when the storage time was less than 4 d, no significant differences were evident between the three O3 treatments. From the 6th day of storage, the lesion depth of the freshly-peeled garlic treated with 85% humidity was significantly higher than that of freshly-peeled garlic exposed to 90% and 95% humidity. Based on these results, 90% humidity was selected as optimal during the O3 treatment of G. intermedia.
3.6. Analysis of the transcriptome differences in the putrefactive fungi in fresh garlic
3.6.1. Sample RNA test results of G. intermedia and A. ochraceus
The RNA of the O3 treated, and untreated Gibberella and ochratoxin were extracted, and the nanodrop UV-Vis spectrophotometer was used to verify the results (Table 1). Fig. 6 shows that the two treatment groups' baselines were stable, and the 5S peak was normal. Furthermore, the purity, concentration, and integrity of the extracted RNA were consistent with subsequent analysis requirements.
Table 1. RNA test results for G. intermedia and A. ochraceus
Sample name
|
Concentration (ug/uL)
|
Volume (uL)
|
Total (ug)
|
OD260/OD280
|
OD260/OD230
|
RIN
|
28S/18S
|
G. intermedia-treated
|
1664
|
45
|
74.88
|
2.16
|
2.47
|
9.1
|
2.3
|
G. intermedia-untreated
|
1236
|
45
|
55.62
|
2.17
|
2.43
|
8.9
|
2.4
|
A. ochraceus-treated
|
1146
|
45
|
51.57
|
2.16
|
2.39
|
7.6
|
1.4
|
A. ochraceus-untreated
|
2052
|
45
|
92.34
|
2.14
|
2.38
|
6.8
|
1.5
|
3.6.2. The quality test results for the transcriptome sequencing for G. intermedia and A. ochraceus
Table 2 shows the quality assessment of the O3-treated and untreated gibberellin and ochratoxin transcriptome. The sequencing data of the processed and untreated G. intermedia and A. ochraceus were good, meeting subsequent analysis requirements.
Table 2. Quality assessment of transcriptome sequencing data for G. intermedia and A. ochraceus
Sample's name
|
Raw reads (M)
|
Clean Reads (M)
|
Clean Bases (Gb)
|
Q20(%)
|
Q30(%)
|
GC (%)
|
G. intermedia-treated
|
69.73
|
68.28
|
6.83
|
96.55
|
87.72
|
50.54
|
G. intermedia-untreated
|
74.71
|
73.09
|
7.31
|
96.45
|
87.44
|
50.45
|
A. ochraceus-treated
|
72.22
|
70.42
|
7.04
|
96.47
|
87.56
|
52.42
|
A. ochraceus-untreated
|
69.73
|
68.19
|
6.82
|
96.50
|
87.64
|
52.31
|
3.6.3. The transcriptome assembly output data for G. intermedia and A. ochraceus
Table 3 shows the results after aggregating the output data, indicating that the assembly quality of G. intermedia and A. ochraceus were good.
Table 3. Assembly result statistics for G. intermedia and A. ochraceus
Strain name
|
Length Range
|
Transcript
|
UniGene
|
G. intermedia
|
200-300
|
6945 (12.95%)
|
3354 (11.38%)
|
300-500
|
5523 (10.30%)
|
2643 (8.97%)
|
500-1000
|
6420 (11.97%)
|
3182 (10.79%)
|
1000-2000
|
11717 (21.84%)
|
6308 (21.40%)
|
2000-3000
|
8479 (15.81%)
|
4883 (16.56%)
|
>3000
|
14557 (27.14%)
|
9111 (30.90%)
|
Total Number
|
53641
|
29481
|
Total Length
|
116080340
|
69638845
|
N50 Length
|
7030
|
3708
|
Mean Length
|
4323
|
2362
|
A. ochraceus
|
200-300
|
6877 (9.03%)
|
3309 (8.22%)
|
300-500
|
6310 (8.28%)
|
2783 (6.92%)
|
500-1000
|
7605 (9.98%)
|
3462 (8.60%)
|
1000-2000
|
12578 (16.51%)
|
6265 (15.57%)
|
2000-3000
|
11463 (15.05%)
|
6015 (14.95%)
|
>3000
|
31336 (41.14%)
|
18409 (45.74%)
|
Total Number
|
76169
|
40243
|
Total Length
|
247108484
|
145109459
|
N50 Length
|
10644
|
5829
|
Mean Length
|
6463
|
3605
|
3.6.4. The functional annotation of genes for G. intermedia and A. ochraceus
The statistics for the gene annotation rate of the transcriptome sequencing data of G. intermedia and A. ochraceus are shown in Fig. 7. The overall annotation rate of the transcriptome sequencing data of G. intermedia and A. ochraceus reached respective levels as high as 99.53% and 89.89%, providing a database for subsequent gene analysis.
3.6.5. The effect of O3 on the gene expression of G. intermedia and A. ochraceus
As shown in Fig. 8, the O3-treated and untreated groups' gene expression levels were compared to explore the effect of O3 treatment on the gene expression of gibberellin (ochratoxin) during the growth process. The results revealed the presence of 2754 (2378) differentially expressed genes (p-value < 0.001 and log2foldchange > 2) between the blank group and the treatment group, among which 1456 (1591) were up-regulated and 1298 (787) were down-regulated.
3.6.6. The GO annotation of the differentially expressed genes for G. intermedia and A. ochraceus
There were 16, 13, and 8 annotation classifications for the biological process, cell composition, and molecular functionality, respectively (Fig. 9). According to the classification of differential gene GO annotations, the gene expression and regulation of Gibberella were influenced by the metabolic process, cell process, and localization of the biological process after O3 treatment. The gene expression and regulation of Gibberella were influenced by the cell, cell part, organelle, membrane, and membrane part of the cell composition. The gene expression and regulation of Gibberella were influenced by the catalytic activity, binding activity, transport activity, and transcriptional regulation activity of the molecular functions. The pathways with more differentially expressed genes include the metabolic process, cell process, localization, cell, cell part, membrane, membrane part, binding, and catalytic activity.
The pathways with more differentially expressed genes include the metabolic process, the cell process, localization, cell, cell part, membrane, membrane part, binding, and catalytic activity. Furthermore, there were 18, 13, and 10 annotation classifications for the differentially expressed genes in the biological process, cell composition, and molecular functionality, respectively. According to the classification of differential gene GO annotation, O3 treatment affects the gene expression and regulation of ochratoxin in the metabolic process, cell process, stress response, localization, cell component tissue or biogenesis, biological process regulation, and biological regulation of the biological process. The pathway affects the gene expression and regulation of ochratoxin in the cell, cell part, organelle, membrane, and membrane part. Furthermore, the pathway affects the gene expression and regulation of ochratoxin in the catalytic activity, binding, transport activity, and transcriptional regulation activity of the molecular functions. The pathways with more differentially expressed genes include the metabolism, cell process, cell, cell part, membrane, membrane part, binding, and catalytic activity.
3.6.7. The significant enrichment of the differential genes of G. intermedia and A. ochraceus
In order to find out the main function of O3 treatment on the inhibition of Gibberella and ochratoxin, go function significance enrichment analysis (p-value < 0.05) was carried out for the differentially expressed genes of O3 treated and no O3 treated Gibberella and ochratoxin, and the results of significance enrichment were shown in Table 4. First, regarding cell composition, the nodes with significant enrichment in Gibberella were the membrane, the cell, the organelle, the macromolecule complex, and nucleoid, while the number of differentially expressed genes was 1, 2, 3, 2, and 1, respectively. The nodes with significant enrichment in the molecular functions were the catalytic activity, binding transport activity, signal sensor activity, and transcriptional regulation activity, while the number of differentially expressed genes was 19, 13, 6, 1, and 1, respectively. There were 18, 7, 9, 1, and 5 differentially expressed genes in the cell process, biological regulation process, localization, stress response, and cell component tissue or biogenesis. Second, regarding the ochratoxin, the nodes with significant enrichment in the cell composition were the cells and organelles, while the number of differentially expressed genes was 9, 10, and 1, respectively. The nodes with significant enrichment in molecular functions were the catalytic activity, binding, signal sensor activity, and structural molecular activity, while the number of differentially expressed genes was 50, 11, 3, and 1, respectively. There were 48, 16, 8, 4, 1, 1, and 6 differentially expressed genes in the biological processes, including the cell process, biological regulation process, tissue or biogenesis of the cell components, metabolic process, and multiple biological processes stress response and localization.
Table 4. The significant enrichment of the GO function of the differentially expressed genes in G. intermedia and A. ochraceus by O3
Strain name
|
GO function
|
Node
|
Number of
up-regulated genes
|
Number of
down-regulated genes
|
G. intermedia
|
Cellular component
|
Membrane part
|
1
|
0
|
Cell
|
1
|
1
|
Organelle part
|
2
|
1
|
Macromolecular complex
|
0
|
2
|
Nucleoid
|
0
|
1
|
Molecular function
|
Catalytic activity
|
9
|
10
|
Binding
|
5
|
8
|
Transporter activity
|
3
|
3
|
Signal transducer activity
|
1
|
0
|
Transcription regulator activity
|
0
|
1
|
Biology process
|
Biological regulation
|
2
|
5
|
Cellular process
|
8
|
10
|
Localization
|
5
|
4
|
Response to stimulus
|
1
|
0
|
Cellular component organization or biogenesis
|
3
|
2
|
A. ochraceus
|
Cellular component
|
Cell
|
7
|
2
|
Organelle part
|
9
|
1
|
Cell part
|
0
|
1
|
Molecular function
|
Catalytic activity
|
33
|
17
|
Binding
|
8
|
3
|
Signal transducer activity
|
3
|
0
|
Structural molecule activity
|
1
|
0
|
Biology process
|
Cellular process
|
34
|
14
|
Biological regulation
|
12
|
4
|
Cellular component organization or biogenesis
|
6
|
2
|
Metabolic process
|
4
|
0
|
Multi-organism process
|
1
|
0
|
Response to stimulus
|
1
|
0
|
Localization
|
2
|
4
|
3.6.8. The annotation of the differentially expressed gene KEGG of G. intermedia and A. ochraceus
The annotation classification map of the KEGG pathway of the O3-treated and untreated gibberellins is shown in Fig. 10(a). Here, 2503 differentially expressed genes were annotated into the KEGG database, 22 metabolic pathways were annotated by the KEGG pathway of which two metabolic pathways belonged to the cell process, two to the environmental information process, and four to the genetic information process, while 13 were metabolic processes, and one denoted the organic system. There were 176 differential gene annotations to the transport and catabolism pathway, 117 to the cell growth and death pathway, 118 to the signal transduction pathway, 64 to the gene transcription pathway, and 167 to the gene transcription pathway during environmental information processing. In the translation process, 171 differential gene annotations were included in the gene folding, classification, and degradation pathways, 77 in the DNA replication and repair pathways, 237 in the carbohydrate metabolism pathway, and 552 in the overall overview pathway.
The annotation classification diagram of the KEGG pathway denoting the differential expression genes between the O3-treated and the untreated Aspergillus ochre is shown in Fig. 10(b). Here, 1934 differentially expressed genes were annotated into KEGG by Aspergillus ochre, including 22 metabolic pathways in total, of which two were due to the cellular process, two due to environmental information processing, four due to genetic information processing, 12 due to the metabolic process, and two due to the organic system. Furthermore, there were 156 differential gene annotations during the process of cell transportation and catabolism, 40 during the process of cell growth and death, 76 during the process of environmental information processing, 35 during the process of genetic information processing, and 107 during the process of gene transcription. In the process of translation, there were 80 differential gene annotations into the gene folding, classification, and degradation pathways, 30 into DNA replication and repair pathways, 229 into the carbohydrate metabolism pathway, 496 into the whole body overview pathway, 182 into the amino acid metabolism pathway, and 123 into the lipid metabolism pathway.
3.6.9. The enrichment of allogeneic KEGG for G. intermedia and A. ochraceus
As shown in Table 5, the statistics regarding the significant KEGG enrichment (p-value < 0.05) of the differentially expressed genes between the O3-treated and untreated Gibberella and ochratoxin were studied, and the metabolic process of the genes involved in the inhibition of Gibberella and ochratoxin growth by O3 was explored. The results indicated that O3 affects the growth of Gibberella via the KEGG pathway process mentioned above, and even causes its death. In addition, O3 mainly affects the growth and death of ochratoxin by impacting its amino acids, overall overview, and lipid metabolism.
Table 5. The significant enrichment of differential genes in KEGG for G. intermedia and A. ochraceus
Strain name
|
KEGG pathway
|
Number of differentially expressed genes
|
Rich Ratio
|
G. intermedia
|
Amino acid metabolism
|
2
|
0.2122
|
Biosynthesis of other secondary metabolites
|
2
|
0.2894
|
Carbohydrate metabolism
|
2
|
0.2243
|
Cell growth and death
|
1
|
0.0974
|
Lipid metabolism
|
3
|
0.4321
|
Membrane transport
|
1
|
0.0957
|
Metabolism of cofactors and vitamins
|
5
|
0.5984
|
Metabolism of other amino acids
|
1
|
0.1103
|
Metabolism of terpenoids and polyketides
|
1
|
0.1875
|
Transcription
|
1
|
0.0920
|
A. ochraceus
|
Aging
|
1
|
0.0852
|
Amino acid metabolism
|
5
|
0.3015
|
Biosynthesis of other secondary metabolites
|
1
|
0.1143
|
Carbohydrate metabolism
|
3
|
0.2313
|
Energy metabolism
|
3
|
0.1887
|
Folding, sorting, and degradation
|
2
|
0.1307
|
Global and overview maps
|
6
|
0.3306
|
Glycan biosynthesis and metabolism
|
2
|
0.1838
|
Lipid metabolism
|
6
|
0.5139
|
Metabolism of cofactors and vitamins
|
3
|
0.2906
|
Metabolism of other amino acids
|
3
|
0.2067
|
Nucleotide metabolism
|
1
|
0.0358
|
Transport and catabolism
|
1
|
0.0345
|