Plant Preservative Mixture
Prevalence of various endophytic bacteria tolerant to PPM™ in papaya, the broad-spectrum microbicide specified for use in plant tissue culture, able to covertly survive on MS-based medium, with implications for contamination management. Plant Preservative Mixture™ was used to establish papaya (Carica papaya) tissue cultures from field explants. Comparing three recommended practices to control endogenous microbial contaminants, axillary shoot tips (1.0-1.5 cm) of cv. Arka Prabhath was treated with 5% PPM™ for 4 h (T1), 50% for 10 min (T2), or 100% for 10 min (T3) and cultured in an MS-based papaya establishment medium (PEM).
At 4-6 weeks, all treatments were unrewarding and the cultures succumbed to microbial contamination (80% in T1) or to the effect of phytotoxicity/contamination (90% in T2 and 95% in T3). Another trial adopting a multi-step surface sterilization treatment (carbendazim-cetrimide-HgCl2) followed by cultivation in PEM supplemented with 0.05% PPM showed 35% evident bacterial contamination compared to 40% in the control. Single colonies of pooled bacterial growths on nutrient agar (NA) with 0.1% PPM incorporated were tested and 60% of isolates were recorded as sensitive to plant preservative mixture (PPM).
Twenty surviving PPM isolates were selected and identified. This showed 85% Gram-positive bacteria, including 80% of the Firmicutes phylum (55% spore-forming Bacillaceae and 25% Staphylococcaceae), 5% Actinobacteria, and 15% Gram-negative Proteobacteria. About 50% of the isolates remained totally occult after cultivation in PEM, while the rest showed slow growth and many showed increased growth after supplementation with host tissue extract. Culture of the isolates in NA supplemented with PPM indicated that 90-95% tolerated 0.05-0.1% PPM and 65% exceeded 0.2% PPM.
The isolates, however, did not show evident growth in PPM-supplemented PEM where the spore-formers survived. The results indicate the prevalence of various PPM™-tolerant endophytic bacteria in papaya, most of which survive covertly on MS-based medium, and the need to take this into account when using PPM™ for contamination management.
Keywords: Carica papaya; Cultural indexing; Microbial contamination; micropropagation; plant tissue culture; Viable but non-culturable bacteria.