- Email: [email protected]
Characterization of a newly discovered Beauveria bassiana isolate to Franklimiella occidentalis Perganda, a non-native invasive species in China
Microbiological Research 167 (2012) 116–120
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.de/micres
Characterization of a newly discovered Beauveria bassiana isolate to Franklimiella occidentalis Perganda, a non-native invasive species in China Junping Wang, Changying Zheng ∗ College of Agronomy and Plant Protection, Qingdao Agricultural university, Chengyang District, Qingdao 266109, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 11 February 2011 Received in revised form 3 May 2011 Accepted 4 May 2011 Keywords: Beauveria bassiana Virulence Heat tolerance Phylogenetic tree, Franklimiella occidentalis Perganda
a b s t r a c t In this study, a new virulent Beauveria bassiana isolate (B. bassiana-CYT5) had been identified as a new member of the species B. bassiana. The B. bassiana-CYT5 isolate was compared with four other virulent B. bassiana isolates and found to be highly infectious and virulent against the Franklimiella occidentalis Perganda. The B. bassiana-CYT5 could approximately 93.08% mortality of F. occidentalis 6 days post inoculation in the concentration of 1 × 108 conidia/mL. The phylogenetic tree based on ITS and partial sequence of elongation factor 1-alpha (EF1-alpha) indicated that B. bassiana-CYT5 isolate was in a cluster of B. bassiana. Furthermore, B. bassiana-CYT5 isolate demonstrated high heat tolerance (60–100% relative germination) between 1-h and 2-h exposure at 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C, respectively. So our results suggested that B. bassiana-CYT5 isolate could be a new efficient biocontrol agent against F. occidentalis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Entomopathogenic fungi (fungal biocontrol agents; BCAs) such as the genus Beauveria and Metarhizium are widely available biological control agents for controlling agricultural pests (Inglis et al. 2001). It is essential for controlling pests to pursuit effective new fungal biological control agents and identification of species and varieties. Because of the direct penetration of the insect cuticle, fungal BCAs can be the most promising microbial biocontrol agents against piercing/sucking insect pests such as thrips and aphids (Shipp et al. 2003). Beauveria species attack many insect pests worldwide. Now, this genus contains six species, namely Beauveria bassiana, Beauveria bassiana cf. clade C, Beauveria brongniartii, Beauveria caledonica, Beauveria vermiconia, and Beauveria amorpha (Glare 2004; Glare et al. 2008; Sevim et al. 2010a,b; Rehner and Buckley 2005). Among these species, B. bassiana is a preferred biological control agent in agriculture in temperate regions (Glare 2004). Some B. bassiana strains have been developed as a biopesticides against thrips and whiteflies in greenhouse (Ugine et al. 2006). The western flower thrip (WFT), Franklimiella occidentalis Perganda is a polyphagous and widely distributed pest and causes substantial economic losses to greenhouse crops via feeding damage and virus transmission such as chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), impatiens necrotic
∗ Corresponding author. E-mail address: [email protected] (C. Zheng). 0944-5013/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2011.05.002
spot virus (INSV), tomato chlorotic spot virus (TCSV) and tomato spotted wilt virus (TSWV) (Jones 2005). It is first reported in China in 2003, with a trend of further spreading (Zhang et al. 2003). The WFT, a non-native invasive species, appears in many areas in China, and is removed from the Catalogue of Quarantine Pest for Import Plants to the People’s Republic of China in 2007. Currently, it is principally controlled with insecticides in China. However, the WFT is difficult to control with insecticides because of its resistance against the major classes of insecticides. It will have negative impact on integrated pest management programmes with chemical control as one of the components. In recent years, therefore, there has been a shift emphasis towards biological control including the use of entomopathogenic fungi (Maniania et al. 2001; Gouli et al. 2009), different bug species (Premachandra et al. 2003; Blaeser et al. 2004; Messelink et al. 2006) and entomopathogenic nemotodes (Premachandra et al. 2003) in other countries. Moreover, different native predators such as Orius sauteri poppius (Zhang et al. 2007a,b) Amblyseius cucumeris (Zhi and Ren 2006) and Chrysopa sinica (Zhang et al. 2007b,c) were investigated in china. But the research on the control of the WFT with entomopathogenic fungi is very limited in China. Here we reported the characterization of entomopathogenic fungi, a member of the species B. bassiana, collected from Drosicha corpulenta Kuwana in Shandong Province, China. In the present study, we have characterized virulence, the morphological properties and heat tolerance of this fungus and determined the nucleotide sequences of the ITS and partial EF1-alpha. We also showed that B. bassiana-CYT5 isolate seems to be a potential biological control agent against the WFT.
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
117
Materials and methods Isolation of fungi and bioassay Fungi were isolated from infected larvae of D. corpulenta and cultured on SDAY medium (dextrose 10 g, peptone 2.5 g, yeast extract 5 g, agar 2 g/100 mL) for 7 days at 25 ◦ C. Conidia were made by scraping the surface of the cultures with a sterile “L” shaped stick into a 10-mL plastic container and suspended in sterile distilled water with 0.05% Tween-80.Conidial suspension were diluted to 1 × 108 conidia/mL. The leaves of purple cabbage (Brassica oleracea var. capitata f. rubra) were dipped into conidial suspension of 1 × 108 conidia/mL for 10 s and then placed into one plastic box (12 mm). Thirty adults of the WFT were selected and put into the plastic box. All boxes were incubated at 25 ◦ C. The control leaves were treated with sterile 0.01% Tween-80. Every treatment contained three replicates. The mortality was recorded at sixth day after inoculation. To confirm whether the WFT died from infection by B. bassiana, the dead adults were laid on the leaves of purple cabbage after being surface-sterilized. The presence of sporulation diagnostic of white muscardine disease caused by B. bassiana was then recorded.
Fig. 1. Mortality of 5 B. bassiana isolates on the adult of the WFT at the concentration of 1 × 108 conidia/mL at sixth day after inoculation. Bars showed standard deviation. Mortality data were corrected according to Abbott’s formula. Data were subjected to a GLM one-way ANOVA for each isolates. Means in each column within each isolates followed by the different letter differ significantly at P < 0.001, Duncan’s multiple range test.
Morphological identification Phylogenetic analysis The adult WFT infected with B. bassiana-CYT5 isolate was studied after 3 or 4-days death in the morphology of synnema by scanning electron microscopy. B. bassiana-CYT5 isolate was cultured on SDAY medium (dextrose 10 g, peptone 2.5 g, yeast extract 5 g, agar 2 g/100 mL) for 7 days at 25 ◦ C. The morphology of synnema taken from the SDAY medium was studied by scanning electron microscopy.
The obtained nucleotide sequences were compared with other B. bassiana species using Blast searches from the NCBI Genbank database. The most similar Genbank sequences to clones were downloaded from Genbank for phylogenetic analysis using the MEGA5 software. Result
Effect of heat on relative germination The isolate was cultured on SDAY in the dark at 25 ◦ C for 7 days. The conidia were harvested and immediately suspended in Tween80 solution (0.01% v/v). The conidial suspension was then exposed to thermal stress in a hot water bath at 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C for up to 4 h. The ongoing experiment methods were done as described (Ying and Feng 2004). Conidial suspensions at 25 ◦ C were used as blank control. Relative germination (%) = (Mt /Mc ) × 100, where Mt is the mean number of germlings of the replicates at exposure time t and Mc is the mean number of germlings of controlreplicates, regardless of exposure time. DNA extraction and sequencing Mycelium for DNA extraction was produced by culturing in quarter strength SDY broth on rotary shaker for 48 h at 25 ◦ C. Genomic DNA were extracted as described (Rehner and Buckey 2005) The fragments of ITS1-5.8S-ITS2 and EF1-alpha were amplified by PCR using ExTaq DNA polymerase (Takara, Japan) and the primes (ITS4:5 -TCCTCCGCTTATTGATATGC3 ITS5:5 -GGAAGTAAAAGTCGTAACAAGG and 1567R:5 EF1T:5 -ATGGGTAAGGARGACAAGAC-3 ACHGTRCCRATACCACCSATC-3 , respectively). The ingredient of PCR (50 L) contained 10× Taq DNA polymerase reaction buffer 5 L, 2 mM dNTPs 5 L, 25 mM MgCl2 5 L, 10 pmol each of the opposing amplification primers, 2 unit Taq DNA polymerase (Tarara, Japan) and 5–20 ng genomic DNA. PCR conditions were as follows: initial denaturation at 94 ◦ C for 5 min, 30 cycles of 94 ◦ C for 1 min, 55 ◦ C for 50 s and 72 ◦ C for 3 min, and final extension at 72 ◦ C for 10 min. PCRs were analyzed on gels and PCR products sequenced.
Bioassay and morphology of B. bassiana-CYT5 The isolates produced the different mortality against the adult of the WFT in comparison with each other (P < 0.001, F(4,5) = 21.492) (Fig. 1). B. bassiana-CYT5 isolate was highly virulent for the adult of the WFT causing approximately 90% mortality 6-days postinoculation in the concentration of 1 × 108 conidia/mL. The other isolates of B. bassiana were significantly less virulent to the WFT. So the result showed that B. bassiana-CYT5 was a high virulent new isolate of B. bassiana against the WFT, a non-native invasive species, in china. The WFT’s body was covered with mycelium and spore ball after 5–6 day post-inoculation. Each spore ball was composed of a cluster of short-globose to flask-shaped conidiogenous cells (Fig. 2a and b), which terminated in a narrow apical extension called a rachis. The rachis elongated in a long zig-zag extension after each conidium is produced (Fig. 2a). The conidia were globose to subglobose (Fig. 2a and b). On SDAY medium, the conidial synnema morphology of B. bassiana-CYT5 was similar (Fig. 2c), but the growth of conidial synnema was worse on the SDAY medium than on the dead adult of WFT. Heat tolerance Variability in conidial thermotolerance was experimented. After 1 h exposure to 37 ◦ C, 38 ◦ C, 39 ◦ C, and 40 ◦ C, respectively, conidia still had a little higher than 100% mean rate of relative germination. High variability in conidial thermotolerance of the B. bassiana-CYT5 isolate was noted after 2 h at 39 ◦ C and 40 ◦ C, ranging from 60% to 30% mean rate of relative germination (P < 0.01, F(3,9) = 6.4) (Fig. 3). However, mean rate of relative germination of B. bassiana-CYT5
118
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
Fig. 3. Mean rate of relative germination of B. bassiana-CYT5 isolate following exposure of aqueous conidial suspensions to 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C for 1, 2, 3 or 4 h. Relative germination was calculated in relation to non-heated controls. Error bars are standard errors of four trials.
isolate at 37 ◦ C and 38 ◦ C slowly declined with increasing exposure time (Fig. 3). Sequence and phylogenetic analysis ITS and EF1-alpha partial sequence were submitted to NCBI Genbank database (accession number: HQ259059 and HQ646366) and analyzed separately to confirm the phylogenetic relation of B. bassiana-CYT5 isolate. The result of Blast search indicated that the ITS and EF1-␣ sequence of CYT5 strain had high similarity with those of other strain of B. bassiana (99%). ITS and EF1-alpha sequences of B. bassiana-CYT5 strain were compared with representative sequences from the study of Rehner and Buckley (2005) and performed for phylogenetic analysis using minimum evolution method of MEGA5 program. As shown in Fig. 4 B. bassiana-CYT5 isolate was most closely related to ARSEF-1185 strain, which was belonging to B. bassiana (clade A). Discussion
Fig. 2. Scanning electron micrographs of B. bassiana-CYT5 isolate. (a) The rachis elongated in a long zig-zag extension (arrow) on the dead adult of the WFT. (b) Spore ball composed of a cluster of short-globose to flask-shaped conidiogenous cells (arrows) on the dead adult of the WFT. (c) Spore ball of B. bassiana-CYT5 isolate on the SDAY medium.
F. occidentalis is a non-native invasive species in China which is extremely polyphagous with over 200 plant species. Entomopathogenic fungi are the most attractive biological agents for thrip control, especially in greenhouses where all necessary conditions (humidity and temperature in particular) for fungal activity are provided. Previously, some authors showed that Metarhizium anisopliae (Maniania et al. 2002), B. bassiana (Jacobson et al. 2001) and Verticillium lecani (Helyer et al. 1995; Ravensberg et al. 1990) could control the WFT. However, investigations related to fungal biological control agent of the WFT have been quite limited in China. In the present study, we performed biochemical molecular characterization and entomopathogenic fungi which have high pathogenicity against the WFT. Virulence and repellency are two factors that must be addressed to improve the efficacy of entomopathogenic fungi (Mburu et al. 2009; Yanagawa et al. 2009). However, it is often true that highly virulent fungal strains are also highly repellent (Staples and Milner 2000). In this study, the new isolate B. bassiana-CYT5 isolated from D. corpulenta was the most virulent and lowest repellency against the WFT when compared with other isolates. So B. bassiana-CYT5 is especially recommended for practical control of thrips as bioinsecticide. In addition, we must consider a fungal fitness to environmental conditions occurring in the pest habitat to evaluate the ability of it to control a pest population. Temperature and humidity are two very important environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
119
cated that B. bassiana-CYT5 isolate was a member of the species within the genus Beauveria. To further confirm identifications of B. bassiana-CYT5, molecular methods were used. Nuclear ITS and EF1-alpha partial sequence have been performed in a number of recent surveys of Beauveria (Rehner and Buckley 2005; Ghikas et al. 2010; Sevim et al. 2010a,b; Campos et al. 2010). Our phylogenetic result from the nuclear ITS sequence was in full agreement with EF1-alpha sequence and B. bassiana-CYT5 isolate belonged to of B. bassiana (clade A). B. bassiana-CYT5 isolate formed a distinct strain, which had a high virulence and repellency against thrips. Acknowledgement The authors are grateful to Dr. Guoyue Yu for reviewing the manuscript. This work was supported by Special Fund for Agroscientific Research in the Public Interest Project No. 200803025. References
Fig. 4. Phylogenetic trees of fungal isolates within Beauveria genus based ITS (a) and EF1-␣ (b) sequences. Representative strains were taken from the study of Rehner and Buckley (2005). The dendrogram was constructed by using minimum evolution method with the Kimura two-parameter model with MEGA5.
insects (Shipp et al. 2003; Fernandes et al. 2008). Relative humidity in greenhouses can, nearly, reach the level that B. bassiana germinates. However, thermotolerance and cold activities are the key and intrinsic characteristics of promising entomopathogenic fungi. B. bassiana grows at a wide temperature range (from 8 to 35 ◦ C) with a maximum thermal threshold for growth at 37 ◦ C (Fargues et al. 1997).The thermal death point for conidia of B. bassiana isolates has been found to be between 45 and 50 ◦ C (Liu et al. 2003). According to our observations, temperature in the greenhouses varies from 5 ◦ C to 38 ◦ C (unpublished data). B. bassiana-CYT5 isolate could attain 70–100% germination after being exposed at 37 ◦ C and 38 ◦ C from 1 h to 4 h. So B. bassiana-CYT5 isolate will be a promising entomopathogenic fungus against piercing/sucking insect pests with high percentage germination in greenhouses. Meanwhile, B. bassiana-CYT5 isolate showed that mean rate of germination at 37 ◦ C, 38 ◦ C and 39 ◦ C for 1 h was higher than that that at 25 ◦ C for 1 h. This phenomenon was not same to the result (Devi et al. 2005) that high temperatures retarded the conidial germination process in B. bassiana. Morphological approaches are important methods in taxonomy of fungi. Strains were identified morphologically based on conidiogenous cell, rachis and conidial shape (Humber 1997). The morphology of synnema by scanning electron microscopy indi-
Blaeser P, Sengonca C, Zegula T. The potential use of different predatory bug species in the biological control of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). J Pest Sci 2004;77:211–9. Campos RA, Boldo JT, Pimentel IC, Dalfovo V, Araújo WL, Azevedo JL, et al. Endophytic and entomopathogenic strains of Beauveria sp to control the bovine tick Rhipicephalus (Boophilus) microplus. Genet Mol Res 2010;9(3):1421–2143. Devi KU, Sridevi V, Mohan Ch.M., Padmavathi J. Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin. J Invertebr Pathol 2005;88:181–9. Fernandes EK, Rangel DE, Moraes AM, Bittencourt VR, Roberts DW. Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria. J Invertebr Pathol 2008;98(1):69–78. Fargues J, Goettel MS, Smits N, Ouedraogo A, Rougier M. Effect of temperature on vegetative growth of Beauveria bassiana isolates from different origins. Mycologia 1997;89:383–92. Ghikas DV, Kouvelis VN, Typas MA. Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi Beauveria bassiana and B. brongniartii. BMC Microbiol 2010;10:174–89. Glare TR. Molecular characterization in the entomopathogenic fungal genus Beauveria. Laimburg J 2004;1:286–98. Glare TR, Reay SD, Nelson TL, Moore R. Beauveria caledonica is a naturally occurring pathogen of forest beetles. Mycol Res 2008;112:352–60. Gouli VV, Gouli SY, Skinner M, Shternshis MV. Effect of the entomopathogenic fungi on mortality and injury level of western flower thrips, Frankliniella occidentalis. Arch Phytopathol Plant Protect 2009;42(2):118–23. Helyer NL, Bmbyn PJ, Richardso PN, Edmondson RN. Control of western flower thrips (Frankliniella occidentalis Pergande) pupae in compost. Ann Appl Biol 1995;127:405–12. Humber RA. Fungi: identification. In: Lacey LA, editor. Manual of techniques in insect pathology. San Diego: Academic Press; 1997. p. 153. Inglis GD, Goettel MS, Butt TM, Strasser H. Use of hyphomycetous fungi for managing insect pests. In: Butt TM, Jackson C, Magan N, editors. Fungi as biocontrol agents: progress problems and potential. New York: CAB International; 2001. p. 23–69. Jacobson RJ, Chandler D, Fenlon J, Russell KM. Compatibility of Beauveria bassiana (Balsamo) Vuillemin with Amblyseius cucumeris Oudemans (Acarina: Phytoseiidae) to control Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) on cucumber plants. Biocontrol Sci Technol 2001;11:381–400. Jones DR. Plant viruses transmitted by thrips. Eur J Plant Pathol 2005;113:119–57. Liu HP, Skinner M, Brownbridge M, Parker BL. Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). J Invertebr Pathol 2003;82:139–47. Maniania NK, Ekesi S, Löhr B, Mwangi F. Prospects for biological control of the western flower thrips, Frankliniella occidentalis with the entomopathogenic fungus, Metarhizium anisopliae, on chrysanthemum. Mycopathologia 2001;155:229–35. Maniania NK, Ekesi S, Lohe B, Mwangi F. Prospects for biological control of the western flower thrips, Frankliniella occidentalis, with the entomopathogenic fungus, Metarhizium anisopliae, on chrysanthemum. Mycopathologia 2002;155(4):229–35. Messelink GJ, Steenpaal SEFV, Ramakers PMJ. Evaluation of phytoseiid predators for control of western flower thrips on greenhouse cucumber. Biocontrol 2006;51(6):753–68. Mburu DM, Ochola L, wangi A, Maniania NK, Njagi PGN, Gitonga LM, et al. Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni. J Insect Physiol 2009;55(9):774–80. Premachandra WTSD, Borgemeister C, Berndt O, Ehlers RU, Poehling HM. Combined releases of entomopathogenic nematodes and the predatory mite Hypoaspis aculeifer to control soil-dwelling stages of western flower thrips Frankliniella occidentalis. Biocontrol 2003;48(5):529–41.
120
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
Ravensberg WJ, Malais M, van der Schaaf DA. Verticillium lecanii as a microbial insecticide against glasshouse whitefly. In: Brighton crop protection conference – Pests and Disease British Crop Protection Council. Lavenham: The Lavenham Press; 1990. p. 265–8. Rehner SA, Buckley EA. Beauveria phylogeny inferred from nuclear ITS and EF1-␣ sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 2005;97(1):84–98. Sevim A, Demir I, Demirba˘g Z. Molecular characterization and virulence of Beauveria spp. from the pine processionary moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae). Mycopathologia 2010a;170(4):269–77. Sevim A, Demir I, Hofte M, Humber RA, Demirbag Z. Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey. Biocontrol 2010b;55(2):279–97. Shipp JL, Zhang Y, Hunt DWA, Ferguson G. Influence of humidity and greenhouse microclimate on the efficacy of Beauveria bassiana (Balsamo) for control of greenhouse arthropod pests. Environ Entomol 2003;32:1154–63. Staples JA, Milner RJ. A laboratory evaluation of the repellency of Metarhizium anisopliae conidia to Coptotermes lacteus (Isoptera: Rhinotermitidae). Sociobiology 2000;36(1):133–48. Ugine TA, Wraight SP, Sanderson JP. Influences of impatiens pollen and exposure to Beauveria bassiana on bionomics of western flower thrips Frankliniella occidentalis. Biol Control 2006;37:186–95.
Yanagawa A, Yokohari F, Shimizu S. The role of antennae in removing entomopathogenic fungi from cuticle of the termite, Coptotermes formosanus. J Insect Sci 2009;9(6):1–9. Ying SH, Feng MG. Relationship between thermotolerance and hydrophobinlike proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents. J Appl Microbiol 2004;97(2): 323–31. Zhang YJ, Wu QJ, Xu BY, Zhu GR. Newly invaded insect pest, Frankliniella occidentalis (Thysanoptera: Thripidae) in Beijing. Plant Protect 2003;29(4): 58–9. Zhang AS, Yu Y, Men XY, Li LL, Sun TL. Predation of Orius sauteri nymph on Frankliniella occidentalis adults. Nat Enem Insect 2007a;29(3):108–12. Zhang AS, Yu Y, Li LL, Zhang SC, Men XY. Functional response and searching rate of Orius sauteri adults on Frankliniella occidentalis nymphs. Chin J Ecol 2007b;26(8):1233–7. Zhang AS, Li LL, Yu Y, Men XY. Functional response and searching rate of Chrysopa sinica larvae on Frankliniella occidentalis nymphs. Acta Phytophylacica Sin 2007c;34(3):247–51. Zhi JR, Ren SX. The Functional response and numerical response of Amblyseius cucumeris (Acari: Phytoseiidae) on Frankliniella occidentalis (Thysanoptera: Thripidae). J South China Agric Univ 2006;27(3):35–8.
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.de/micres
Characterization of a newly discovered Beauveria bassiana isolate to Franklimiella occidentalis Perganda, a non-native invasive species in China Junping Wang, Changying Zheng ∗ College of Agronomy and Plant Protection, Qingdao Agricultural university, Chengyang District, Qingdao 266109, Shandong Province, China
a r t i c l e
i n f o
Article history: Received 11 February 2011 Received in revised form 3 May 2011 Accepted 4 May 2011 Keywords: Beauveria bassiana Virulence Heat tolerance Phylogenetic tree, Franklimiella occidentalis Perganda
a b s t r a c t In this study, a new virulent Beauveria bassiana isolate (B. bassiana-CYT5) had been identified as a new member of the species B. bassiana. The B. bassiana-CYT5 isolate was compared with four other virulent B. bassiana isolates and found to be highly infectious and virulent against the Franklimiella occidentalis Perganda. The B. bassiana-CYT5 could approximately 93.08% mortality of F. occidentalis 6 days post inoculation in the concentration of 1 × 108 conidia/mL. The phylogenetic tree based on ITS and partial sequence of elongation factor 1-alpha (EF1-alpha) indicated that B. bassiana-CYT5 isolate was in a cluster of B. bassiana. Furthermore, B. bassiana-CYT5 isolate demonstrated high heat tolerance (60–100% relative germination) between 1-h and 2-h exposure at 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C, respectively. So our results suggested that B. bassiana-CYT5 isolate could be a new efficient biocontrol agent against F. occidentalis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Entomopathogenic fungi (fungal biocontrol agents; BCAs) such as the genus Beauveria and Metarhizium are widely available biological control agents for controlling agricultural pests (Inglis et al. 2001). It is essential for controlling pests to pursuit effective new fungal biological control agents and identification of species and varieties. Because of the direct penetration of the insect cuticle, fungal BCAs can be the most promising microbial biocontrol agents against piercing/sucking insect pests such as thrips and aphids (Shipp et al. 2003). Beauveria species attack many insect pests worldwide. Now, this genus contains six species, namely Beauveria bassiana, Beauveria bassiana cf. clade C, Beauveria brongniartii, Beauveria caledonica, Beauveria vermiconia, and Beauveria amorpha (Glare 2004; Glare et al. 2008; Sevim et al. 2010a,b; Rehner and Buckley 2005). Among these species, B. bassiana is a preferred biological control agent in agriculture in temperate regions (Glare 2004). Some B. bassiana strains have been developed as a biopesticides against thrips and whiteflies in greenhouse (Ugine et al. 2006). The western flower thrip (WFT), Franklimiella occidentalis Perganda is a polyphagous and widely distributed pest and causes substantial economic losses to greenhouse crops via feeding damage and virus transmission such as chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), impatiens necrotic
∗ Corresponding author. E-mail address: [email protected] (C. Zheng). 0944-5013/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2011.05.002
spot virus (INSV), tomato chlorotic spot virus (TCSV) and tomato spotted wilt virus (TSWV) (Jones 2005). It is first reported in China in 2003, with a trend of further spreading (Zhang et al. 2003). The WFT, a non-native invasive species, appears in many areas in China, and is removed from the Catalogue of Quarantine Pest for Import Plants to the People’s Republic of China in 2007. Currently, it is principally controlled with insecticides in China. However, the WFT is difficult to control with insecticides because of its resistance against the major classes of insecticides. It will have negative impact on integrated pest management programmes with chemical control as one of the components. In recent years, therefore, there has been a shift emphasis towards biological control including the use of entomopathogenic fungi (Maniania et al. 2001; Gouli et al. 2009), different bug species (Premachandra et al. 2003; Blaeser et al. 2004; Messelink et al. 2006) and entomopathogenic nemotodes (Premachandra et al. 2003) in other countries. Moreover, different native predators such as Orius sauteri poppius (Zhang et al. 2007a,b) Amblyseius cucumeris (Zhi and Ren 2006) and Chrysopa sinica (Zhang et al. 2007b,c) were investigated in china. But the research on the control of the WFT with entomopathogenic fungi is very limited in China. Here we reported the characterization of entomopathogenic fungi, a member of the species B. bassiana, collected from Drosicha corpulenta Kuwana in Shandong Province, China. In the present study, we have characterized virulence, the morphological properties and heat tolerance of this fungus and determined the nucleotide sequences of the ITS and partial EF1-alpha. We also showed that B. bassiana-CYT5 isolate seems to be a potential biological control agent against the WFT.
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
117
Materials and methods Isolation of fungi and bioassay Fungi were isolated from infected larvae of D. corpulenta and cultured on SDAY medium (dextrose 10 g, peptone 2.5 g, yeast extract 5 g, agar 2 g/100 mL) for 7 days at 25 ◦ C. Conidia were made by scraping the surface of the cultures with a sterile “L” shaped stick into a 10-mL plastic container and suspended in sterile distilled water with 0.05% Tween-80.Conidial suspension were diluted to 1 × 108 conidia/mL. The leaves of purple cabbage (Brassica oleracea var. capitata f. rubra) were dipped into conidial suspension of 1 × 108 conidia/mL for 10 s and then placed into one plastic box (12 mm). Thirty adults of the WFT were selected and put into the plastic box. All boxes were incubated at 25 ◦ C. The control leaves were treated with sterile 0.01% Tween-80. Every treatment contained three replicates. The mortality was recorded at sixth day after inoculation. To confirm whether the WFT died from infection by B. bassiana, the dead adults were laid on the leaves of purple cabbage after being surface-sterilized. The presence of sporulation diagnostic of white muscardine disease caused by B. bassiana was then recorded.
Fig. 1. Mortality of 5 B. bassiana isolates on the adult of the WFT at the concentration of 1 × 108 conidia/mL at sixth day after inoculation. Bars showed standard deviation. Mortality data were corrected according to Abbott’s formula. Data were subjected to a GLM one-way ANOVA for each isolates. Means in each column within each isolates followed by the different letter differ significantly at P < 0.001, Duncan’s multiple range test.
Morphological identification Phylogenetic analysis The adult WFT infected with B. bassiana-CYT5 isolate was studied after 3 or 4-days death in the morphology of synnema by scanning electron microscopy. B. bassiana-CYT5 isolate was cultured on SDAY medium (dextrose 10 g, peptone 2.5 g, yeast extract 5 g, agar 2 g/100 mL) for 7 days at 25 ◦ C. The morphology of synnema taken from the SDAY medium was studied by scanning electron microscopy.
The obtained nucleotide sequences were compared with other B. bassiana species using Blast searches from the NCBI Genbank database. The most similar Genbank sequences to clones were downloaded from Genbank for phylogenetic analysis using the MEGA5 software. Result
Effect of heat on relative germination The isolate was cultured on SDAY in the dark at 25 ◦ C for 7 days. The conidia were harvested and immediately suspended in Tween80 solution (0.01% v/v). The conidial suspension was then exposed to thermal stress in a hot water bath at 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C for up to 4 h. The ongoing experiment methods were done as described (Ying and Feng 2004). Conidial suspensions at 25 ◦ C were used as blank control. Relative germination (%) = (Mt /Mc ) × 100, where Mt is the mean number of germlings of the replicates at exposure time t and Mc is the mean number of germlings of controlreplicates, regardless of exposure time. DNA extraction and sequencing Mycelium for DNA extraction was produced by culturing in quarter strength SDY broth on rotary shaker for 48 h at 25 ◦ C. Genomic DNA were extracted as described (Rehner and Buckey 2005) The fragments of ITS1-5.8S-ITS2 and EF1-alpha were amplified by PCR using ExTaq DNA polymerase (Takara, Japan) and the primes (ITS4:5 -TCCTCCGCTTATTGATATGC3 ITS5:5 -GGAAGTAAAAGTCGTAACAAGG and 1567R:5 EF1T:5 -ATGGGTAAGGARGACAAGAC-3 ACHGTRCCRATACCACCSATC-3 , respectively). The ingredient of PCR (50 L) contained 10× Taq DNA polymerase reaction buffer 5 L, 2 mM dNTPs 5 L, 25 mM MgCl2 5 L, 10 pmol each of the opposing amplification primers, 2 unit Taq DNA polymerase (Tarara, Japan) and 5–20 ng genomic DNA. PCR conditions were as follows: initial denaturation at 94 ◦ C for 5 min, 30 cycles of 94 ◦ C for 1 min, 55 ◦ C for 50 s and 72 ◦ C for 3 min, and final extension at 72 ◦ C for 10 min. PCRs were analyzed on gels and PCR products sequenced.
Bioassay and morphology of B. bassiana-CYT5 The isolates produced the different mortality against the adult of the WFT in comparison with each other (P < 0.001, F(4,5) = 21.492) (Fig. 1). B. bassiana-CYT5 isolate was highly virulent for the adult of the WFT causing approximately 90% mortality 6-days postinoculation in the concentration of 1 × 108 conidia/mL. The other isolates of B. bassiana were significantly less virulent to the WFT. So the result showed that B. bassiana-CYT5 was a high virulent new isolate of B. bassiana against the WFT, a non-native invasive species, in china. The WFT’s body was covered with mycelium and spore ball after 5–6 day post-inoculation. Each spore ball was composed of a cluster of short-globose to flask-shaped conidiogenous cells (Fig. 2a and b), which terminated in a narrow apical extension called a rachis. The rachis elongated in a long zig-zag extension after each conidium is produced (Fig. 2a). The conidia were globose to subglobose (Fig. 2a and b). On SDAY medium, the conidial synnema morphology of B. bassiana-CYT5 was similar (Fig. 2c), but the growth of conidial synnema was worse on the SDAY medium than on the dead adult of WFT. Heat tolerance Variability in conidial thermotolerance was experimented. After 1 h exposure to 37 ◦ C, 38 ◦ C, 39 ◦ C, and 40 ◦ C, respectively, conidia still had a little higher than 100% mean rate of relative germination. High variability in conidial thermotolerance of the B. bassiana-CYT5 isolate was noted after 2 h at 39 ◦ C and 40 ◦ C, ranging from 60% to 30% mean rate of relative germination (P < 0.01, F(3,9) = 6.4) (Fig. 3). However, mean rate of relative germination of B. bassiana-CYT5
118
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
Fig. 3. Mean rate of relative germination of B. bassiana-CYT5 isolate following exposure of aqueous conidial suspensions to 37 ◦ C, 38 ◦ C, 39 ◦ C and 40 ◦ C for 1, 2, 3 or 4 h. Relative germination was calculated in relation to non-heated controls. Error bars are standard errors of four trials.
isolate at 37 ◦ C and 38 ◦ C slowly declined with increasing exposure time (Fig. 3). Sequence and phylogenetic analysis ITS and EF1-alpha partial sequence were submitted to NCBI Genbank database (accession number: HQ259059 and HQ646366) and analyzed separately to confirm the phylogenetic relation of B. bassiana-CYT5 isolate. The result of Blast search indicated that the ITS and EF1-␣ sequence of CYT5 strain had high similarity with those of other strain of B. bassiana (99%). ITS and EF1-alpha sequences of B. bassiana-CYT5 strain were compared with representative sequences from the study of Rehner and Buckley (2005) and performed for phylogenetic analysis using minimum evolution method of MEGA5 program. As shown in Fig. 4 B. bassiana-CYT5 isolate was most closely related to ARSEF-1185 strain, which was belonging to B. bassiana (clade A). Discussion
Fig. 2. Scanning electron micrographs of B. bassiana-CYT5 isolate. (a) The rachis elongated in a long zig-zag extension (arrow) on the dead adult of the WFT. (b) Spore ball composed of a cluster of short-globose to flask-shaped conidiogenous cells (arrows) on the dead adult of the WFT. (c) Spore ball of B. bassiana-CYT5 isolate on the SDAY medium.
F. occidentalis is a non-native invasive species in China which is extremely polyphagous with over 200 plant species. Entomopathogenic fungi are the most attractive biological agents for thrip control, especially in greenhouses where all necessary conditions (humidity and temperature in particular) for fungal activity are provided. Previously, some authors showed that Metarhizium anisopliae (Maniania et al. 2002), B. bassiana (Jacobson et al. 2001) and Verticillium lecani (Helyer et al. 1995; Ravensberg et al. 1990) could control the WFT. However, investigations related to fungal biological control agent of the WFT have been quite limited in China. In the present study, we performed biochemical molecular characterization and entomopathogenic fungi which have high pathogenicity against the WFT. Virulence and repellency are two factors that must be addressed to improve the efficacy of entomopathogenic fungi (Mburu et al. 2009; Yanagawa et al. 2009). However, it is often true that highly virulent fungal strains are also highly repellent (Staples and Milner 2000). In this study, the new isolate B. bassiana-CYT5 isolated from D. corpulenta was the most virulent and lowest repellency against the WFT when compared with other isolates. So B. bassiana-CYT5 is especially recommended for practical control of thrips as bioinsecticide. In addition, we must consider a fungal fitness to environmental conditions occurring in the pest habitat to evaluate the ability of it to control a pest population. Temperature and humidity are two very important environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
119
cated that B. bassiana-CYT5 isolate was a member of the species within the genus Beauveria. To further confirm identifications of B. bassiana-CYT5, molecular methods were used. Nuclear ITS and EF1-alpha partial sequence have been performed in a number of recent surveys of Beauveria (Rehner and Buckley 2005; Ghikas et al. 2010; Sevim et al. 2010a,b; Campos et al. 2010). Our phylogenetic result from the nuclear ITS sequence was in full agreement with EF1-alpha sequence and B. bassiana-CYT5 isolate belonged to of B. bassiana (clade A). B. bassiana-CYT5 isolate formed a distinct strain, which had a high virulence and repellency against thrips. Acknowledgement The authors are grateful to Dr. Guoyue Yu for reviewing the manuscript. This work was supported by Special Fund for Agroscientific Research in the Public Interest Project No. 200803025. References
Fig. 4. Phylogenetic trees of fungal isolates within Beauveria genus based ITS (a) and EF1-␣ (b) sequences. Representative strains were taken from the study of Rehner and Buckley (2005). The dendrogram was constructed by using minimum evolution method with the Kimura two-parameter model with MEGA5.
insects (Shipp et al. 2003; Fernandes et al. 2008). Relative humidity in greenhouses can, nearly, reach the level that B. bassiana germinates. However, thermotolerance and cold activities are the key and intrinsic characteristics of promising entomopathogenic fungi. B. bassiana grows at a wide temperature range (from 8 to 35 ◦ C) with a maximum thermal threshold for growth at 37 ◦ C (Fargues et al. 1997).The thermal death point for conidia of B. bassiana isolates has been found to be between 45 and 50 ◦ C (Liu et al. 2003). According to our observations, temperature in the greenhouses varies from 5 ◦ C to 38 ◦ C (unpublished data). B. bassiana-CYT5 isolate could attain 70–100% germination after being exposed at 37 ◦ C and 38 ◦ C from 1 h to 4 h. So B. bassiana-CYT5 isolate will be a promising entomopathogenic fungus against piercing/sucking insect pests with high percentage germination in greenhouses. Meanwhile, B. bassiana-CYT5 isolate showed that mean rate of germination at 37 ◦ C, 38 ◦ C and 39 ◦ C for 1 h was higher than that that at 25 ◦ C for 1 h. This phenomenon was not same to the result (Devi et al. 2005) that high temperatures retarded the conidial germination process in B. bassiana. Morphological approaches are important methods in taxonomy of fungi. Strains were identified morphologically based on conidiogenous cell, rachis and conidial shape (Humber 1997). The morphology of synnema by scanning electron microscopy indi-
Blaeser P, Sengonca C, Zegula T. The potential use of different predatory bug species in the biological control of Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). J Pest Sci 2004;77:211–9. Campos RA, Boldo JT, Pimentel IC, Dalfovo V, Araújo WL, Azevedo JL, et al. Endophytic and entomopathogenic strains of Beauveria sp to control the bovine tick Rhipicephalus (Boophilus) microplus. Genet Mol Res 2010;9(3):1421–2143. Devi KU, Sridevi V, Mohan Ch.M., Padmavathi J. Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin. J Invertebr Pathol 2005;88:181–9. Fernandes EK, Rangel DE, Moraes AM, Bittencourt VR, Roberts DW. Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria. J Invertebr Pathol 2008;98(1):69–78. Fargues J, Goettel MS, Smits N, Ouedraogo A, Rougier M. Effect of temperature on vegetative growth of Beauveria bassiana isolates from different origins. Mycologia 1997;89:383–92. Ghikas DV, Kouvelis VN, Typas MA. Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi Beauveria bassiana and B. brongniartii. BMC Microbiol 2010;10:174–89. Glare TR. Molecular characterization in the entomopathogenic fungal genus Beauveria. Laimburg J 2004;1:286–98. Glare TR, Reay SD, Nelson TL, Moore R. Beauveria caledonica is a naturally occurring pathogen of forest beetles. Mycol Res 2008;112:352–60. Gouli VV, Gouli SY, Skinner M, Shternshis MV. Effect of the entomopathogenic fungi on mortality and injury level of western flower thrips, Frankliniella occidentalis. Arch Phytopathol Plant Protect 2009;42(2):118–23. Helyer NL, Bmbyn PJ, Richardso PN, Edmondson RN. Control of western flower thrips (Frankliniella occidentalis Pergande) pupae in compost. Ann Appl Biol 1995;127:405–12. Humber RA. Fungi: identification. In: Lacey LA, editor. Manual of techniques in insect pathology. San Diego: Academic Press; 1997. p. 153. Inglis GD, Goettel MS, Butt TM, Strasser H. Use of hyphomycetous fungi for managing insect pests. In: Butt TM, Jackson C, Magan N, editors. Fungi as biocontrol agents: progress problems and potential. New York: CAB International; 2001. p. 23–69. Jacobson RJ, Chandler D, Fenlon J, Russell KM. Compatibility of Beauveria bassiana (Balsamo) Vuillemin with Amblyseius cucumeris Oudemans (Acarina: Phytoseiidae) to control Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) on cucumber plants. Biocontrol Sci Technol 2001;11:381–400. Jones DR. Plant viruses transmitted by thrips. Eur J Plant Pathol 2005;113:119–57. Liu HP, Skinner M, Brownbridge M, Parker BL. Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). J Invertebr Pathol 2003;82:139–47. Maniania NK, Ekesi S, Löhr B, Mwangi F. Prospects for biological control of the western flower thrips, Frankliniella occidentalis with the entomopathogenic fungus, Metarhizium anisopliae, on chrysanthemum. Mycopathologia 2001;155:229–35. Maniania NK, Ekesi S, Lohe B, Mwangi F. Prospects for biological control of the western flower thrips, Frankliniella occidentalis, with the entomopathogenic fungus, Metarhizium anisopliae, on chrysanthemum. Mycopathologia 2002;155(4):229–35. Messelink GJ, Steenpaal SEFV, Ramakers PMJ. Evaluation of phytoseiid predators for control of western flower thrips on greenhouse cucumber. Biocontrol 2006;51(6):753–68. Mburu DM, Ochola L, wangi A, Maniania NK, Njagi PGN, Gitonga LM, et al. Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni. J Insect Physiol 2009;55(9):774–80. Premachandra WTSD, Borgemeister C, Berndt O, Ehlers RU, Poehling HM. Combined releases of entomopathogenic nematodes and the predatory mite Hypoaspis aculeifer to control soil-dwelling stages of western flower thrips Frankliniella occidentalis. Biocontrol 2003;48(5):529–41.
120
J. Wang, C. Zheng / Microbiological Research 167 (2012) 116–120
Ravensberg WJ, Malais M, van der Schaaf DA. Verticillium lecanii as a microbial insecticide against glasshouse whitefly. In: Brighton crop protection conference – Pests and Disease British Crop Protection Council. Lavenham: The Lavenham Press; 1990. p. 265–8. Rehner SA, Buckley EA. Beauveria phylogeny inferred from nuclear ITS and EF1-␣ sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 2005;97(1):84–98. Sevim A, Demir I, Demirba˘g Z. Molecular characterization and virulence of Beauveria spp. from the pine processionary moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae). Mycopathologia 2010a;170(4):269–77. Sevim A, Demir I, Hofte M, Humber RA, Demirbag Z. Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey. Biocontrol 2010b;55(2):279–97. Shipp JL, Zhang Y, Hunt DWA, Ferguson G. Influence of humidity and greenhouse microclimate on the efficacy of Beauveria bassiana (Balsamo) for control of greenhouse arthropod pests. Environ Entomol 2003;32:1154–63. Staples JA, Milner RJ. A laboratory evaluation of the repellency of Metarhizium anisopliae conidia to Coptotermes lacteus (Isoptera: Rhinotermitidae). Sociobiology 2000;36(1):133–48. Ugine TA, Wraight SP, Sanderson JP. Influences of impatiens pollen and exposure to Beauveria bassiana on bionomics of western flower thrips Frankliniella occidentalis. Biol Control 2006;37:186–95.
Yanagawa A, Yokohari F, Shimizu S. The role of antennae in removing entomopathogenic fungi from cuticle of the termite, Coptotermes formosanus. J Insect Sci 2009;9(6):1–9. Ying SH, Feng MG. Relationship between thermotolerance and hydrophobinlike proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents. J Appl Microbiol 2004;97(2): 323–31. Zhang YJ, Wu QJ, Xu BY, Zhu GR. Newly invaded insect pest, Frankliniella occidentalis (Thysanoptera: Thripidae) in Beijing. Plant Protect 2003;29(4): 58–9. Zhang AS, Yu Y, Men XY, Li LL, Sun TL. Predation of Orius sauteri nymph on Frankliniella occidentalis adults. Nat Enem Insect 2007a;29(3):108–12. Zhang AS, Yu Y, Li LL, Zhang SC, Men XY. Functional response and searching rate of Orius sauteri adults on Frankliniella occidentalis nymphs. Chin J Ecol 2007b;26(8):1233–7. Zhang AS, Li LL, Yu Y, Men XY. Functional response and searching rate of Chrysopa sinica larvae on Frankliniella occidentalis nymphs. Acta Phytophylacica Sin 2007c;34(3):247–51. Zhi JR, Ren SX. The Functional response and numerical response of Amblyseius cucumeris (Acari: Phytoseiidae) on Frankliniella occidentalis (Thysanoptera: Thripidae). J South China Agric Univ 2006;27(3):35–8.