Inconspicuous endophytic bacteria mimicking latex exudates in shoot-tip cultures of papaya

Shoot-tip cultures of papaya (Carica papaya L.) cv. Surya were initiated from field-grown plants after extensive surface sterilization for their use in micropropagation and to assess endophytic bacterial associations with apparently clean cultures. Out of 150 explants cultured, 40% of them displayed microbial contamination with obvious colony growth on tissue culture medium during the first in vitro passage of 1 month. Among the apparently clean 90 cultures, 12% showed cloudiness of medium at the base of the explant resembling latex exudates. These 11 cultures, upon indexing, i.e., testing the cultures for any covert bacteria by transferring traces of tissue culture medium to enriched bacteriological media, revealed inconspicuously associated endophytic bacteria in them, whereas the remaining cultures that had clear medium proved to be index-negative. Each of the above 11 cultures yielded a single organism, which included 10 Gram-negative isolates (5× Ralstonia mannitolilytica, 2× Paenibacillus sp., 2× Sphingomonas sp., 1× Pantoea sp.) and 1 Gram-positive isolate (Lysinibacillus fusiformis) as per 16S rRNA gene sequence homology analysis. Up on inoculation of index-negative papaya cultures with the above organisms on Murashige and Skoog based tissue culture medium at 25 °C, all the endophytes except Pantoea sp. remained inconspicuous or non-detectable in vitro, and they affected the survival and growth of papaya cultures as per visual assessment. The organisms tended to grow actively on tissue culture medium and become expressive with increase in incubating temperature (30-37 °C) or with the supply of host tissue extract. The study has brought out that the hazy exudates from the explant often encountered with plant tissue cultures at culture initiation could be resulting from inconspicuously growing endophytic bacteria, with possible threat to various tissue culture applications. Early identification of such cultures through indexing helps in containing the problem, prevent lateral spread of contaminants and offer more security to in vitro germplasm and culture exchange. Tissue culture system also helped in isolating some novel endophytic bacteria associated with papaya shoot-tips.