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

Abstract

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.

In Vitro Collection for the Safe Storage of Grapevine Hybrids and Identification of the Presence of Plasmopara viticola Resistance Genes

“…The contamination of plant tissue cultures by different microorganisms such as bacteria and fungi is one of the major concerns in micropropagation protocols [ 53 , 54 , 55 ]. Sometimes, even apparently healthy-looking cultures may harbor endogenous contaminants (endophytes) that may not always be apparent in the early stages of tissue cultures [ 56 , 57 , 58 ]. Some plants are hosts for a wide range of endophytes, which may only arise in tissue cultures after several rounds of subcultures and/or after the cultures have undergone some stress [ 56 , 59 , 60 ].…”

“…Sometimes, even apparently healthy-looking cultures may harbor endogenous contaminants (endophytes) that may not always be apparent in the early stages of tissue cultures [ 56 , 57 , 58 ]. Some plants are hosts for a wide range of endophytes, which may only arise in tissue cultures after several rounds of subcultures and/or after the cultures have undergone some stress [ 56 , 59 , 60 ]. Therefore, given the high levels of contaminants we experienced during in vitro initiation, and to minimize the risk of endophyte contamination, we further screened for the presence of these contaminants in apparently clean tissue culture-initiated explants using a specialized medium that encourages the proliferation of endogenous contaminants.…”

“…The effectiveness of a propagation protocol depends largely on the rate of shoot multiplication [ 52 ]. The shoot multiplication rate (MR) reflects the number of new progeny plants produced from a single explant [ 56 , 72 ]. We found that the concentration of phytohormones and the acidity of the culture medium have a great effect on the MR and the quality of in vitro stock cultures ( Table 4 ).…”

“…Therefore, these results suggest that it is not necessary to supplement the culture medium with PPM TM during the multiplication phase. Although the use of PPM TM can be effective in controlling endophyte contamination [ 56 , 73 , 74 ], it also has been shown to have a negative impact on in vitro plant growth, and the tolerance levels and response to PPM TM vary greatly with species [ 75 , 76 , 77 ]. In order to evaluate its potential for controlling microbial contaminants, future research may include PPM TM only during the in vitro establishment of grapevine cultures.…”

“…Medium 523, a general medium for growth of bacteria, is composed 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 of 6.9 [ 61 ]. Plates were incubated at room temperature to encourage proliferation of endogenous pathogens [ 56 ]. Therefore, cloudiness of the medium or growth of colonies on medium 523 after 2 weeks indicates contamination of the accessions.…”