Indole acetic acid and lipopeptide-producing endophytic bacteria from Taxus chinesis: toxicity evaluation of metabolic products

Endophytes play an important role in plant growth and development. Some one can produce auxins, ACCs, iron carriers, and so on to help plants grow and resist unhealthy growth environments. In addition, they can produce certain antimicrobial substances to resist pests and diseases. Among them, Bacillus is the most common beneficial endophytic bacterium in plants. In this paper, 20 IAA-producing strains were screened from endophytic bacteria isolated from Taxus chinensis var. mairei plant tissues by high-performance liquid-chromatography (HPLC). Based on the 20 IAA-producing strains, LC-TOF-MS technology was used to screen lipopeptide-producing Bacillus sp. As a result, three strains (KLBMPTC01, KLBMPTC10, and KLBMPTC29) of Bacillus-producing lipopeptides with abundant contents and species were obtained. According to the situation of the IAA and lipopeptides produced by these strains, KLBMPTC10 was selected as the experimental strain for later toxicological tests. In an Ames test and oral toxicity experiments in mice, we did not detect mutagenicity and other physiological toxicity. This is hoping to provide a theoretical basis for forest resource protection and biofertiliser production therewith.


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An endophyte is an endosymbiont, often a bacterium or fungus(1), that lives within Preparation of KLBMPTC10 extract and in vivo toxicity in 180 mice 181 The experiments were performed on ICR mice (female: male, 50:50) (weighing 18-182 22 g) obtained from the Xuzhou Medical College (Jiangsu, China). The animals were 183 housed at 25 ± 5 ℃ at 60 ± 5 % relative humidity, and subjected to a 12 h light-dark cycle  Strain KLBMPTC10 extraction was dealt with as described in experiment 190 "Fermentation, extraction, and evaluation of IAA-production ability" and freeze-dried. 191 Bacitracin was used as standard substance to determine the content of bacitracin in each 192 sample by external standard method. Then the dried extract was dissolved in sterile 193 distilled water and specimens with bacitracin at concentrations of 5, 10, 20, 50, and 100 194 µg/ml prepared for acute oral toxicity and Ames studies (37).

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Ames test 196 The test strains of TA97, TA98, TA100, and TA102 used in this study were all 197 mutant strain of Salmonella which they cannot synthesize the necessary amino acid-198 histidine for standard bacterial culture (38). Before the experiment, the characteristics of 199 the tested strains were identified to ensure that they met the requisite experimental 200 standards. An Ames test was carried out according to the standard protocol without the 201 S9 mix as described by Grúz et al. (39). Furthermore, 2-acetamidofluorene, Dexon, and 202 sodium azide are three known positive mutagens with genotoxicity(40). And in this test, 203 we used 20 µg/mL 2-acetamidofluorene, 50 µg/mL Dexon, and 20 µg/mL sodium azide 204 as the positive control, the solvent group was DMSO and the experimental group is 205 KLBMPTC10 bacterial extracts with concentrations of 5, 10, 20, and 50 µg/mL 206 respectively. The blank control group was also set to observe spontaneous revertant counts.

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There were three replicates established of each treatment. Colonies were counted after 2 208 days of incubation at 37 ℃ using a bacterial colony counter (35).

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Acute oral toxicity study 210 To test the toxicity of strain KLBMPTC10, 36 mice were divided into six groups 211 (female and male mice in each): six mice in each group were individually housed in cages.

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Animals were fasted overnight before the experiment, and were administered with a 216 single dose of the extraction of KLBMPTC10 (20 mg/kg)/vehicle and any change in 217 weight measured every seven days with a count of the number of poisoned and dead mice 218 on each day. These tests were performed in duplicate (37). At the end of the study, all the 219 surviving animals were sacrificed for blood analysis. Blood samples were collected from 220 the heart and blood analysis was conducted at the weekend of the first and second week 221 (7 and 14 days) after treatment. The non-heparinised blood was allowed to coagulate and then centrifuged to obtain serum. The serum was assayed for alanine aminotransferase 223 (ALT), aspartate aminotransferase (AST), urea, uric acid, and creatinine (35).

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The experiments were conducted in triplicate. Data were reported as the mean ± SD 226 for triplicate determinations. ANOVA and Tukey's tests were undertaken to identify 227 differences among means. Statistical significance was set at P < 0.05, and the results were 228 expressed as mean ± SD.

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In this study, a total of 31 isolate strains were identified from the inner tissues of respectively. In leaf specimens, the five isolates belonged to Bacillus (four strains) and 240 Sphingomonas (one strain). The specific information of the isolates is shown in Table 1 241 and their taxonomic status is shown in Figure 1. it can be seen that three endophytic bacteria showed significant differences in the 259 production of two typical lipopeptide compounds (surfactin and iturin A2) which were 260 typical lipopeptide compounds in Bacillus. Among them, the surfactin-production ability 261 of strain KLBMPTC29 was better than that of other two strains, the content of which was 262 24.47±4.05%. The ability of strain KLBMPTC10 to produce iturin A2 was greater than 263 that of the other two strains, and the content thereof was 26.06± 2.68%.

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In vivo toxicity of KLBMPTC10 extraction on mice bacteria" and "IAA-production and lipopeptide-production properties of the isolates", we 267 selected strain KLBMPTC10 to evaluate the safety of mice. Firstly, we observed the  (47,48). In this study, we first isolated endophytic bacteria from Taxus chinensis and identified them with 16S rDNA, and then we screened the IAA 311 producing ability of these strains. The results showed that we isolated a high IAA 312 producing Bacillus from the roots of Taxus chinesis. Its IAA production in testing 313 conditions is higher than other Bacillus strains(49, 50). In addition, we used HPLC-314 TOFMS to test the ability the ability of these IAA producing strains to produce lipopeptide 315 compounds which may help reduce the damage of some diseases or insect pests and is 316 environmentally friendly. And we found that KLBMPTC10 is a high lipopeptide producer 317 strain and the quantitative results showed that the ability of producing iturin A2 was better 318 than other strains, and the content was 26.06 ± 2.68%. And iturin compounds have 319 significant inhibitory activity on fungal growth and it has been widely used in agriculture 320 (51) .

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In addition, microbes are harmless to humans and animals, which is the most 322 important evaluation index to evaluate their potential application. So according to the 323 above characteristics evaluation results of strain KLBMPTC10, and to further understand 324 its potential value, we also undertook Ames and acute oral toxicity, testing in mice to 325 evaluate its safety when used on mammals. Ames test can evaluate possible mutagenic 326 effects caused by chemicals in vitro in short term (52). We can consider it as a mutagen if 327 bacterial extract of strain KLBMPTC10 induces back-mutation colonies that exceed twice 328 the number of spontaneous back-mutations (41). ALT, AST, urea, uric acid data in mice 329 can assess whether liver and kidney functions have been damaged (53). In this study, the 330 results of Ames test and acute oral toxicity bacterial extract of strain KLBMPTC10 has 331 not mutagenicity and non-toxic to mice. And this shows that the strain KLBMPTC10 has great potential value in application to forest resource protection and biofertiliser peroxidation products in the Ames test. Mutation Research/Genetic Toxicology and