1. Entomopathogenic Fungi on Hemiberlesia pitysophila Chengqun Lv*, Baoling Huang, Mengji Qiao, Jiguang Wei, Bo Ding. 2011. Entomopathogenic Fungi on Hemiberlesia pitysophila. PLoS ONE.

2. Introduction

3. Entomopathogenic fungi (EPF) are the fungi capable of invading, parasitizing, and causing insects sick or death. More than 60% of naturally-occurred insect epidemics are caused by pathogenic fungi, including EPF, one of the major controlling nature factor of insect populations.

4. Introduction More and more studies using combined methods of community ecology and insect mycology on EPF biodiversities. Especially for those forest ecosystems with large number of EPF species and qualities. For example, Wang et al. found 7 entomogenous fungal species in 4 genera and explored the population dynamics of B. bassiana.

5. Introduction H. pitysophila is an exotic, extremely harmful quarantine pest for pines including P. massoniana. An outbreak may cause a decline of pine volume growth by 2.548 m 3, which is about 3.2- fold of the damage caused by pine caterpillars. The forest may die out in 3 to 5 years.

6. Introduction In this study, we noticed a peculiar natural death of H. pitysophila in the epidemic areas of Guangxi province. We isolated and cultured entomogenous fungi from naturally died H. pitysophila, performed morphological taxonomic and molecular systematic identification, finally we found five entomopathogenic species of Pestalotiopsis.

7. Materials and Methods

8. Cadaver collection of H. pitysophila Collect cadavers seal in kraft paper bags 3 batches (random 300~400 bodys/batch) disinfect and isolate fungi in the laboratory

9. Materials and Methods Isolation of entomogenous fungi and their taxonomic identification by morphology surface sterilized with 75% ethanol （ 15 seconds ） Identification by morphology washed with sterile water placed in 9cm culture dishes (containing PDM ) PDM=potato dextrose medium: Potato 200g Dextrose 20g Agar 15- 20g Distilled water 1000 ml transferred to a new PDM plate cultured at 28 ℃ till fungal colonies appeared preserved on slant at 4 ℃

10. Materials and Methods Identification of entomogenous fungi by Molecular taxonomy DNA of all fungal strains as template to amplify internal transcribed spacer (ITS) All ITS amplicons were sequenced by Sangon Biotech (Shanghai) Co., Ltd. ITS4 (59-TCCTCCGCTTATT- GATATGC-39) ITS5 (59-GGAAGTAAAAGTCGTAA- CAAGG-39) selected the most similar sequences to compare with Genbank

11. Materials and Methods Virulence of Pestalotiopsis spp. culture suspensions were diluted to 10 8 cells/L using randomized block experiments to examine the toxicity (3 repeats) water was used as control inoculated on liquid PDM on a shaker (72 hours) sprayed on the crown of 5 trees of P. massoniana to infect with H. pitysophila chose the most frequently 5 strains in H. pitysophila

12. Materials and Methods Sample collection and Mortality calculation of H. pitysophila Live: shiny and plump Dead: dark and shrivelled

13. Materials and Methods Sample collection and Mortality calculation of H. pitysophila  Mortality = Dead/Total body counts (dead+live) We also adjusted the mortality to:  Adjusted mortality= Calculated mortality - natural mortality  The difference of the adjusted mortality among the 5 strains was tested using one- way ANOVA.  p<0.05 was considered statistically significant

14. Results

15. Morphological taxonomy of entomogenous fungi on H. pitysophila

16. Results Taxonomic identification of entomogenous fungi on H. pitysophila by ITS sequencing 9 strains : 6 species of 4 genera. 5 strains : genus. 1 strain : family.

17. Results Composition of entomogenous fungi 93.3 % 16.7 % 46.7 % 40% 46.7 % 26.7 % 33.3 %

18. Results Phylogenetic tree construction of entomopathogenic Pestalotiopsis Pestalotiopsis spp. had insect host besides plant. Pathogenic fungi Endophytic fungi

19. Results Virulence of entomopathogenic fungi  The 5 tested strains of Pestalotiopsis showed remarkable protection of P. massoniana by killing H. pitysophila

20. Results Virulence of entomopathogenic fungi  all these 5 strains could be used to control H. pitysophila with the same efficacy.

21. Conclusion

22. Conclusion 5 strains that were classified as species of Pestalotiopsis, which has been considered plant pathogens and endophytes, were the dominant entomopathogenic fungus of H. pitysophila. 5 strains that were classified as species of Pestalotiopsis, which has been considered plant pathogens and endophytes, were the dominant entomopathogenic fungus of H. pitysophila. Molecular phylogenetic tree showed that entomopathogenic Pestalotiopsis spp. were similar to plant Pestalotiopsis, but not to other pathogens and endophytes of its host plant P. massoniana. Molecular phylogenetic tree showed that entomopathogenic Pestalotiopsis spp. were similar to plant Pestalotiopsis, but not to other pathogens and endophytes of its host plant P. massoniana. Our findings suggest a potential and promising method of H. pitysophila bio-control. Our findings suggest a potential and promising method of H. pitysophila bio-control.

23. Discussion

24. Numerous studies have shown that the relationship between plants and Pestalotiopsis spp. can be parasitic, symbiotic and saprophytic. Numerous studies have shown that the relationship between plants and Pestalotiopsis spp. can be parasitic, symbiotic and saprophytic. Pestalotiopsis is somewhat specific for H. pitysophila. Pestalotiopsis is somewhat specific for H. pitysophila. Pestalotiopsis can be used as antagonistic microbe to control H. pitysophila. Pestalotiopsis can be used as antagonistic microbe to control H. pitysophila. It is not clear why Pestalotiopsis spp. are specific for killing H. pitysophila. The future study may be focused on the micro-ecological environment and some specific metabolic pathways in H. pitysophila. It is not clear why Pestalotiopsis spp. are specific for killing H. pitysophila. The future study may be focused on the micro-ecological environment and some specific metabolic pathways in H. pitysophila.

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