Influence of native endophytic bacteria on the growth and bacterial crown rot tolerance of papaya (Carica papaya)

Abstract

Bacterial crown rot (BCR) is a re-emerging disease of papaya (Carica papaya) in Southeast 26 Asia caused by a gram-negative, facultative anaerobic bacterium, known as Erwinia 27 mallotivora. Ultrastructural observations using SEM revealed colonization of bacterial 28 community common in a BCR-tolerant genotype, before onset of tisue regeneration or 29 'regrowth'. This observation led to the hypothesis that papaya-associated bacteria might play a 30 role in BCR tolerance. Isolation, characterization and identification using 16S rRNA sequence 31 of bacteria associated with the tolerant 'regrowth' genotype resulted to the discovery of two 32 endophytic bacteria, of genera Kosakonia (isolate EBW) and Sphingomonas (isolate EBY). 33Papaya root-associated Bacillus isolates were also identified to belong to the B. 34 amyloliqufaciens clade (isolate BN, BS) and B. thuringiensis clade (isolate BT). Separate 35 inhibition assays indicated that, papaya latex and autoclaved culture metabolites (ACMs) from 36 isolated bacteria promptly (between 18-30h) and efficiently inhibited (60-65%) E. mallotivora 37 proliferation in vitro. Moreover, when ACMs from BN and EBW were inoculated in surface-38 sterilized papaya seeds, it variably retarded seed germination (20-60% reduction), depending 39 on plant genotype, but significantly stimulated plant growth and biomass accumulation, at 40 around two-fold increase. Interestingly, ACMs from all isolates, especially EBW, significantly 41 reduced BCR incidence and severity in susceptible genotype at around two-fold. Finally, these 42 results indicated that tolerance of papaya against E. mallotivora infection is probably 43 influenced by host innate properties such as tissue regrowth and pathogen inhibition by plant 44 latex, and acquired properties including pathogen antagonism, growth promotion and defense 45 biopriming through metabolites from beneficial endophytic and root-assocaited bacteria. 47Keywords: bacterial crown rot, defense biopriming, Bacillus, endophytic bacteria 48 49 82 perhaps a mechanism for resistance or tolerance (Magdalita et al., 2016). Breeding for 83 resistance and development of management strategies against the bacterial crown rot disease 84 are already in its early stages in the Philippines. Regrowth hybrid lines with good marketable 85 qualities and with resistance to papaya ringspot disease are currently under preliminary field 86 trials and back-cross F3 progenies are now in the pipeline (Magdalita et al., 2015). 88However, there are no reports yet regarding the events in pathogenesis up to the recovery of 89 papaya from the crown rot disease. Regrowth tissue examination was done initially in this study 90 to gain insight to probable role of beneficial microbes in disease tolerance. This was followed 91 by antimicrobial assays using papaya latex, culture metabolites from bacterial endophytes and 92 root-associated microbes and priming for disease resistance. Antimicrobial activity and 93 defence biopriming of beneficial microbes were thought to be acquired m...