**4. Discussion**

Fungicide resistance in different pathogens has been a major problem in crop protection worldwide in two decades. The extensive use of fungicides to control pathogens in same area for several seasons creating this problem; as a result the efficacy of fungicides decreasing and resistance induced into the pathogens against a specific or multiple fungicides is increasing. Therefore, it is necessary to evaluate the resistance risk in the lab before new fungicides are widely practice into the field. In Guangxi province, grey mould, caused by the fungal pathogen *B. cinerea*, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In the present study, 112 isolates of *B. cinerea* collected from major tomato production area of Guangxi province, China and screened with different concentrations of iprodione and found that toxicity steadily increased in mutants. This is considered as the first report to assess iprodione sensitivity of *B. cinerea* collected from Guangxi Province China. Previously sensitive isolates of *B. cinerea* on tomato were detected in Germany [38]. All strains were sensitive to iprodione from 0.07 to 0.87 μg/mL with an average of 0.47 μg/mL. These results are similar to *B. cinerea* collected from strawberry, a procymidone and zoxamide sensitive strain from Hubei province having EC50 value of 0.25 μg/mL and 0.360 μg/mL, respectively [22]. No 100% sensitive strain was discovered in our collected samples because iprodione is a site specific fungicide with high efficacy, less toxicity and lower application rate. The high dozes or sustained application of fungicides leads to put selection pressure to develop fungicide resistance [39]. In order to solve this problem, there was an urgen<sup>t</sup> need to develop effective resistance managemen<sup>t</sup> strategies.

High frequencies of *B. cinerea* resistant isolates have been recorded not only iprodione but also to various groups of fungicides including DC, MBC, and PP across the globe [40–42]. Although field resistant mutants of iprodione was documented in Northeatern part (21%), Henan (8%), and also in Anhuai (5%) of China [18,43]. This study was showed low frequency of resistant mutants as compared to the previous one, but with the passage of time resistance level will increase with rapid and continuous application of DC's fungicides. In the present work, UV irradiation used to detect specific iprodione resistant mutants to assess the risk from emergence strain. After stability and sensitivity testing the EC50 of UV mutants (M0, M1) exceeded 1 μg/mL which was higher 1025.74 μg/mL and 674.48 μg/mL respectively than corresponding wild strain (W0). The cell membrane permeability was significantly increased in M1 and wild type than M0 which indicate M0 is more stable. In contrast CE and PG enzyme activity were higher in M0. Polygalactronase family genes (*Bcpgx1* and *Bcpg3*) involved in pathogenicity expression were higher in M-ipro than wild type. These findings were distinction with the Guo et al. [44] found that resistant strains when continuously dealt with fungicides, pathogens lose

its potential and viability resulted in failure of infection. The virulence of M0 like as wild type while M1 results supported the previous findings of Chen et al. [45] mutant strain of *Verticillium dahliae* lose its pathogenicity by the repressed of cytochrome p450 gene expression. Comparative genomic studies have been frequently conducted to understand the expression of selected genes against di fferent chemicals. However, inadequate information is available due to a lack of whole genomic sequencing (RNA seq data) and suitable methodologies for comparative transcriptome. In recent studies, transcriptome analysis has been widely applied on fungicidal resistant plant pathogens, including Fusarium spp. [40], *B. cinerea* [37], and *Penecillium didgitatum* [46]. According to our transcriptome analysis the expression of lysophospholipase genes, transmembrane transporter genes, MF (multifunctional genes), MFS (super family genes) encoding, amino acid and carbohydrate metabolism genes were clearly upregulated in field mutant (M0, M-ipro) than wild type (W0, W-ipro) with or without application of iprodione (Supplementary Table S15 Excel sheet). Many microorganisms produce phospholipases heterogeneous groups of enzymes, either secreted or induced intracellularly by physical disruption of the cellular membrane [47]. Among them lysophospholipases are key enzymes that hydrolyze the esters linkages in glycephosopholipids and contribute to detoxification of potentially cellular lysophospholipids that facilitate the survival of fungi in vivo, cell wall integrity, proliferation expression of virulence, fungal cell signaling and immunomodulatory pathways [48]. Here, the expression of lysophopholipase genes (*BCIN\_13g03170*, *BCIN\_02g08890*) were superiorly upregulated in M-ipro vs. W-ipro than M0 vs. W0 (Figure 7) contribute to iprodione resistance. *BCIN\_02g08890* is highly upregulated in mutant strain with or without fungicide and absent in wild type.

Plant cell wall mainly composed of polysaccharides with less amount of glycoproteins esters, mineral contents, phenolic compounds and enzymes [49]. The predominant polysaccharides are cellulose, hemicellulose galacto (mannans, xylans, and xyloglucans), and pectin. Carbohydrate-degrading enzymes of pathogens constitute a key factor involved in the metabolic breakdown of glycoconjugates, oligosaccharides, and polysaccharides of host plant cell wall components during infection or invasion [50]. Cell wall degrading enzymes are abundantly found in *B. cinerea* [51]. Recent studies revealed that cellulase, xylanase, and pectinase (glucanase) enzymes functioned as a virulence factor in phytopathogens and were recognized as PAMPS by plants to trigger the PTI responses, during host plant–pathogen interactions [52].

In this study, we analyzed the enzymatic activity of cellulase, polygalactrose, and polymethylgalactrose in mutant and wild type *B.cinerea*. Enzymatic assays and gene expressions of cellulase (*BCIN\_12g06630*, *BCIN\_16g03020*) and xylanase A (*BCIN\_03g03480*, *Bcxyn11A*) were more upregulated in mutant than wild type (Figure 7). Cellulase catalyzes the degradation of the β-1,4- glycosidic bonds in cellulose [53]. Cellulase is an elicitor in plant–pathogen interactions but its enzymatic activity is independent of its elicitor. In contrast, it was previously reported that xylanase activity promotes the necrotic infection of *B. cinerea* into plant tissues [54]. *Bcxyn11A*, an endo-β-1,4-xylanase degrades plant cell wall xylan contents, and is required for successful infection. Furthermore, a small nanogram of xyn A was su fficient as an elicitor in *S.lycopersci* and *N. benthamiana* [55]. In contrast, not all fungal xylanases have been conclusively involved in pathogenicity and virulence [51]. The *B. cinerea* equipped with di fferent patterns of endopolygalactrase (*Bcpg*) genes and exopolygalactrase (*Bcpgx*) genes to degrade pectate machinery of host cell. In the current study, the expression of *Bcpg2* and *Bcpgx1* genes were highly upregulated in (M-ipro vs. W-ipro) and there is no change in *Bcpg1* and *Bcpg4* in all treatments. *Bcpg2* is a necessary gene during primary infection and lesion expansion in tomato [56]. A major result of our work is reported that there is no diversification in virulence genes of mutant and wild type before and after iprodione application. *B. cinerea* secretes several genes of the aspartic proteinase (AP) family to perform proteolytic activity. A functional analysis of our results showed that there is no change in the expression of *Bcap* genes of wild and mutant type after iprodione application (Figure 6). No significant di fference was found in *Bcap1-5* genes mutants and the wild type strain of B05.10.

The resistance of fungi is sturdily associated with multiple mechanisms, including (1) the nonsynonyms mutation in the target protein encoding genes, (2) the upregulation of the target proteins, and (3) the overexpression of transporting and membrane encoding genes. Fungal e fflux pumps such as cytochrome P450 are the most versatile natural bio-catalyst genes and constitute a large superfamily related to the detoxification of fungicides, insecticides, and xenobiotics under mild conditions [57,58].

The mechanism of resistance to iprodione was associated with point mutations in the tubulin gene that changes the structure of the fungicide binding site to decrease sensitivity according to Grabake et al. [28]. According to the genome sequencing information, two tubulin genes showed mutation. A point mutation at codons 593 (A198E) and 599 (F200Y) in *bctubA* gene and at 646 codon (R216C) in *bctubB* were detected from resistant strain M0, and a similar mutation was reported in a field isolate of *B. cinerea* resistant to benzimidazole that had a mutation at points A198E and F200Y [59]. A novel point mutation at codon 646 (R216C) was detected in *bctubB* in mutant (M0). Contrarily, point mutation variations at single codon I365S/N/R of the *Bos1* gene were responsible for dicarboximide (iprodione) low resistance, as reported from France, England, Israel, Japan, New Zealand, Italy, Switzerland, and the United States. Iprodione reduced DNA, RNA synthesis in the germinating fungal spore and inhibited the enzymatic acivity of NADH cytochrome c reductase, thereby preventing lipid and membrane synthesis and ultimately mycelium growth [60]. Wang et al. [14] reported that the resistance in *B. cinerea* to fenhexamid mainly relied on the mutation of *BCIN\_16062* encoding P450 gene. In a hypersensitive strain of *Candidas albicans*, *CaALK8* gene was promoted and confers multidrug resistance [61]. The expression of *BCIN\_01g03510* and *BCIN\_13g05140* were upregulated in mutant (M0) relative to wild type (W0, W-ipro) (Figure 6). Glutathione Stransferases are multifunctional detoxification enzymes that regulate the cell functions, countering oxidative stress and signal transduction with several resistance mechanisms [62,63]. Our findings revealed that *BCIN \_08g01800* was highly downregulated in wild type (W1-ipro) after iprodione application while other genes showed similar expression. Remarkably, transmembrane proteins in fungal e fflux systems (ABC and MFS transporter genes) have been reported to provide protection for fungal cells against antibiotics and fungicides found in the environment [64]. Furthermore, these transporters determine the baseline of sensitivity or resistance to fungicides [65]. Several genes encoding transmembrane transporters were identified from the RNA sequencing data (Figure 6). Most of them showed higher expression in mutant strain than wild type. Particularly, two MFS encoding genes *Bcstl1* and *BCIN\_08g01780* were highly upregulated in mutant (M-ipro) after iprodione treatment than M0 and wild type (W-ipro, W0). Intriguingly, *BCIN\_14g01090* gene logfold2 value were intensely upregulated before and after iprodione application in mutant (M-ipro, M0) and downregulated in (W-ipro, W0). The high expression of drug e fflux transporter has been reported in di fferent isolates of *B. cinerea* against di fferent classes of fungicides [66,67]. On the other hand, Grabke et al. [17] reported the overexpression of MFS encoding genes of *B. cinerea* conferring a low level of resistance to iprodione in strawberry. Contrarily, MFS genes contributed to resistance in *Fusarium* spp against prochloraz and carbendazim fungicides [40,46]. Likewise, *BCIN\_14g04470* and *BCIN\_12g02430* in fungal e fflux were highly upregulated in mutant strain (M0) relative to wild type (W0).
