Effects of Rice Seed Surface Sterilization with Hypochlorite on Inoculated Burkholderia vietnamiensis

Abstract

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10 2 -to 10 4 -fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin-and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin-and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.The use of hypochlorite salts for disinfection dates back to the mid-18th century. Since that time, chlorination has been the most widely used bactericidal treatment for conventional disinfection of municipal drinking water for prevention of epidemic diseases such as cholera and typhoid, and it is still the most widely used method for disinfecting water (18). Hypochlorite is also routinely used as a sanitizer for domestic uses, as well as in food-processing plants to remove surface contaminants which can alter food quality or lead to food-borne diseases (2,3,23,29).Hypochlorite is known to be a very effective to killer of bacteria; even micromolar concentrations are enough to reduce bacterial populations significantly (27). However, little is known about the exact mechanisms of bacterial killing by this sanitizer. When diluted in water, the hypochlorite salts used [NaOCl, Ca(OCl) 2 , LiOCl, and KOCl] lead to formation of HOCl, whose concentration is correlated with bactericidal activity (27). Bacterial killing by HOCl may be due at least in part to lethal DNA damage (13, 42). However, HOCl itself is so reactive that it is unlikely to penetrate cells and reach the DNA; rather, it seems that the bactericidal activity is due to formation of secondary products, as hypochlorous acid reacts avidly with a wide variety of subcellular compounds (membranes, proteins, etc.) (10, 18). In particular, HOCl reacts with NH 4 ϩ and organic amines to form highly toxic chloramines, which also are strong oxidizing and chlorinating compounds and could be the actual killing agents. These chloramin...

Isolation, Diversity, and Antimicrobial Activity of Rare Actinobacteria from Medicinal Plants of Tropical Rain Forests in Xishuangbanna, China

“…By our tentative experiments, the in- crease in the number of CFU over that for the control was obvious (see Table S1 in the supplemental material). This was consistent with the effects of sterilization of rice seed surfaces with 2.0% sodium thiosulfate instead of water rinses (59) and with the fact that thiosulfate can suppress the detrimental effects of hypochlorite on skin (34). It is also known that an alkaline environment favors the growth of actinomycetes but not that of endophytic fungi.…”

Analysis of the Bacterial Diversity Associated with the Roots of Maize (Zea mays L.) through Culture-Dependent and Culture-Independent Methods

“…Aliquots of 0.1 ml were taken from the different dilutions and plated in duplicate on nutrient agar (NA: meat extract 3 g, soy peptone 5 g, NaCl 8 g, agar-agar 15 g) to count colony forming units (CFUs) of the rhizoplane. Afterwards, roots were washed with sterile distilled water and surface-sterilized by immersion in 1.3% sodium hypochlorite (15 min) followed by four rinses with sterile distilled water, an immersion in 2% sodium thiosulfate to remove the residual sodium hypochlorite [15] and, a final rinse with sterile distilled water. Surface sterilization was considered to be achieved by the absence of CFUs in NA plates.…”

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

“…After drying at room temperature, the tissue segments were surface-sterilized by immersing in 0.1% Tween 20 for 1 min, followed by 5% (available Cl -) NaClO for 4 min (leaf samples) or 6 min (stem and root samples). The samples then were rinsed in 2.5% (w/v) Na 2 S 2 O 3 for 10 min to remove the residual chlorine (Miche and Balandreau 2001;Qin et al 2009) and they were washed three times with sterile H 2 O. The last step was to immerse the samples in 70% (v/v) ethanol for 4 min (leaf samples) or 6 min (stem and root samples), then followed by a minimum of three washes with sterile water.…”