| More and more attention has been paid to the studies on bacterialsymbionts and their interaction with hosts. Numerous evidences indicate that theinteraction between bacterial symbionts and fungus have been involved in manyscientific biological questions which have tightly related with many domainsincluding agriculture, forestry, environmental science, food production and medicine.Furthermore, many scholars believe that bacteria harbouring in fungi is not anaccidental ecological phenomena, but a result of long-term coevolution, in which theycould adapt to each other then enter into interdependence finally. Entomophthoraleanfungi, which could cause high-frequency epizootic to keep the pest population at alow level, has considered as an important biological control agents as well asscientific research object. However, the viewpoint on whether there are bacterialsymbionts harbouring in Entomophthoralean fungi or not have not been concernedtotally, to say nothing of effective proofs. In this study, we sought to prove thisbacterial symbionts viewpoint through the Pandora neoaphidis, an obligated aphidpathogenic fungi. Studies involved in the biological diversity information of P.neoaphidis using16S rRNA library construction technology, isolation andclassification of bacterial symbionts species, location of cultured bacterial symbiontsusing fluorescence in situ hybridization and confirmation on the universalcharacteristic for Entomophthoralean fungi by constructing the symbiotic bacteria16SrRNA gene library from different fungus samples. Additionally, bioassays werecarried out to verify the correlation between biological diversity of bacterialsymbionts and virulence of P. neoaphidis. These results could provide new insights toexplore the intrinsic mechanism of interaction between the symbiotic bacteria andEntomophthorales fungi.1.16S rRNA gene library construction of P. neoaphidis bacterial symbiotsUsing16S rRNA technology, we constructed the P. neoaphidis symbiotic bacterial16S rRNA gene library and obtained total9species which mainly belonged to theOrder Proteobacteria. Accordingly, the highest proportion of bacterial symbiont wasclass Gammaproteobacteria which counted for62.0%, the proportion of classAlphaproteobacteria and Betaproteobacteria accounted for14.3%and2.4%,respectively, while the proportion of class Firmicutes and Bacteroidetes accounted for 4.8%and16.7%, respectively, Acinetobacter species has the highest ratio of42.9%inall bacterial symbionts. Therefore, we could confirm the hypothesis that bacterialsymbionts have harboured in P. neoaphidis and also have abundant biologicaldiversity.2. Isolation, classification and FISH verification of P. neoaphidis symbioticbacteriaWe have isolated and cultured symbiotic bacteria from P. neoaphidis liquid hyphaeusing traditional method. Identified by16S rRNA sequencing and Biochemicalmethod, the symbiontic bacteria was considered as Acinetobacter calcoaceticus.Furthermore, expected band with425bp sizes could be amplified from P.neoaphidis total DNA template using Acinetobacter specific primer pairs, P-rA1andP-rA2. Those findings were consistent with the results of16S rRNA gene library insection1. According specific probes stemed from the acquired sequences of bacterial16S rRNA gene, we detected the intracellular bacteria and obtained the FISH imagescaptured by confocal laser scanning microscope. Visible Image displayed that thebacteria have harboured in P. neoaphidis hypha, thereby we could verify theAcinetobacter sp was one of the symbiotic bacteria of P. neoaphidis.3.16S rRNA gene library construction of bacterial symbiots from differentEntomophthoralean fungi samples and its relevant function analysisWe obtained total25bacterial symbiont species in21genera through analyzed thebacterial complete16s rRNA clone libraries derived from different samples. Thesefungal samples included the P. neoaphidis isolate ARSEF5403(differentgeographical host), P. nouryi isolate ARSEF8931(different specie in same genus), P.neoaphidis isolate F98028+(isolation process with PSN antibiotic treatment), P.neoaphidis isolate F98028(isolation process without PSN antibiotic treatment) andF98028+pc (primary conidia discharged from P. neoaphidis isolate F98028+). After wemade a statistic classification for the possible bacterial symbionts usingbioinformation software, we found that these bacterial symbionts belonged to theclass of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,Flavobacteria, Firmicutes, Actinobacteria and Bacteroidetes, repectivly. Therefore,these results suggest that bacterial symbiosis phenomenon be common in the Pandoragenus and these bacterial symbiots have abundant species population diversity aswell.Comparing with the bacterial symbiots of P. neoaphidis F98028+and its primary conidia, we found that some groups of bacteriail symbiots from P. neoaphidis couldtransmit vertically to its primary conidia. In addition, we found these possiblebacterial symbionts in class Gammaproteobacteria had a strong resistance toantibiotics when we attempt to analyze the antibiotic impact on symbionts of P.neoaphidis during rejuvenation. Bioassay results showed that the LC50value of P.neoaphidis isolate F98028was significantly higher than P. neoaphidis isolateF98028+. So we could infer that the virulence of P. neoaphidis rise up afterhost-rejuvenation-cycle. Besides, the virulence of P. neoaphidis and diversity index ofbacterial symbionts have a positive correlation. Reasons to explain may be that smallpart of bacterial symbiots would be regained through re-infecting aphid host, and thenrestored its original virulence that weakened by repeatly subculture or antibiotictreatment.4. Conclusion and implicationsActually, the symbiotic bacteria phenomenon we studied could strongly supportedthe hypothesis that P. neoaphidis harbour symbiotic bacteria, and it could have auniversal characteristic in the all Entomophthoralean fungi. This finding wouldprovide a deep insight for understanding the biological function mechanism ofEntomophthoralean fungi. |
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