**1. Introduction**

Grey mold disease caused by *Botrytis cinerea* is one of the most destructive disease on more than 200 plant species including various economically important crops like tomato, grapevines, pepper, cucumber and strawberry [1,2]. The annual economic losses of *B. cinerea* is more than \$10 billion worldwide including fresh fruits and vegetables [3]. *B. cinerea* is a necrotrophic fungal plant pathogen of pre and post-harvest diseases with broad host range reproduces sexually and asexually [4]. *B. cinerea* produced micro and macro conidia on the surface of host plant cells [5,6]. These one-celled spores borne on multiple branches expressed its obvious symptoms as greyish to light brown mold leaves, stem, flowers and fruits of host plant [7]. Grey mold is more protruding during persistent rainy, heavy dew, and foggy weather around temperature 18–24 ◦C [8].

*B. cinerea* has a unique ability to survive in di fferent environmental conditions in the form of conidia and seclerotia that make this fungus very destructive and hazardous of resistance development [1,9,10]. The tremendous results against *B. cinerea* have been attained by applying various agrochemicals on tomato, cucumber, vine grapes etc. Every year more than half billion-dollar cost of fungicides are sprayed against di fferent pests [2]. Chemical control of grey mold currently approached by seven classes of fungicides including anilinopyrimidines (APs), dicarboximide (Dc's), methyl benzimidazole, carbamates (MBCs), hydroxyanilides (HAs), quinone outside inhibitors (QoIs), succinate dehydrogenase inhibitors (SDHIs), and phenylpyrroles (PPs) [11,12]. Extensive use of agrochemicals induces resistance in plant pathogens in field. Nowadays we selected those site specific fungicides that have high e fficacy, low toxicity and little human health risk [13]. However, these characteristics somewhat o ffset by their susceptibility to resistance development [14]. Iprodione, a dicarboximide (Dc) fungicide, has been used commercially for more than 30 years to control a wide variety of fungal pathogens. Among dicarboximides (Dcs), iprodione is a broad spectrum contact fungicide used as a foliar application and seed protectant for many fruits and vegetable crops. It has both preventive and curative action [15]. It was first manufactured in 1990. The mode of action of iprodione is to obstruct the synthesis of RNA and DNA during the germination of many fungal spores as well as lower the activity of enzyme NADH cytochrome c reductase, so the production of lipids and membrane restricted, ultimately mycelial growth inhibited. It can be used as a wettable powder, granules dispersed in water, flow able for all crops at the rate of 450–750 g/<sup>L</sup> [16].

Dc-resistant strains (iprodione) have been reported in many plant pathogenic fungal species [17]. Previously Hamada et al. [18] obtained iprodione resistant mutants of *Rhizoctonia cerealis* collected from wheat in China, *Botryosphaeria dothidea* from pistachio in California [19], *Alternaria* isolates collected from pistachio in California [20], and *B. cinerea* from strawberry and tomato in Hubei province, China [21].

The point mutation in the two components of histidine kinase genes (Bcos1) responsible for resistance to iprodione has been identified in *B. cinerea* isolated from strawberry fruit [21]. Substitution at codon I365 was dominant to cause resistance against iprodione among various strains of *B. cinerea* [22–24]. BcNoxA and BcNoxB catalytic subunits are responsible for pathogenicity and the formation of spores, which provide a favorable environment to fungal sclerotic to survive under adverse environmental conditions [25]. Various isolates of *B. cinerea* may possess low, moderate, or high levels of resistance to iprodione [26]. Mostly field isolates possess moderate to low level resistance, whereas laboratory mutants have high level resistance [27].

*B. cinerea* is a famous plant pathogen for its aptitude to become resistant against di fferent fungicides. Although resistance against a di fferent group of fungicides induced by di fferent mechanisms like structural alteration in binding sites of pathogens reduces the a ffinity of fungicides, up regulation of fungicide target genes, decomposition of active ingredients reduces the e fflux of fungicide

concentration [28]. The study of genomics, transcriptome, bioinformatics and proteomic provide a new array to explore resistance attributes. Rendering RNA sequencing the most e ffective and direct way to explore resistance genes in mutant species of *B. cinerea*.

Guangxi province of China is one of the major tomato producing area [29] and facing several challenges of fungal diseases including *B. cinerea*. Iprodione, fludioxonil, and tebuconazole fungicides have been widely used against fungal diseases in Guangxi province for many years. Thus it is necessary to study the sensitivity of *B. cinerea* against these fungicides and asses the resistance in isolates for these chemicals before they are widely used to control *B. cinerea.* In this study, *B. cinerea* isolates collected from tomato plants in Guangxi Province, China. The key objectives of the present study were to: (i) determine the prevalence and frequency of *B.cinerea*. (ii) determine the iprodione sensitivity of *B. cinerea* isolates; (iii) preliminarily evaluate the risk of *B. cinerea* resistance to iprodione and to characterize the iprodione induced mutants; (iv) asses the fitness stability and pathogenicity of iprodione resistant mutants; and (v) investigate the molecular mechanism responsible for the development of resistance in *B. cinerea* against iprodione.

#### **2. Material and Methods**

#### *2.1. Collection of Samples and Chemicals*

There were one hundred and twelve di fferent isolates of *B. cinerea* collected from di fferent locations of Tian Dong, Tian Yang County, Baise City, Guangxi province, a southern region of China during 2016–2018 (Supplementary Table S1). Single spore isolation was accomplished from diseased leaves and fruits, as described by Fernández et al. [30]. All isolates were stored at 20 ◦C on dried filter paper [31]. Iprodione (96.7%) active ingredient (a.i) fludioxonil (99.2% a.i.) tebuconazole (97% a.i.); original drug, (Shandong Weifang Runfeng Chemical Co., Ltd., Jinan, Shandong, China) used in this experiment. Iprodione (0.103 g) was dissolved in acetone (10 mL) for the preparation of the stock solution and stored at 4 ◦C at dark for further use.

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

To evaluate the drug sensitivity, all strains preliminarily tested at 0.1, 1, 10, 100 μg/mL di fferent concentrations of iprodione based upon previous findings of Grabke et al. [17]. The particular above mentioned concentrations in acetone were amended into PDA medium to examine the inhibitory rate. The inhibitory e ffect of acetone on mycelial growth of *B. cinerea* was 0.00001% in the sensitivity analysis of iprodione content which is ignorable. The mycelial plug of 5 mm diameter of 3d old *B. cinerea* colony was placed in the center of the 90 mm petri plate that contains iprodione amended media. These plates incubated at 23 ◦C for 3 days and radial mycelial growth (colony diameter) of each isolate measured in Petri plate by using a scale in the perpendicular direction and 5 mm original plug subtracted from the whole measurement. The experiment preliminarily repeated thrice, and each treatment had 3 replicates with control (only PDA medium). Those isolates that grew successfully on iprodione amended PDA were considered as resistant and failed as sensitive. Data processing system statistics (DPS version 7.05, Zhejiang, China) was used to analyze the e ffect of di fferent concentrations of fungicides on *B. cinerea* mycelial growth inhibition rate to inhibit the rate of probability, the value of the ordinate (*y*), the concentration of the agen<sup>t</sup> to the value of the abscissa (*x*), obtained virulence linear regression equation *y* = a + b*<sup>x</sup>*, e ffective medium concentration (EC50) value, and correlation coe fficient (*r*).

#### *2.3. Evaluation of B. cinerea Mutants Resistant to Iprodione*

After measuring the colony diameter of *B. cinerea*, the plates were further analyzed to obtain resistant mutants induced by iprodione. The method of UV mutagenesis induced resistance into drug sensitive strains. The mycelial plug of two resistant strains was placed in 1 μg/mL PDA medium containing iprodione and incubated at 23 ◦C. After 3 days incubation, these plates put in preheated, 20W UV lamp, 25 cm irradiation. After applying the treatment, the plates were kept at the incubator in the dark for ten days. After observation, each isolate was transferred to a higher concentration of drug-containing culture plates and repeat the above UV-induced colonies taken at edge cake. The resistant strains were plugged into the PDA medium and continuously cultured for 15 generations and measured as described above. Only stable resistant mutants selected for further analysis (Supplementary Table S2).

#### *2.4. Characteristics of Iprodione Mutants and Sensitive Isolates*

Mycelial growth, sporulation, virulence, cell membrane permeability, osmotic potential, and enzymatic activities were performed to compare the fitness characteristics between the sensitive and resistant strains. Mycelial colony diameter and sporulation were assessed with or without iprodione amended PDA medium. The mycelial colony diameter measured perpendicularly after 24, 48, 96, and 72 h and sporulation after 12, 13, 14, and 15 days of incubation at 23 ◦C.

Mycelium growth assay was conducted on fungicide-free PDA. Mycelial plugs were cut from the borders of 3-day-old colony and transferred to the center of PDA plates. Four plates for each isolate were incubated at 23 ◦C in the dark and colony diameter was measured at two perpendicular directions after 60 h of incubation.

The virulence was assessed on detached tomato leaves as previously described by Fan et al. [32]. Fresh tomato leaves were washed with double distilled water then disinfected by dipping in 75% ethanol for 1–2 min followed by three washings of double distilled water and allow them to dry on filter paper at room temperature. Leaves were placed in a 15 cm petri dish and cover the petioles with a wet cotton ball for moisture. Each leaf was punctured with a sterile lancet (Yangzhou Shuangling Medical Appliance Co., Ltd., Shuangling, China) in the middle as previously applied by Fan et al. [32] than placed in 5 mm mycelial plug on top of the wounds. Lesion diameter recorded with the help of measuring tape from each leaf after 4 days of incubation in the dark at 23 ◦C.

Osmotic sensitivity was measured to evaluate the cell wall elasticity by adding 10, 20, 40, and 80 mg/mL NaCL in PDA medium after 3 days of incubation at 23 ◦C. Mycelial growth inhibition rate (MGIR) calculated by the formula MGIR (%) = (CK-N)/(CK-5) × 100, whereas CK (mm) is the control plate colony diameter, N (mm) is that of a plate containing NaCl amendment. To determine cell membrane permeability, the wild type and its mutant strains were first incubated in 100 mL potato dextrose broth in a conical flask. These conical flasks were placed in a continuous shaker at a temperature 23 ◦C for 3 days. A (5 mm) eight mycelial plug of *B. cinerea* was added in each 250 mL conical flask contains 20 mL solution of iprodione with a concentration of 0, 1, 5, and 10 μg/mL. The conductivities were detected after 0 h, 0.5 h, 1 h, 2 h, and 3 h with the help of DDS-11A conductivity meter (Nanjing T-Bota Scietech Instruments & Equipment Co., Ltd., Nanjing, China) after each treatment. The final conductivities were measured via boiling the mycelium in water for 5 min. Each treatment has three replications. The relative permeability was calculated by using this formula: Relative permeability (%) = (Ct − C0)/C × 100 whereas C0: initial conductivity value; Ct: conductivity value at a certain moment; C: After boiling treatment [14].

#### *2.5. Enzymatic Activities of Iprodione Resistant Mutant and Its Corresponding Wild Type*

Polyglacturonase (PG), Polymethylglacturonase (PMG) and Cellulase (CE) Performed by DNS (3,5-dinitrosalicylic acid) Method Previously Described by Jiang et al. [33]. All three isolates were grown on potato dextrose broth media (PDB) at 120 r/min Shaker culture at 23 ◦C for 3 days. 0.1 g mycelium was grounded in liquid nitrogen in precooled pestle and mortar. Add 5 mL of sodium acetate bu ffer (pH 5.5) and centrifuge at 16,000 rpm for 20 min. The supernatant was collected and stored at 4 ◦C for further enzyme analysis. The substrate used to estimate the PG activity was 1% polygalacturonic acid in 50 mM sodium acetate bu ffer. The reaction mixture contains 0.5 mL sample volume, 0.5 mL substrate and 1 mL sodium acetate bu ffer in an eppendorf tube. The mixture was incubated in water bath at 37 ◦C for 1 h. After incubation add 1.6 mL DNS and boil for 5 min. The absorbance was measured at 540 nm by using a spectrophotometer (Multiskan GO, ThermoFisher Scientific, Boston, MA, USA). The boiled enzyme was used as a control. The standard curve was drawn by taking different concentrations of galacturonic acid. The activity of CE and PMG was measured by using the above mentioned procedure except substrate. For PMG and CE 1% pectin added in 50 mM sodium acetate buffer and 1% CMC dissolved in 50 mM citric acid-sodium citrate buffer respectively. Total proteins were determined by coomassie brilliant blue method [34]. Standard solutions of proteins were prepared by Bovine serum mg/mL also used as control without sample (Supplementary Tables S3 and S4).

#### *2.6. Transcriptome Analysis of Wild and Mutant Strain*

To explore whole sequence analysis, highly iprodione resistant mutant M0 and its wild type W0 were selected according to their fitness stability. Three biological repeats of wild type and mutant were grown in 100 mL of PDB under 0 μg/mL iprodione treatment grouped as (W0, M0). Similarly, three biological repeats of wild type and mutant were grown in 100 mL of PDB amended with 1 μg/mL iprodione and cultured at 220 rpm for 48 hr at 28 ◦C in the dark grouped as (W-ipro, M-ipro). After 2 days mycelia of all treatments were collected, washed with double distilled water, frozen in liquid nitrogen and stored in freezer at −80 ◦C. Untreated wild type (W0), mutant (M0) samples used as a control.

Total RNA was extracted according to the RNA isolation kit (TRIzole reagent, Invitrogen, Carlsbad, California, CA, USA). The purification of RNA was estimated with the Nano Photometer spectrophotometer at 260/280 nm (IMPLEN, California, CA, USA) and integrity was evaluated by assay kit (Nano 6000, California, CA, USA) using Bioanalyzer 2100 system (Agilent Technologies, California, CA, USA). The library generation and RNA sequencing was carried out by staff at I-sanger cloud platform. The further library was prepared by using an NEB-Next Ultra RNA illumina platform [35]. The Illumina platform converts the sequenced image signal to a text signal via CASAVA base calling and stores it in fastq format as raw data. Quality assessment was performed on raw data of each sample including base quality, base error rate and base level distribution statistics to obtain high quality clean reads by using FASTQ for subsequent analysis. The clean reads were mapped into a transcript and compared to a reference genome using Tophat2 alignment software. Some transcripts without annotation to the reference genome were called new transcripts.

### Differential Gene Expression

We identified differentially express genes of two phenotypical groups of strains (Mutant and wild type, with or without fungicide) by using DEseq2 [36]. To estimate the gene expression level fragment per Kilobase of exon model per million fragments mapped (FPKM) tool was used. The statistical difference among genes was analyzed using the recommended Benjamini-Hochberg correction method (*p*-value ≤ 0.05) for controlling the false discovery rate (FDR). Eventually, the fold change (log fold2) and FDR values used as a key indicator the expression amount of different genes among samples and represent a heat map. Functional annotation of genes was performed as described by Wang et al. [14] and Cai et al. [37]. GO enrichment of differential expression of genes was implemented by the GO seq R packages (1.10.1) based on Wallenius' non-central hyper-geometric distribution. KEGG pathways enrichment statistical analysis was performed by KOBAS software.

#### *2.7. Quantitative Real Time PCR Analysis*

For the confirmation of differential gene expression levels attained from the RNA sequencing data analysis, the qRT-PCR investigation was carried out. Total RNA was extracted from mutant and wild type with and without treatment of EC50 concentration of iprodione 1 μg/mL according to the kit instructions (TaKaRa Biotechnol. Co., Ltd., Dalian, China). For the preparation of reverse transcription first Single stranded cDNA was synthesized according to labelled kit instructions (TaKaRa Biotechnol. Co., Ltd., Dalian, China). The expression of seven genes were studied. Moreover, one control gene UBQ used as a reference gene. The primers were designed by using oligo software v7.37 and the

specificity was confirmed by blast against *B. cinerea.* B.010 genome. The sequence of all primers were listed in (Supplementary Table S5). The length of primers fragment were between 19–23 base pair with melting temperature 80 ◦C. 1 μg RNA of each sample was first treated with RNase Free dHH2O and 4 × gDNA wiper Mix (Nanjing Nuo Weizan Biotechnology Co., Ltd., Nanjing, China) for removal of contaminated DNA in the extract. For the preparation of reverse transcription reaction system, the reaction mixture consisted of template cDNA 2 μL, reverse primer 0.8 μL (5 μM), forward primer 0.8 μL (5 μM) and ChamQ SYBR Color qPCR Master Mix 16.5 μL (TaKaRa Biotechnol. Co., Ltd., Dalian, China) of total volume 20 μL. The qRT-PCR reaction was conducted in a thermal cycler (ABI 7500, Hangzhou Langji Scientific Instruments Co, Ltd., Hangzhou, China) with initial temperature 95 ◦C for 5 min, 40 cycles include melting at 95 ◦C for 5 s annealing for 30 s and finally extension at 72 ◦C for 40 s. Three biological repeats of each treatment were performed with triplicate of each gene reaction vs. reference gene. Then changing in fold expression of different genes of the mutant and wild type was evaluated by using algorithm 2−ΔΔCT value. All qRT-PCR data were analyzed by using Light Cycler® 480 software version 1.5.1 (Roche Diagnostics Corporation, Indianapolis, IN, USA).

#### *2.8. DNA Extraction, Cloning and Sequence Analysis of the Tubulin Genes*

For DNA extraction wild type W0 and its mutant M0, mycelium was cultured on potato dextrose broth (PDB) and incubated for 48 h at 28 ◦C under shaking condition (200 rpm). Mycelia was harvested and washed with sterilized water and ethylenediamine tetra acetic acid (EDTA). The DNA was extracted by cetyl trimethylammonium bromide (CTAB) method [6]. The specific primers β-TUB (F-5--TGAAGGTATGGACGAGAT-3-) (R-5--GCATCCTGGTATTGTTGA-3-) under accession number (XM\_001560987.1) and α-TUB (F-5-GTTGGAGTTCTGTGTCTA-3-) (R-5-GTGGTCAAGATGGAGTTA-3-) under accession number (XM\_001555875.1) were used to amplify the complete coding sequence (CDS) of two Tubulin genes *bctubA* and *bctubB*. Three biological replicates of each strain used for DNA extraction and the PCR reactions were conducted three times independently for each sample. The amplified PCR products were purified using a PCR Purification Kit (TIANGEN, Beijing, China), ligated into the pMD18-T Vector (TaKaRa Biotechnol. Co., Ltd., Dalian, China), and then sequenced by Sangon (Guangzhou, China). The exon sequences of the *bctubA* and *bctubB* genes were translated into amino acid sequences and aligned using DNAMAN8.0 software (Lynnon Biosoft, Quebec, Canada) to check the mutation point.

#### *2.9. Statistical Data Analysis*

All values related sensitivity, osmotic potential, enzymatic activities are mean of three replicates was analyzed using statistical software (DPS version 7.05, Zhejiang, China). The LSD test was used to determine significant differences (α = 0.05). Pearson's correlation coefficients were calculated to evaluate the correlation of gene expression obtained by RNA-seq and qRT-PCR using Origin 9.0 software (Origin Lab, Newyork, USA). In the SAM method, the delta value was set to obtain an average. A false discovery rate (FDR) of 5% and the fold change cut-off value was established as 1.5. In LIMMA analysis, genes with a fold change >1.5 and *p* < 0.05 were considered as differentially expressed. Only the genes identified as differentially expressed by both SAM and LIMMA were considered.
