Effect of Plant Preservative MixtureTM on Endophytic Bacteria Eradication from In Vitro-Grown Apple Shoots

Endophytic contaminants are a common problem for the in vitro propagation of woody plants and have significant economic repercussions for the conservation of plant genetic resources and commercial micropropagation. In this study, first, the microbial contamination that appeared around the base of in vitro-grown apple shoots was identified as Bacillus megaterium. Then, plant preservative mixture (PPMTM) was used as a bactericidal agent in plant tissue culture. Its efficacy for eradicating endophytic B. megaterium in in vitro cultures of apple was tested. In vitro-contaminated shoots were grown in tissue culture medium supplemented with 0.2% v/v PPMTM for 12 weeks and then transferred to medium without any PPMTM and cultured for 24 weeks. This study showed that PPMTM is an effective agent for controlling the growth of B. megaterium. Our results highlight the species-specific response of apple shoots to PPMTM. PPMTM was effective in controlling endogenous microbial contaminations from apple varieties ‘Golden Delicious’, ‘Landsberger Renette’, ‘Suislepper’, and ‘Aport krovavo-krasnyi’; meanwhile, in ‘KG 7’ and ‘Gold Rush’, all the plants grown in the absence of PPMTM were still bacterially contaminated, even though they were pre-treated for 12 weeks in PPMTM-supplemented medium. These results therefore suggest the essentiality of further testing of extended incubation of PPMTM in these cultivars that had outbreaks of bacterial contamination.


Introduction
Micropropagation is one of the advanced methods for vegetative propagation of plants [1][2][3]. In vitro tissue culture supplements conventional propagation methods in the production of healthy, disease-free plants and allows the propagation of a large number of progeny plants with desirable traits throughout the year in a short time and reduced space [3][4][5][6][7][8]. In addition, micropropagation plays a key role in the conservation of plant genetic resources [1,9,10].
A micropropagation protocol proceeds through a series of stages, starting with the establishment of aseptic cultures [3]. Avoiding microbial contamination of plant tissue cultures is critical to successful micropropagation [11]. Despite extensive surface sterilization and the use of best practices for culture maintenance, the contamination of plant tissue may be a persistent problem during micropropagation [12][13][14]. This source of contamination is known as endophytes, which are of particular concern because they can survive surface sterilization [14]. Endophyte contamination causes culture losses and directly affects the efficiency of micropropagation protocols [13]. These microorganisms live within plant tissues and may not be visible until after several rounds of subculture and/or after the cultures have undergone some stress [12][13][14][15]. For example, many plant species have shown controlling endogenous contaminants, its impact on plant growth should be tested before using it at a commercial level.
In view of the reported efficacy of PPM™ for controlling endogenous microbial contaminants, this study was taken up to assess the effectiveness of PPM TM at the concentration recommended by its manufacturer in eradicating endophytes from contaminated, in vitrogrown apple shoots. Currently, there are no previously published studies on PPM TM use to eradicate endophytes from in vitro-grown apple cultures.

Results and Discussion
Endophytic contaminants have been a common problem for the in vitro propagation of woody plants and have significant economic repercussions for the conservation of plant genetic resources and commercial micropropagation [14,53,54]. Although different strategies have already been described to minimize the deleterious effects of endophytes, the effectiveness of the treatment may be dependent on the plant species and cultivar, the source of materials, propagation methods, and explant tolerance [29,32]. Culture media containing antimicrobial compounds may suppress or eliminate microbial growth [32,[42][43][44]. Although some success has been reported using antibiotics, they often suppress the growth of bacteria rather than eliminate them from in vitro cultures [55][56][57][58]. In this study, as an alternative to using antibiotics, PPM TM was tested for controlling the endophyte contaminants present in in vitro cultures of apple.
The microbial contaminants that appeared around the base of the in vitro-grown apple shoots and on the surface of the medium were identified as Bacillus megaterium de Bary. Strains identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) had a score varying from 1.7 to 2.2, indicating reliable identification (Supplementary Table S1). B. megaterium is a relatively harmless Grampositive, rod-shaped bacterium found in diverse habitats but commonly in soil [59][60][61][62][63]. This bacterium has already been identified as an endophytic contaminant in several plant species such as cotton [64], sweet corn [64], coffee [65], ginger [66], black pepper [67], Ilex paraguariensis [68], etc.
Previously, bacterial strains of the genera Bacillus were recognized as a safe group, producing substances that are beneficial for plant development [69][70][71]. B. megaterium isolates are known to promote plant growth [72][73][74][75] and biocontrol ability against plant pathogens [67,[76][77][78][79][80]. Recently, Wang et al. [81] showed that B. megaterium (WL-3 strain) had a strong ability to control late blight (Phytophthora infestans), one of the most destructive diseases in potato. They observed the antagonistic effect of B. megaterium against potato late blight, as well as the promotion of potato plant growth in plant tissues in vitro [81]. Similarly, working on the mulberry tree, Ou et al. [82] found that B. megaterium (HGS7 strain) consistently exhibited antagonistic activity against ten phytopathogens in vitro, especially for Sclerotinia sclerotiorum and Scleromitrula shiraiana. In addition to the antifungal traits, they also found that B. megaterium had strong tolerance to abiotic stress both in vitro and in planta [82]. Several studies have shown that endophytes are common in tissue culture and demonstrated their beneficial effects as plant growth-promoting agents [13,23,[26][27][28][83][84][85][86]. Interestingly, working on apple plants grown in vitro, Tamošiūnė et al. [87] noted contrasting effects of bacterization with endophytic bacteria strains of Bacillus and Pseudomonas spp. on shoot biomass accumulation and the proliferation of auxiliary shoots. They observed that the stimulating or suppressing effect on shoot growth and proliferation was related to the specific interaction between plant host and endophytic bacteria strain [87].
Herein, latent or non-latent infections by B. megaterium did not affect the growth of the apple shoot cultures; however, when shoot tips, apparently from clean in vitro plants, were exposed to the cryopreservation procedure, endophyte outbreaks arose and compromised the shoot tip regrowth (Supplementary Figure S1). The negative impact of endophytes on the recovery of the cryopreserved shoot tips was previously reported [30][31][32][33]. Recently, Bettoni et al. [49] found that the addition of 1.5% PPM TM (v/v) to pretreatment and preculture media was effective at controlling endophytic bacteria in cryopreserved Vitis shoot tips. They observed that the addition of PPM TM prior to shoot tip excision was essential to eliminate endogenous microbial contaminants during the recovery process [49]. Beneficial effects for Vitis, however, may not be evident if PPM TM (1.5% v/v) is added only to the regrowth medium; despite still having a suppressive effect on the growth of contaminants, it has also been shown to have a negative impact on shoot tip regrowth (J.C. Bettoni, personal communication). Therefore, to effectively carry out future cryopreservation experiments, we are faced with the task of eliminating endogenous microbial contaminants by B. megaterium from in vitro apple plants. In vitro-contaminated shoots were grown in Murashige and Skoog [88] (MS)-based tissue culture medium with the incorporation of 0.2% v/v PPM TM for two cycles of 6 weeks each (Experiment 1, E1) and then transferred to the same medium but without PPM TM and cultured for an additional four cycles of 6 weeks each (Experiment 2, E2). It was possible to control the identified endophyte contaminants using PPM TM ; however, the efficacy of the treatments varied according to the apple cultivar as well as the PPM TM incubation period (Table 1). Table 1. Percentage of aseptic shoots of in vitro cultures of apple (Malus spp.) grown for 12 weeks (two passages of 6 weeks each) in culture medium supplemented with 0.2% v/v Plant Preservative Mixture (PPM™) (Experiment 1, E1) and, subsequently, in pre-treated shoots from E1 grown in culture medium without PPM™ for 24 weeks (four passages of 6 weeks each) (Experiment 2, E2).

Species
Genotypes Aseptic Shoots (%) x Data represent mean ± standard error (SE). z Controls comprising shoot segments were excised from six-weekold cultures with the presence of bacteria and cultured on basal medium without PPM TM for three passages of 6 weeks each (total of 18 weeks of culture). Values followed by different letters within each section were significantly different at p ≤ 0.05 using Tukey's mean separation test.
The presence of bacterial colonies was assessed by placing pieces of the base of the shoots that were subjected to PPM TM treatment in bacterial indexing medium 523 ( Figure 1). This is a rich medium containing sucrose as the energy source, vitamins from yeast extract, and amino acids from hydrolyzed casein, so it would support the growth of many bacteria. Bacterial colonies were observed in 100% of the shoots derived from control plants (without PPM TM treatment) after 3 weeks on medium 523, suggesting the effectiveness of this bacteriological medium in indexing B. megaterium. Notably, our results indicate that PPM TM controlled B. megaterium in shoots cultured in PPM TM -supplemented medium (0.2% v/v) for two cycles of 6 weeks (E1) for all cultivars tested. Aseptic shoot levels ranging from 62.5% to 100% were obtained across six apple cultivars; on average, 79.2% of in vitro shoots in E1 were free of bacteria (Table 1, E1). Shoots derived from E1 were then transferred to medium without any PPM TM and cultured for an additional four subculture cycles of 6 weeks each (total of 24 weeks) before being observed for bacterial growth (E2). After 24 weeks of culture, bacterial contamination was completely controlled in in vitro shoots of apple varieties 'Golden Delicious' and 'Landsberger Renette', while 62.5% of 'Suislepper' and 75% of 'Aport krovavo-krasnyi' shoots were found to be free of bacterium, suggesting that the bacterium was either suppressed to very low levels or eliminated. Interestingly, in contrast, we found that apple varieties 'KG 7' and 'Gold Rush' grown in the absence of PPM TM had outbreaks of bacterial contamination around the base of the shoots and on the surface of the medium (Table 1, E2). Similarly, aiming to control bacterial contamination in in vitro cultures of switchgrass, Wang et al. [89] found that the growth of six endophytic bacterial strains was completely suppressed from cultures grown in a medium containing 0.2% PPM TM ; however, the effectiveness of eradicating the bacteria was only achieved when PPM TM was used throughout the micropropagation process; otherwise, in the absence of PPM TM , an outbreak of bacterial contamination was observed. Therefore, the addition of 0.2% PPM TM to the growth media during the entire cultivation process may be a possible alternative to suppress the outbreak of bacterial contamination in 'KG 7' and 'Gold Rush' apple cultures.
62.5% of 'Suislepper' and 75% of 'Aport krovavo-krasnyi' shoots were found to be free of bacterium, suggesting that the bacterium was either suppressed to very low levels or eliminated. Interestingly, in contrast, we found that apple varieties 'KG 7' and 'Gold Rush' grown in the absence of PPM TM had outbreaks of bacterial contamination around the base of the shoots and on the surface of the medium (Table 1, E2). Similarly, aiming to control bacterial contamination in in vitro cultures of switchgrass, Wang et al. [89] found that the growth of six endophytic bacterial strains was completely suppressed from cultures grown in a medium containing 0.2% PPM TM ; however, the effectiveness of eradicating the bacteria was only achieved when PPM TM was used throughout the micropropagation process; otherwise, in the absence of PPM TM , an outbreak of bacterial contamination was observed. Therefore, the addition of 0.2% PPM TM to the growth media during the entire cultivation process may be a possible alternative to suppress the outbreak of bacterial contamination in 'KG 7' and 'Gold Rush' apple cultures. Several studies have demonstrated the positive effect of PPM TM to control endophyte contamination when it is used at the proper concentration, which was indicated to be different depending on the plant species [32,43,45,47,48,50,[90][91][92][93]. Here, we used the PPM TM manufacturer's concentration instructions for controlling endogenous contamination in woody plants. The results obtained in the present study clearly demonstrate that the effectiveness of the proposed procedure for eradicating endophytic contamination was cultivar-specific. Therefore, future research is needed to access the effect of different incubation periods of in vitro shoots of apple 'KG 7' and 'Gold Rush' in growth medium supplemented with PPM TM at different concentrations to control B. megaterium contamination. Furthermore, the effectiveness of PPM TM has been described as dependent on the plant species tested and, mainly, which endophytic bacteria are present in the plant tissue [45,48,91].
Although the use of PPM TM can be effective in controlling endophyte contamination, it also has been shown to have a negative impact on in vitro plant growth [45,50,[92][93][94][95]. The tolerance levels and response to PPM TM treatments vary greatly with species, type of plant tissue, exposure duration, and concentration, so preliminary testing is necessary for successful treatment, especially in sensitive materials [29,[93][94][95]. While in Several studies have demonstrated the positive effect of PPM TM to control endophyte contamination when it is used at the proper concentration, which was indicated to be different depending on the plant species [32,43,45,47,48,50,[90][91][92][93]. Here, we used the PPM TM manufacturer's concentration instructions for controlling endogenous contamination in woody plants. The results obtained in the present study clearly demonstrate that the effectiveness of the proposed procedure for eradicating endophytic contamination was cultivar-specific. Therefore, future research is needed to access the effect of different incubation periods of in vitro shoots of apple 'KG 7' and 'Gold Rush' in growth medium supplemented with PPM TM at different concentrations to control B. megaterium contamination. Furthermore, the effectiveness of PPM TM has been described as dependent on the plant species tested and, mainly, which endophytic bacteria are present in the plant tissue [45,48,91].
Although the use of PPM TM can be effective in controlling endophyte contamination, it also has been shown to have a negative impact on in vitro plant growth [45,50,[92][93][94][95]. The tolerance levels and response to PPM TM treatments vary greatly with species, type of plant tissue, exposure duration, and concentration, so preliminary testing is necessary for successful treatment, especially in sensitive materials [29,[93][94][95]. While in chrysanthemum, the addition of 0.2% (v/v) PPM TM to the growth medium completely inhibited shoot regeneration from leaf explants [95], in walnut, 21.7% of clean shoots were obtained after combined treatment of shaking explants at 5% (v/v) PPM TM for 10 min followed by 0.2% (v/v) PPM TM in the initiation medium, although the percentage of necrosis increased significantly to 60% compared to 10% in media without PPM TM [29]. In this study, no phytotoxicity to the in vitro plants from the PPM TM -supplemented medium was observed. Shoots grown on medium supplemented with PPM TM for 12 weeks were healthy and showed average multiplication rates of 3.9 across the six cultivars that were equivalent  Table S2). The results from these experiments demonstrate that the addition of 0.2% (v/v) PPM TM to the culture medium for 12 weeks is a viable alternative for reducing, and in some cases controlling, endogenous contaminants from shoot cultures of apple after in vitro establishment, without any negative impact on the growth of in vitro cultures. The determination of the suitable PPM™ concentration and incubation period is an essential requirement for those apple cultivars that showed outbreaks of bacterial contamination when shoots were transferred to medium without any PPM™.

Isolation, Cultivation, and Identification of Endophytic Bacteria from In Vitro Plants
Shoots were isolated from six-week-old cultures and used for the isolation of bacterial contaminants. Initially, apparent bacterial contamination was detected visually, collected, and aseptically streaked in Petri dishes (100 mm × 15 mm) containing bacteriological growth medium 523, a general media for the growth of bacteria composed of 8 g L −1 casein hydrolysate (Sigma-Aldrich, St. Louis, MO, USA), 10 g L −1 sucrose, 4 g L −1 yeast extract, 2 g L −1 monopotassium phosphate (KH 2 PO 4 ), 0.15 g L −1 magnesium sulfate (MgSO 4 7H 2 O), and 6 g L −1 Gelrite™ at pH 6.9 [97]. Plates were incubated at room temperature. After 48 h of incubation, the developed colonies were purified by repeating isolation. Solitary colonies of contamination were collected using swabs and subsequent immersion into a tube containing Amies medium [98]. To obtain pure culture, all samples were cultivated on esculin-gelatin medium with incubation at 37 • C for 48 h under anaerobic conditions [99]. Thereafter, isolates were identified by MALDI-TOF/MS (Bruker Daltonik GmbH, Bremen, Germany), as described by Kozhakhmetova et al. [99]. MALDI-TOF/MS scores of ≥1.7 were accepted for species assignment, and scores below 1.7 were considered unreliable [99]. Further micropropagation experiments were carried out with indexed shoots.

Elimination of Contaminant Endophytic Bacteria Using PPM TM
Eight shoot segments (2 cm in length) for each cultivar were excised from six-week-old cultures with the presence of bacteria ( Figure 2) and placed in individual test tubes (150 mm × 25 mm) with basal medium supplemented with 0.2% v/v PPM TM (Plant Cell Technology, Washington, D.C., USA) and cultured in standard culture conditions. After 6 weeks of culture, shoot segments were transferred to fresh MS basal medium with 0.2% v/v PPM TM and cultured for another 6 weeks. After 12 weeks of culture on medium with PPM TM (two passages of 6 weeks each; E1), eight shoot segments for each cultivar were then transferred to basal medium without PPM TM and cultured for four passages of 6 weeks Plants 2022, 11, 2624 7 of 12 each (total of 24 weeks of culture; E2). The control (C) comprised shoot segments excised from six-week-old cultures with the presence of bacteria and cultured on MS basal medium without PPM TM for three passages of 6 weeks each (total of 18 weeks of culture). Shoot segments from E1, E2, and the control were then indexed for endophyte contamination using 523 detection medium [53]. For this, the basal parts of in vitro shoots (approx. 5 mm) were placed onto 100 × 15 mm Petri plates containing 25 mL of bacteriological growth medium 523 at a density of 8 shoots per plate. The Petri plates with shoots were incubated at 24 ± 1 • C under a photoperiod of 16 h light (40 µM m −2 s −1 ) for 2-3 weeks. In the absence of bacteria, the medium remains transparent, while the turbidity of the medium and the growth of colonies of microorganism near the base of the shoots indicate contamination.
old cultures with the presence of bacteria ( Figure 2) and placed in individual test tubes (150 mm × 25 mm) with basal medium supplemented with 0.2% v/v PPM ТМ (Plant Cell Technology, Washington, D.C., USA) and cultured in standard culture conditions. After 6 weeks of culture, shoot segments were transferred to fresh MS basal medium with 0.2% v/v PPM ТМ and cultured for another 6 weeks. After 12 weeks of culture on medium with PPM ТМ (two passages of 6 weeks each; E1), eight shoot segments for each cultivar were then transferred to basal medium without PPM ТМ and cultured for four passages of 6 weeks each (total of 24 weeks of culture; E2). The control (C) comprised shoot segments excised from six-week-old cultures with the presence of bacteria and cultured on MS basal medium without PPM ТМ for three passages of 6 weeks each (total of 18 weeks of culture). Shoot segments from E1, E2, and the control were then indexed for endophyte contamination using 523 detection medium [53]. For this, the basal parts of in vitro shoots (approx. 5 mm) were placed onto 100 × 15 mm Petri plates containing 25 mL of bacteriological growth medium 523 at a density of 8 shoots per plate. The Petri plates with shoots were incubated at 24 ± 1 °C under a photoperiod of 16 h light (40 µ M m −2 s −1 ) for 2-3 weeks. In the absence of bacteria, the medium remains transparent, while the turbidity of the medium and the growth of colonies of microorganism near the base of the shoots indicate contamination.

Assessment of the Impact of PPM ТМ on Shoot Multiplication
The multiplication rate (MR) was calculated in shoots grown for 12 weeks (two passages of 6 weeks each) in MS basal medium with 0.2% v/v PPM ТМ and control without any PPM ТМ . Data were recorded during the subcultures. MR was determined by the formula: MR = a/bc, where a is the number of new shoots, b is the number of initial shoots, and c is the number of subcultures.

Statistical Analysis
Each experiment was performed with two replicates of eight shoots in each treatment, and the whole experiment was repeated twice. Data were expressed as the mean values ± standard error (SE). Least significant differences were calculated using one-directional ANOVA and Tukey's mean separation test (p ≤ 0.05) using the software package SYSTAT 12.0 [100].

Assessment of the Impact of PPM TM on Shoot Multiplication
The multiplication rate (MR) was calculated in shoots grown for 12 weeks (two passages of 6 weeks each) in MS basal medium with 0.2% v/v PPM TM and control without any PPM TM . Data were recorded during the subcultures. MR was determined by the formula: MR = a/bc, where a is the number of new shoots, b is the number of initial shoots, and c is the number of subcultures.

Statistical Analysis
Each experiment was performed with two replicates of eight shoots in each treatment, and the whole experiment was repeated twice. Data were expressed as the mean values ± standard error (SE). Least significant differences were calculated using onedirectional ANOVA and Tukey's mean separation test (p ≤ 0.05) using the software package SYSTAT 12.0 [100].

Conclusions
Although endophytic bacteria remain a continuing threat to plant tissue culture, techniques for controlling, and in some cases eliminating, contamination are available. In this study, application of the recommended concentration of PPM TM (0.2% v/v) over 12 weeks of culture proved be effective to control the growth of B. megaterium in apple tissue cultures. Our results highlight the cultivar-specific response of apple shoots to PPM TM . These results therefore suggest the essentiality of further testing of extended incubation of PPM TM in these cultivars that had outbreaks of bacterial contamination. It also provides a scope for extending PPM TM application to other types of plants and endogenous contaminants.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11192624/s1, Table S1: Result overview of identification of isolates by MALDI-TOF/MS; Table S2: Assessment of the impact of PPM TM on shoot multiplication (multiplication rate = MR) of in vitro shoots of apple (Malus spp.) accessions grown in MS-based tissue culture medium with incorporation of 0.2% v/v PPM TM for two cycles of 6 weeks each or without any PPM TM ; Figure S1: Apple (Malus spp.) in vitro cultures exhibiting microbial contamination. Shoot tips exhibiting contamination 45 days after cryopreservation; bacterial colony growth was developed around shoot tips and on the surface of the medium during the recovery process.