As one of the three deciduous fruit trees, pear is an important fruit crop grown throughout the temperate zone. Pyrus communis L. cv. “Doyenne du Comice” is very popular among consumers because of its gorgeous appearance, rich fragrance, soft and juicy flesh, high commodity value, and health care value (López et al., 2001). However, the fruit decay caused by pathogenic agents has seriously limited the post-harvest shelf life.

Pathogen-induced decay is the most crucial reason for post-harvest fruit loss among many other factors such as environmental conditions, sprouting, quality loss, and overripening (Sommer, 1985; Buchholz et al., 2018). Disease losses in pears are mainly caused by fungi, including blue mold caused by Penicillium expansum, gray mold caused by Botrytis cinerea, bitter rot caused by Glomerella cingulate, and Mucor rot caused by Mucor piriformis (Mari et al., 2003; Sardella et al., 2016; Luciano-Rosario et al., 2020). In recent years, there has been an increasing number of reports of fungal infections in pears on different cultivars worldwide. The interactions between pathogens, including fungi and bacteria, and plants have been extensively studied, but much remains to be explored about the diversity of fruit microbiome during post-harvest storage, especially the pathogens causing fruit decay (Willersinn et al., 2015; Buchholz et al., 2018).

To date, various chemical and physical methods have been used to maintain the quality of fruits and to reduce the damage of pathogens (Wassermann et al., 2019; Aslam et al., 2020). By competing with ethylene for binding receptors, 1-methylcyclopropene (1-MCP), as an ethylene action inhibitor, delays ethylene-mediated physiological and biochemical responses related to fruit ripening. Thus, 1-MCP has been widely used to store and preserve fruits and vegetables as a new fresh-keeping agent (Jiang et al., 2004; Khan and Singh, 2007; Zhang et al., 2012; Li et al., 2013). Several reports have shown that 1-MCP reduces fruit decay in various fruits under proper concentration (Dou et al., 2005; Xu et al., 2017; Min et al., 2018). However, these studies mainly focused on the inhibition effect of 1-MCP on some particular pathogens or diseases; the impact of 1-MCP on the microbial diversity of post-harvest fruits remains unclear. Therefore, we anticipate that this study will contribute to a deeper understanding of 1-MCP using post-harvest fruit storage.

Initially, the effect of fresh-keeping agents on fruit microorganisms was studied using traditional approaches, such as pathogen isolation, colony counts of bacteria and molds, and denaturing gradient gel electrophoresis (DGGE) technology (Sheffield et al., 1989; Feroz et al., 2016; Lin et al., 2018). However, these methods required re-culturing microorganisms in a nutrient medium, which led to the loss of many slow-growing but important microorganisms. The microbiome can greatly delineate the composition, structure, and diversity of microbial populations in various environments. The development of DNA sequencing technology has made the microbiome an efficient and direct means to explore biological diversity in post-harvest fruit (Droby and Wisniewski, 2018; Zhang et al., 2020; Taîbi et al., 2021).

In the present study, we have performed a DNA metabarcoding approach to investigate the fruit microbiome changes induced by 1-MCP treatment in post-harvest pear storage. Furthermore, the relationship among the disease incidence, fruit quality, physiological characteristics, and microbial composition was also demonstrated after the 1-MCP treatment in the pear fruit.

As a specific inhibitor of ethylene receptor, 1-MCP can bind to ethylene receptor in plant cells, thereby preventing ethylene-dependent responses such as fruit ripening and senescence and, thus, extending shelf-life and maintaining fruit quality (Hassan and Mahfouz, 2010). Several studies have shown that 1-MCP can effectively reduce post-harvest fruit decay. For instance, 1-MCP may help preserve tomato fruit by reducing fruit decay caused by Alternaria alternata, B. cinerea, and Fusarium spp. (Su and Gubler, 2012). In jujubes, 1-MCP effectively suppressed the growth of blue mold-induced fruit rot and significantly reduced the incidence of natural decay (Zhang et al., 2012). To examine the effect of 1-MCP on the decay of d’Anjou pear fruit, the application of 1-MCP was found to reduce bull’s-eye rot, Phacidiopycnis rot, stem end gray mold at 300 nl L^–1, and snow-mold rot at 30 nl L^–1 (Spotts et al., 2007). Another study found that 1-MCP treatment significantly reduced post-harvest decay of peach fruit and that disease development was reduced when inoculated with P. expansum (Liu et al., 2005). In apples, the effect of 1-MCP treatment on post-harvest gray mold and its possible mechanisms were investigated (Zhou et al., 2016). In the current research, 1-MCP substantially reduced the disease incidence of pear (P. communis L.cv. “Doyenne du Comice”) by increasing disease resistance, demonstrating that it had a remarkable capacity to mitigate post-harvest diseases of pear fruit.

The next-generation Illumina-based sequencing was used in this study to explore the microbial community of pear fruit treated with or without 1-MCP for 0–15 days of storage. Many fungi and bacteria associated with fruit decay were detected, and their abundance varied among different treatments (Figures 2, 3). In our research, we found the two most common phyla, Ascomycota and Basidiomycota, in fungi associated with pear fruit samples, both of which are known to cause post-harvest fruit diseases (Ren et al., 2019b; Wassermann et al., 2019; Zambounis et al., 2020). The class Dothideomycetes was the most abundant class in both treatments and its abundance increased with the storage time in the present research, which is in line with the studies of endophytic mycobiota in “Jingbai” pear and microbial communities associated with apple and blackcurrant fruits (Vepštaitë-Monstavičë et al., 2018; Ren et al., 2019a). Several families in the Pleosporales order of fungi have infected living plants and caused serious plant diseases (Zhang et al., 2009; Wang et al., 2020). Pleosporaceae is the largest family in Pleosporales, which was characterized as the most abundant family in this study. Members of this family have been reported as plant parasites or saprobes occurring on plant leaves, stems, or fruits (Ariyawansa et al., 2015). At genus level, fungi such as Alternaria, Botryosphaeria, Penicillium, and Fusarium were found to grow significantly during storage in this study, which have been detected in a variety of diseased fruits in previous studies (Campenhout et al., 2017; Parveen et al., 2018; Zhao X. et al., 2019; Chen et al., 2020). In comparison with 1-MCP-treated group, Botryosphaeria and Penicillium were found highly abundant in the control group after 15 days of storage, indicating that 1-MCP may alleviate the disease caused by Botryosphaeria and Penicillium. The growth patterns of most dominant microorganisms indicated that their abundance increased with the extension of storage time. The main reason for this growth pattern is that the decay-causing pathogens constantly proliferated in the fruit.

Moreover, a high number of Proteobacteria were found in many orchard samples including fruits, bark, leaves, and rootstocks by metagenomics (Martins et al., 2013; Liu et al., 2018; Wassermann et al., 2019; Jo et al., 2020). Proteobacteria are usually classified into five groups based on rRNA sequences, which are denoted by the Greek letters α, β, γ, δ, and ε. In the present study, two classes of proteobacteria such as Alphaproteobacteria and Gammaproteobacteria were found to be more prevalent across all the samples (Figure 4), which is consistent with recent studies indicating that bacteria in this taxon are closely related to plant disease (Zhang et al., 2018; Wang et al., 2019). In the current study, the abundance of family Acetobacteraceae increased significantly both in control and 1-MCP treatment, indicating that the members in this family played an important role in fruit decay. Further results showed that the Gluconobacter genus, an important member of the family Acetobacteraceae, increased significantly after 15 days of storage. In accordance with the present results, previous studies have demonstrated that genus Gluconobacter were closely related to the fruit decay in apple, pear, and apricot (Van Keer et al., 1981; Liu et al., 2020). However, Bevardi, Frece (Bevardi et al., 2013) reported the biocontrol abilities of Gluconobacter oxydans strain isolated from the apple surface against post-harvest infecting fungus P. expansum. Current research indicates that members of this genus may grow on the surface of pears as an antagonistic or saprophytic bacterium, but more isolation and identification studies are required to validate this hypothesis.

Biomarker discovery is one of the most important means being used for reflecting the lifestyle and disease of various hosts (Di Paola et al., 2011; Segata et al., 2011; Zhimo et al., 2021). It has been proven that Random Forest can effectively, stably, and accurately classify microbial community samples (Yatsunenko et al., 2012; Chen et al., 2021). Botryosphaeria and Fusarium were identified as biomarkers in this study to distinguish between 1-MCP treatment and untreated control pear fruits. Notably, members of these two fungal genera have been reported as the pathogens that cause fruit decay in a variety of fruits (Rittenburg, 1983; Garibaldi et al., 2012; Sever et al., 2012; Tang et al., 2012; Zhao X. et al., 2019; Zhao Z.Y. et al., 2019).

Furthermore, correlation matrix revealed a few positive and negative correlation patterns among the dominant fungal and bacterial genera. Lactobacillus and Muribaculaceae were found highly correlated in the microbial community of pear, which seemed to be consistent with other research that found Lactobacillus plantarum restored the gut microbiota imbalance by manipulating relative abundances of certain bacteria in Muribaculaceae (Gao et al., 2021). Gluconobacter and Botryosphaeria were also found to be correlated during the process of fruit decay, indicating that these pathogens could coexist and may be functionally related (Tournas and Katsoudas, 2012). In addition, there were several negative correlation patterns among the microbes, for instance, Alternaria and Malassezia, Alternaria and Asticcacaulis, Gluconobacter and Aquabacterium, Gluconobacter and Asticcacaulis, and Gluconobacter and Sphingomonas, which may be due to the high abundance of Alternaria and Gluconobacter inhibiting the growth of other microorganisms.

In order to show the effect of 1-MCP on fungi, fluorescence staining experiment was conducted to determine the membrane damage of spores caused by 1-MCP. The results showed that 1.0 μl L^–1 of 1-MCP had little effect on the integrity of fungal cell membrane, suggesting that 1-MCP does not inhibit the development of fungi by damaging cell integrity of spores. Several studies have shown that 1-MCP decreased fruit firmness during storage but was delayed (Villarreal et al., 2010; Moises et al., 2017; Cheng et al., 2019), which has also been confirmed by the present findings (Figure 5). Fruit softening is closely related to the activities of cell wall hydrolases, such as polygalacturonase (PG), pectin methyl esterase (PME), and β-galactosidase (β-Gal). It has been reported that 1-MCP significantly lessened the fruit decay by reducing the activities of PG, PME, and β-Gal (Zhang et al., 2019). Therefore, 1-MCP is likely to minimize fruit decay by inactivating the pectinase activity in fruit, maintaining high fruit hardness, and preventing pathogen infection, rather than inhibiting pathogens directly. The RDA findings in this study obviously revealed the relationship between fruit quality and microbial community after 1-MCP treatment. Botryosphaeria, Penicillium, and Fusarium were shown to be positively correlated with disease incidence and respiration rate, and negatively correlated with fruit firmness (Figure 6), suggesting that these fungal genera are involved in fruit decay and quality change during storage.

Findings in this study suggest that 1-MCP treatment effectively reduced the disease incidence and induced multiple changes in fungal and bacterial microbiota in “Doyenne du Comice” pear fruit. In contrast to the control, decay-causing fungi such as Botryosphaeria, Penicillium, and Fusarium were suppressed by 1-MCP treatment and their quantity was positively correlated with TA and respiration rate but negatively correlated with fruit firmness. Thus, this study investigated the microbiome responses to 1-MCP during storage and revealed a relationship between fruit decay and microbial composition in “Doyenne du Comice” pear fruit.

The datasets generated for this study can be found in the Sequence Read Archive (Accession: PRJNA739857).

YZ and CG performed the experiments and analyzed the data. JG designed the study and provided the funding. YZ, MM, and JG wrote the manuscript. YC, CW, and YG did the visualization and reviewed the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.