**3. Results**

#### *3.1. Sensitivity of B. cinerea to Iprodione*

One hundred and twelve samples of *B. cinerea* were collected from different locations in tomato production area and tomato fields, Baise City, Guangxi Province, China. The sensitivity of B. *cinerea* was checked on PDA medium amended with iprodione at 0.01 μg/mL. The inhibition rate of all samples ranged from 7.69–74.35% with an average of 50.70% (Figure 1). The EC50 of iprodione against all samples ranged from 0.07 to 0.87 μg/mL with an average of 0.47 μg/mL, indicating these *B. cinerea* isolates were susceptible to iprodione. The value of EC50 of all isolates indicated that 0.47 μg/mL was

an appropriate threshold concentration to assess iprodione resistance in the consequent experiments. Among them 5 isolates were highly sensitive to EC50 for 0.134 μg/mL iprodione and five isolates were moderately sensitive to EC50 for 0.434 μg/mL iprodione (Supplementary Tables S6–S9).

**Figure 1.** The inhibition rate of *B. cinerea* isolates against iprodione (0.01 μg/mL) collected from different areas of Guangxi Province China.

#### *3.2. In Vitro Iprodione-Induced B. cinerea Mutants*

UV radiation is a toxic mutagen and was expected to decrease the viability of the cells but also increase the probability of the emergence of mutants of drug sensitive strains. Five iprodione sensitive isolates of *B. cinerea* exposed to different concentrations of iprodione fungicide with 20W UV lamp were continuously cultured at 28 ◦C to induce rapid growth of mutants. The only strain B67 showed two mutants M1 and M0 respectively. The EC50 of these mutants were 674.48 μg/mL and 1025.74 μg/mL, respectively, and 597.63 and 906.94 times than that of wild isolate (EC50 was 1.12 μg/mL). However, other isolates showed higher sensitivity to iprodione and did not produce any mutant used as a control. These two mutants were continuously sub-cultured for 1, 5, 10, and 15 generations on drug free PDA medium for stability test (Supplementary Table S2).

#### *3.3. Morphology and Physiology of Mutants*

#### 3.3.1. Iprodione Resistant Mutant's Mycelium Growth Rate and Sclerotia Formation

The results showed that mycelial growth of wild type on PDA medium is significantly higher than both mutants after 5 days at 28 ◦C (Figure 2A). Wild type (W0) showed maximum sclerotia formation on PDA after 8 days; in petri dish edges are produced around a small contiguous black sclerotia; did not spread throughout the surface of the medium. M0 produced dark grey to black sclerotia after 12 days and spread over the medium and M1 after 14 days produced sclerotia over the surface of the medium (Figure 2B). The mutant M1 after 10 days began to produce small contiguous black sclerotia circles at the edge of the dish and after 12 days spread on the petri dish. When PDA medium was amended with 100, 500, 600, and 1000 μg/mL iprodione, mutant M0 showed significantly high mycelial growth than M1 and wild type W0 (Figure 2C). Mutant M0 after 12 days began to produce fewer black sclerotia circles at the edge of the dish with 5 μg/mL iprodione containing media. In contrast, wild type failed to produce any (Supplementary Figure S1).

**Figure 2.** Colony morphology of *B. cinerea* Wild type (W0) and its mutant (M0, M2); (**A**) Mycelial growth of mutants (M0, M1) and its wild type on PDA medium after 5 days; (**B**) Sclerotia formation of wild type (W0) and its mutants (M0,M1) on PDA medium after 8, 12 and 14 days respectively; Sporulation: mean number (×<sup>10</sup>6) seclerotia per square centimeter, (**C**) Mycelial growth of wild type (W0) and its mutants (M0, M1) after exposed 100, 500, 600 and 1000 μg/mL to iprodione on PDA medium at 28 ◦C for 3 days.

3.3.2. Cell Membrane Permeability Osmotic Sensitivity Pathogenicity and Enzymatic Activity of Mutants and its Wild Type

Cell membrane permeability at four different concentrations (0, 1, 5, 10 μg/mL) of iprodione were measured, the relative rate of infiltration with the extension of the processing time increases and gradually stabilized in M1, indicating that the cell membrane permeability is significantly higher in M1 and wild strain than M0 (Supplementary Figure S2). Whereas, there was a no significant difference in osmotic potential of wild type and its mutants (Supplementary Figure S3). Protein concentration was calculated according to the sample suction photometric method. The remarkably highest protein

contents were in W0 (1023.97 μg/mL) than M0 and M1 mutant (941.38, 908.02 μg/mL respectively (Supplementary Table S10). Enzymatic activities of both mutants and wild type were also measured. Although PG and CE activity was higher in M0 than M1 and wild type W0. (Supplementary Table S11). In pathogenicity assays, detached tomato leaves inoculated with both mutants (M0, M1) or wild type strain showed typical symptoms and lesions by W0 and M0, while the only PDA or control plants and M1 remained asymptomatic after 24 h of inoculation (Figure 3). These results showed that mutant M0 pathogenicity and enzymatic activity is more vulnerable than M1.

**Figure 3.** Virulence of *B. cinerea* Wild type (W0) and its mutnats (M0, M1) on detached tomato leaves after 24 h of inoculation.

### 3.3.3. Cross Resistance

The sensitivity of both *B. cinerea* mutants (M0, M1) were also determined against tebuconazole and fludioxinil using a discriminatory dose. Both iprodione mutants showed positive cross resistance against these fungicides.

#### *3.4. Transcriptomic Data Analysis of Iprodione-Resistant Mutant and Its Wild Type after Exposed to Iprodione In Vitro*

The results revealed that transcriptomic sequencing of twelve iprodine resistant mutant and wild type samples generating 187,062,138 raw reads after the screening and filtration of raw reads, a total of 186,026,964 clean reads were obtained. The percentage nucleotide quality score of more than 20 (Q20) was noted as high as 97.75%, and the percentage of Guanine and Cytosine GC (%) among all nucleotides was obtained as 47.33% (Table 1). According to Illuminia platform, the contrast efficiencies of mapped reads, uniquely mapped reads and multi mapped reads were 95.91%, 0.52%, and 95.2%, respectively, as compared to the reference genome (Table 1). A total number of transcripts was 20,375 and its length varied from 201 to1800 bp with an average 1000 bp (Supplementary Figure S4). On the basis of existing reference genome based assembly is performed by using compare software. To check the presence of novel transcripts, we combined the RNA-seq data of 4 samples with 3 biological repeats to identify novel transcripts, which are not assembled in the database. New transcript is obtained by comparing it with known transcript and further classified into 12 different class codes (Supplementary Figures S5 and S12). Out of 20,375 transcripts, 13,639 complete matches of intron chain, 3574 potentially novel isoforms, 1229 unknown intergenic transcript, and 372 generic exonic overlaps with a reference transcript were obtained during analysis. The raw data is submitted to NCBI under SRA number SRP254522.


**Table 1.** Details of raw and clean data of twelve transcriptomes of *B. cinerea* and the reference genomes.

#### 3.4.1. Functional Annotation of Transcripts and Unigenes

Almost all transcripts (13,703) and unigenes (11,698) sequences were alligned to the NCBI and annotated at least one of these six databases, Non Reductase (NR), SwissProt, Protein family (Pfam), Gene ontology (GO), Clustre of Orthologous Groups of Proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Supplementary Figure S6). The database indicated that maximum transcripts (13,687; 99%) and unigenes (11,685; 99%) were alligned by NR, whereas more than 50% of transcripts and unigenes were aligned to the COG, Pfam, and Swissprot databases. The minimum number of transcripts vs. unigenes was annotated by KEGG 33%. Moreover, 14 transcripts and 12 unigenes remained unannotated (Supplementary Table S13 Excel sheet).

#### 3.4.2. Discovery of New Genes

A total of 1024 new genes were discovered according to the above mentioned six databases. COG annotated 150 new genes in 14 di fferent categories, GO 160 new genes in 19 di fferent compartments (Figure 4), and KEGG 17 in 12 di fferent disciplines. Furthermore, GO database is secondarily classified into three categories, namely molecular functions (47), cellular components (57), and biological processes (56) of new genes were explored. The highest number of new genes were involved in binding, metabolism, and cellular processes. COG annotated a maximum 84 new genes, which were poorly characterized, and 44 genes were involved in the repair, replication, and recombination of RNA. KEGG aligned the highest number of genes in the biosynthesis of the secondary metabolism process.

**Figure 4.** Histogram of KEGG pathways.

3.4.3. Differential Genes Expression of Iprodione Resistant Mutant and its Wild Type After Exposure to Iprodione

After obtaining the clean reads, the differential expression of unigenes was analyzed by using software DESeq2. A total of 281 unigenes were expressed in mutant type (M0), including 166 up-regulated and 115 downregulated with or without iprodione treatment. Meanwhile, wild type (W0) showed 99 unigenes expressions in which 85 were up-regulated and 14 downregulated (Table 2).


**Table 2.** The total number of DEG'S in wild type and its mutant with or without iprodione.

The analysis showed that mutant and wild type shared 19 DEGs and 262 and 80 unique DEGs were detected in mutant and wild type respectively when exposed to iprodione. Overall, 1897 DEGs were detected after iprodione exposure and 1707 without iprodione between mutant and wild type. Furthermore, M-ipro vs. W-ipr0 and M0 vs. Wo share 1192 common unigenes and 720 unigenes are upregulated in mutant corresponding to wild type after iprodione treatment (Supplementary Figure S7). These results demonstrated that the DEGs pattern significantly changed in mutant and wild type with or without iprodione exposure, suggesting that some compounds may be specific to produce resistance in mutants against iprodione treatment.To understand the mechanism of resistance in *B cinerea* against iprodione, the gene function, expression level, and expression difference were analyzed in gene set analysis. Genes of the same function were located on one transcript, particularly within the three loci easy to annotate by gene ontology (GO) rather than those situated on different transcripts. The genes related to metabolic process, localization, ATP binding, transmembrane transport antibiotic activity, and the cellular process were most abundant in mutant type (M-ipro vs. M0) relative to wild type (W-ipro vs. W0) (Figure 5).

To elucidate the difference between mutant and wild type, the expression pattern of four treatments were divided into a hierrachial clustering analysis (Supplementary Figure S8). Out of 1912 genes, we focused our attention on highly expressed genes in M-ipro that were more or less related to iprodione resistance and assembled them into 12 small clusters according to their functions (Figure 6). In clusture I, MFO (multifunctional genes) were analyzed (*BCIN\_06g07150*, *BCIN\_09g01190*, *BCIN\_07g01720*, *BCIN\_02g04800*, *Bcape1*) highly expressed in M-ipro and involved in molecular and biological functions of *B. cinerea*. Among the five genes of aspartic proteinase family (*bcap1*, *bcap4*, *bcap6*, *bcap8, bcap10*), only *bcap8* log2 value was significantly high in M-ipro, while the remaining genes did not show any significant difference in both mutant and wild type with or without iprodione treatment. In cluster II genes, set ABC transporter genes (*BcatrD*, *Bcbfr1*) were highly expressed in mutant type M-ipro than M0 and downregulated in wild type (W-ipro, W0). Various cytochrome p450 coding genes were expressed in a comprehensive data base and assembled in cluster III. Almost all genes were depressed in wild type (W-ipro, W0). *BccpoA90*, *Bccyp51*, and *BCIN\_15g04350* expression was high in M0 and M-ipro (Figure 6) except *BCIN\_02g00240*. Genes that were involved in amino acid metabolism exhibited high variability among mutant (M-ipro, M0) and wild type (W1, W0). Cluster IV has transmembrane transporter genes which were also expressed in both wild and mutant type. Only *BCIN\_14g04470* and *BCIN\_12g02430* were highly downregulated in W-ipro and W0 and upregulated in M0 and M-ipro.

**Figure 6.** Variability in altered genes of wild type and its corresponding mutant with or without iprodione application represented in the form of Heat map. Genes with the same annotation modulated in a similar group. Twelve major groups are displayed in the heat map (Group I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII). Group VII have four subgroups I (Glycosyl family), II (Cellulase genes). III (Polygalacturose genes) and IV (carbohydrate metabolism).

In contrast, many more genes had no significant difference among all treatments (Supplementary Table S14 Excel sheet). Moreover, the maximum high expression was detected in carbohydrate metabolism, glycosyl family, polyglacturose family and cellulose related genes attributed in cluster VI. *BCIN\_02g04690* log fold 2 values significantly high in M0 and low in W0 related to carbohydrate

binding. In cluster VII several genes were upregulated belongs to zinc finger proteins in mutant type (M-ipro, M0). Intriguingly *BCIN\_10g0230*, *BCIN\_09g00280*, and *BCIN\_14g04850* genes were highly upregulated in M0 rather than M-ipro and downregulated in wild type (W-ipro, W0). Drug sensitive proteins were highly downregulated in wild type after exposure to iprodione pooled in cluster VIII. *BCIN\_15g04850* and *BCIN\_13g05140*, highly upregulated in mutant without iprodione treatment instead of its application (Figure 6). Glutathione-S transferase encoding genes, i.e., GSt enzymes play an important role to detoxify the chemicals. *BCIN\_08g01800* gene regulating glutathione enzyme was more expressed in mutant without iprodione application than wild type. Clusture X, XI represents the lysophospholipase and super family genes (MFS). Two sugar transporter genes (*BCIN\_09g04610*, *BCIN\_14g01090*) of super family upregulated after exposure to iprodione fungicide in the resistant mutant. *BCIN\_13g03170* lysophospholipase gene expression log fold 2 value was −0.629 in W0, showed decrease −1.072 in W-ipro while expression level was increased in mutant after exposure to iprodione (Table 3). On the basis of these results, the presence of resistance in mutant strain may not be due to particular resistant gene against chemical or inactivation of enzymes and metabolic process. These data base also suggested that synergistic and combination of several genes belong to different functions or families generate resistance in mutants against a particular drug or multi drugs.



#### *3.5. qRT-PCR Amplification of Some Specific Genes*

In order to verify the transcriptomic analysis of mutant and wild type *B. cinerea*, a total of eight highly expressed encoding genes of lysophospholipase (*BCIN\_13g03170*), drug sensitivity (*BCIN\_04g01200*), cytochrome p450 (*Bccyp51*), cellulase (*BCIN\_12g06630*), glutathione-S- transferase (*BCIN\_08g01800*), oxaloacetate acetyl hydrolase (*bcoah*), cellulase (*BCIN\_12g06630*), glycosyl family (*BCIN\_12g01530*), and reference gene *UBQ* have been selected for expression analysis of RT-qPCR. Among these genes gluthathione-S transferase gene (*BCIN\_18g01800*) highly upregulated in mutant type with or without iprodione application (Figure 7C). Moreover, two lysophopholipase genes (*BCIN\_13g01370*, *bcoah*) expression in the mutant (M-ipro) were highly upregulated as compared to M0 and wild type (W-ipro, W0) (Figure 7A,B). The drug sensitivity and cytochrome familyP450 genes (*Bccyp51*, *BCIN\_14g01200*) were downregulated in wild type after iprodione treatment (Figure 7E,F). The cellulase gene (*BCIN\_08g01800*) was highly expressed in all treatments (Figure 7G).

**Figure 7.** Validation of seven DEG's log fold 2 value of RNA seq by qRT-PCR of wild type and its corresponding mutant with or without iprodione treatment. (**A,B**) lysopholipase genes, (**C**) Glutathione-S transferase gene, (**D**) Glycosyl family carbohydrate metabolism, (**E**) Drug sensitivity genes, (**F**) Cytochrome P450 family, (**G**) Cellulase genes, (**H**) Pearson correlation of log fold 2 value of qRT-PCR and RNA sequencing of wild type and its mutant after iprodione application. The mRNA abundance was normalized by using the reference gene UBQ and relative expression (log fold 2) was valued as 2−ΔΔCT. All values of qRT-PCR represents as mean ± SD (*n* = 7).

#### *3.6. Detection of Mutations in Tubulin Genes in Iprodione Mutants*

Genome sequencing of wild type (W0) and its mutant (M0) showed two tubulin genes encoding *bctubA* and *bctubB*. The coding region of *bctubA* had 1985 nucleotides encoding 661 amino acids, which was 65% match with *bctubA* B05.10 strain of *B. cinerea* (Gene Bank accession number: XP\_024546500.1). While the coding region of *bctubB* gene had 1919 nucleotides encoding 639 aminoacids was match with *bctubB* (*B. cinerea* B05.10) (GeneBank accession number: XP\_024546928.1). The *bctubA* gene in mutant (M0) replaced TTC and GAT codon at position 593 and 599 by TTA and GAA, resulting in replacement of phenyl alanine into leucine (transversion C/A) and aspartic acid into glutamic acid (transversion T/C) respectively. Whereas, in *bctubB* gene GAT codon at position 646 replaced by AAT and aspartic acid converted into asparagine (transition G/A).(Figure 8) No point mutation was found in wild type as compared to control (*B. cinerea* B05.10 strain).

**Figure 8.** Amino acid sequence alignment of *B. cinerea* tubulin genes in wild type (W0) and its mutant (M0), (**A**): Mutation variation in *bctubA* gene at position 593 and 599, (**B**): Mutation variation in *bctubB* gene at position 646.
