Apple (Malus domestica Borkh.), one of the most economically important fruits widely consumed worldwide, has been suffering from apple ring rot caused by Botryosphaeria dothidea, which dramatically affects its quality and yield. In the present study, we demonstrated that Pseudomonas protegens, isolated from Chinese leek (Allium tuberosum), significantly suppressed the mycelial growth and propagation of B. dothidea, respectively, further displayed a considerably inhibitory effect on the apple ring rot of postharvest fruits. In addition, P. protegens significantly improved the total soluble solid/titrable acidity (TSS/TA) ratio and soluble sugar/titrable acidity (SS/TA) ratio and drastically maintained the fruit firmness. Further analysis manifested that P. protegens substantially induced the defense-related genes such as MdGLU, MdPAL, MdPOD, MdCAL, and transcription factors related to the resistance to B. dothidea, including MdWRKY15, MdPUB29, MdMyb73, and MdERF11 in apple fruits. Meanwhile, P. protegens considerably restrained the expressions of the pathogenicity-related genes in B. dothidea, including the BdCYP450, BdADH, BdGHY, BdATS, Bdα/β-HY, and BdSTR. By inference, P. protegens inhibited the apple ring rot on postharvest fruits by activating the defense system of apple fruit and repressing the pathogenic factor of B. dothidea. The study provided a theoretical basis and a potential alternative to manage the apple ring rot on postharvest fruits.

Apple (Malus domestica Borkh.) is one of the most widely produced and economically important fruits in temperate regions (Chen et al., 2021). China is the largest producer in the world, with harvested areas of 1.91 million hectares (41.01% of the world total) and 40.50 million tons (46.85% of the world total) in 2020 (FAOSTAT, 2022).1 In recent years, apple fruit has been among the most widely consumed fruits in the world since they are available the whole year, inexpensive, and convenient to consume. Above all, apple contains a range of nutrients with different biological activities, including polyphenols, vitamins, minerals, lipids, proteins/peptides, and carbohydrates (Koutsos and Lovegrove, 2015; Pollini et al., 2021), which are beneficial for our health, as per the saying, “an apple a day keeps the doctor away” (Davis et al., 2015).

Unfortunately, due to the lack of resistant varieties, continuous cropping, and poor field management, apple orchards in China often suffer from pests and diseases. Apple ring rot, caused by Botryosphaeria dothidea, is one of the most destructive apple diseases worldwide, including in China, Japan, South Korea, United States, Australia, and South Africa (Wang et al., 2018). B. dothidea infects apple trees, resulting in fruit rot, twig dieback, stem and branch canker, and tree death (Dong and Guo, 2020). Besides, B. dothidea often infects the apple fruits during the early growth stages, remains latent, and causes fruit rot during ripening or storage (Yu et al., 2022). The decayed fruit incidence caused by the disease usually ranges between 10 and 20% each year and may reach 70% in seasons with conditions conducive to fungal development (Zhao et al., 2016). According to an investigation, the average occurrence of the disease was as high as 77.6% in 88 apple orchards across seven main apple production areas in China (Guo et al., 2009). Therefore, apple ring rot has seriously impeded the sustainable and healthy development of the apple industry in China.

Currently, chemical fungicides are still the primary strategies for controlling apple ring rot disease (Dai et al., 2017; Song et al., 2018; Fan et al., 2019). However, these fungicides were potentially hazardous to human health, other non-target organisms, and the natural environment. In addition, pathogenic microorganisms may evolve fungicide resistance (Yin and Qiu, 2019). Given the above-described concerns, there is an urgent need to research and develop alternative measures for preventing and controlling the disease.

Biological control is a safe way to control pests and pathogens. Antagonistic microorganisms showed tremendous potential for substituting chemical fungicides to manage plant pathogens (El-Hasan et al., 2017; Fan et al., 2020; Zhao et al., 2021). It is reported that Pseudomonas spp. have been widely studied for their biocontrol potential. For example, P. chlororaphis significantly reduced the postharvest gray mold on Chinese cherry (Wang C. et al., 2021). P. fluorescens drastically suppressed the blue mold disease of postharvest citrus fruits (Wang Z. et al., 2021). P. segetis markedly reduced soft rot symptoms in potatoes (Rodriguez et al., 2020). P. aeruginosa considerably controlled several pathogens in tomatoes, potatoes, taro, and strawberries (Ghadamgahi et al., 2022). P. putida strongly reduced the common bean rust disease severity (Abo-Elyousr et al., 2021). P. synxantha significantly reduced brown rot incidence and severity on peaches (Aiello et al., 2019). P. parafulva effectively controlled the soybean bacterial pustule (Kakembo and Lee, 2019). In addition, P. protegens had intense antifungal activity against various plant pathogens, including Heterobasidion abietinum, Heterobasidion annosum, Heterobasidion irregulare, and Heterobasidion parviporum (Pellicciaro et al., 2021), Calonectria pseudonaviculata (Yang and Hong, 2018), Rhizoctonia solani (Jing et al., 2020), Fomes fomentarius, Ganoderma lucidum, Phellinus pini, Phellinus tuberculosus, Sclerotinia sclerotiorum, Alternaria tomatophila (Prigigallo et al., 2021), Alternaria alternata, Aspergillus niger, Penicillium expansum, Neofusicoccum parvum (Andreolli et al., 2019), Botrytis cinerea, and Monilinia fructicola (Zhang et al., 2020). In our preliminary study, we isolated an endophytic bacterium P. protegens from Chinese leek (Allium tuberosum). The present study demonstrated the antifungal activity against B. dothidea and the inhibitory effect on ring rot on postharvest apple fruits. Further, we primitively uncovered the underlying mechanism from the apple fruit aspect and the pathogen B. dothidea aspect to provide a theoretical basis and an alternative for controlling the apple ring rot on postharvest fruits.

Apple fruits (Malus domestica, cv. Fuji) were purchased from the local market. Fruits in uniform size and color, free of visible disease and mechanical injuries, were selected for the experiments. The endophytic bacterium P. protegens isolation NSJ-2101 and the pathogen fungus B. dothidea isolate LW-1801 were cultured on the nutrient agar (NA) and potato dextrose agar (PDA) medium, respectively, and kept in our laboratory.

We conducted the experiments using the PDA and potato dextrose broth (PDB) medium.

The PDA medium (20 ml) was poured into a 9-cm-diameter Petri dish. One mycelium disc (0.5 cm in diameter) of the fungus B. dothidea was inoculated in the center of the Petri dish. Then one disc of P. protegens was inoculated on each side 2 cm from the fungal disc (Pp). Sterilized water (50 μL) was used as the control (CK). All the Petri dishes were inverted and incubated at 28°C in the dark for 3 days. The diameters of the fungal colony were measured daily to evaluate the inhibition of P. protegens on the mycelial growth B. dothidea. After that, P. protegens-treated mycelia and the control mycelia were carefully picked from the Petri dishes and observed under a microscope (EVOS Auto2, Thermo Fisher Scientific, United States) with × 40 magnification. The experiments were repeated three times, and five replicates were included for each sample in each experiment.

Various amount (2.5, 5, and 10 ml) of P. protegens culture (OD600 = 0.6) was added into a 150 ml Erlenmeyer flask, respectively. PDB medium was appended until it reached a total volume of 50 ml, making the concentrations of the P. protegens 5, 10, and 20%. 50 ml of PDB medium was used as a control. Then, one mycelium disc (0.5-cm-diameter) of B. dothidea was put into the Erlenmeyer flask. The Erlenmeyer flask was incubated at a constant temperature shaking incubator at 28°C, shaking at 200 rpm for 2 days. After centrifugation, the precipitate was collected and weighed to evaluate the suppression of P. protegens on the propagation of B. dothidea. The experiments were performed in triplicates.

According to the different ways of inoculating fungus B. dothidea, we performed two experiments to test the control effect of P. protegens on apple ring rot.

The apple fruits were surface-sterilized with 1.5% NaOCl for 5 min, washed thrice with distilled water, and air-dried. Then a tiny wound (3 mm width × 5 mm deep) was created at the equator in each apple fruit using a sterile needle. Next, 20 μl of P. protegens (OD600 = 0.6) was added into the small cavity. One hour later, a B. dothidea mycelial disc (3 mm in diameter) was inoculated into the wound. Again, 20 μl of the nutrient broth (NB) medium was used as a control. Finally, all the apple fruits were packed into a black plastic box and incubated at 28°C under dark conditions for 3 days. The disease spot diameter was measured to evaluate the inhibitory effect of the P. protegens on the apple ring rot incidence on fruits. The experiment was repeated ten times.

First, the apple fruits were surface-sterilized and air-dried, as in experiment 1. Then the apple fruits were immersed in P. protegens culture (OD600 = 0.6) for 15 min and soaked in B. dothidea (OD600 = 0.6) culture for another 15 min after they were air-dried at room temperature for 1 h (Pp + Bd). In addition, the apple fruits that were soaked in NB medium for 15 min, air-dried at room temperature for 1 h, then dipped in B. dothidea culture for 15 min were used as the control (Bd). Next, all the fruits were packed in plastic bags and incubated at 28°C. Finally, the disease symptom, such as mycelial cluster and disease spots, were observed and recorded on 1, 3, 5, 7, and 9 days to evaluate the inhibitory effect of P. protegens on apple ring rot. The experiment was performed in triplicate.

To fully confirm whether P. protegens affected the internal quality of apple fruit, we designed four treatments (CK, Pp, Bd, and Pp + Bd). (1) CK: The apple fruits were soaked in PDB medium for 15 min. (2) Pp: The apple fruits were soaked in P. protegens culture (OD600 = 0.6) for 15 min (Pp). (3) Bd and (4) Pp + Bd were described above. All the fruits were packed in plastic bags and set in an incubator at 28°C. Then, fruits were sampled and peeled 9 days later. Fruit firmness was measured at the fruit’s equator with the FHM-5 fruit hardness tester (Takemura Electric Works Ltd., Tokyo, Japan). The pulp at the fruit equator was sampled to determine other internal quality indexes, including total soluble solid (TSS), soluble sugar (SS), titratable acidity (TA), vitamin C (VC), total soluble solid/titrable acidity (TSS/TA) ratio, and soluble sugar/titrable acidity (SS/TA) ratio. TSS content was determined using a PAL-1 type sugar concentration detector (ATAGO, Japan). SS, TA, and VC were determined using the anthrone colorimetric, NaOH titration, and 2, 6-dichloroindophenol colorimetric, respectively (Yang L. et al., 2020). Three replicates were included for each sample at each sampling time point.

To determine whether the reduction of the fruit disease symptom was caused by the P. protegens induction of the fruit defense system, we detected the expressions of the defense-related genes and the transcription factors in apple fruits. The defense-related genes contained phenylalanine ammonia-lyase (MdPAL; XM_008368428.3), glucanase (MdGLU; XM_029095631.1), peroxidase (MdPOD; XM_029099288.1), and catalase (MdCAT; XM_008375181.3). The transcription factors included MdWRKY15 (XM_008395555.3), apple U-box E3 ubiquitin ligase 29 (MdPUB29; XM_008350166.3), MdMYB73 (XM_008379837.3), and apple ethylene response factor 11 (MdERF11; NM_001301117.1), which were reported to enhance the resistance of apple fruits to B. dothidea.

According to previously described methods, the four treatments (CK, Pp, Bd, and Pp + Bd) were performed on apple fruits. All the fruits were packed in plastic bags and incubated at 28°C. One day later, the fruits were sampled and peeled around the equator with a sterilized paring knife. All the samples were stored at –80°C for later qRT-PCR.

We examined several published pathogenicity-related genes to verify whether the reduced disease symptom in the apple fruits was also associated with the repressed expression of vital gene in B. dothidea by P. protegens. These genes included cytochrome p450 protein (BdCYP450; BOTSDO04127), alcohol dehydrogenase (BdADH; BOTSDO00477), glycoside hydrolase (BdGHY; BOTSDO02303), aminoacyl-tRNA synthetase (BdATS; BOTSDO01704), alpha/beta hydrolase (Bdα/β-HY; BOTSDO04588) and sugar transporter (BdSTR; BOTSDO09350).

A mycelium disc (0.5 cm in diameter) of B. dothidea was inoculated on a layer of sterilized cellophane placed on the newly prepared PDA medium. The Petri dish was incubated at 28°C in an inverted position for 2 days in the dark. Then P. protegens (2 ml; OD600 = 0.6) was sprayed on the fungal mycelia (Pp). Sterilized water (2 ml) was used as the control (CK). The Petri dishes continued to be incubated in the same condition. Six hours later, the P. protegens-treated and the control mycelia were sampled for qRT-PCR.

The abovementioned gene sequences of the apple and the mycelium were retrieved from the apple genome (ASM211411v1) and the B. dothidea genome (ASM1150312v2) that were downloaded from the National Center for Biotechnology Information (NCBI) genome website.2 Then, the special primers were designed using Primer Primer 5 according to the respective gene sequences (Supplementary Table 1) and synthesized in Sangon Biotech (Sangon Biotech, Shanghai, China).

The total RNA of the prepared apple and the mycelia samples were extracted using RNAprep Pure Plant Plus Kit [Tiangen Biotech., Beijing, China]. And the cDNA was synthesized using HiScript RIII RT SuperMix for qPCR (CgDNA wiper; Vazyme Biotech, Nanjing, China). qRT-PCR was performed using ABI7500 Thermal Cycler (Applied Biosystems, Foster City, CA, United States) to detect the relative expressions of the genes mentioned above. The MdActin and the BdTubulin were used as the apple’s and mycelia’s internal reference genes, respectively. The relative expression was calculated by 2–ΔΔCT method (Livak and Schmittgen, 2001). The experiment was performed in three replicates.

Analysis of variance (ANOVA) was conducted using SAS 8.0 software (SAS Institute Inc., Cary, NC, United States). A least significance difference test (LSD) was applied to determine the significance between different treatments (p