IOBC/wprs Bulletin Vol. 28(2) 2005

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Isolation of Beauveria brongniartii from soil: Are the available isolation tools neutral? Insect Pathogens and Insect Parasitic Nematodes: Melolontha IOBC/wprs Bulletin Vol. 28(2) 2005 pp. 25-29 Jürg Enkerli 1 , Priska Moosbauer 1,2 , Franco Widmer 1 , Silvia Dorn 2 , Siegfried Keller 1 1 Swiss Federal Research Station for Agroecology and Agriculture, Reckenholzstrasse 191, 8046 Zürich, Switzerland; 2 Institute of Plant Sciences/Applied Entomology, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland Abstract: For Beauveria brongniartii the fungus used in biological control of Melolontha melolontha, three techniques are routinely applied to monitor or isolate B. brongniartii from soil after BCA application. These are baiting with the host M. melolontha (MB), baiting with Galleria mellonella (GB) or plating soil suspensions on selective medium (SM). It has never been investigated whether any of the three isolation methods may select for certain B. brongniartii strains or whether they are equally suited for the isolation of different strains. We performed two experiments to address this question. First, the virulence of 30 genetically different isolates from Jenaz (eastern Switzerland) were tested in a bioassay with M. melolontha. The collection of 30 isolates consisted of 3 groups of ten isolates, each group consisting of isolates collected with one of the three isolation technique. Although, the virulence of single isolates was significantly different across all isolates the average virulence among the three groups was not significantly different, suggesting that the isolation techniques did not select for isolates with different virulence against M. melolontha. For the second experiment conidia of six B. brongniartii isolates of which always two were obtained with the same isolation techniques were mixed into B. brongniartii free soil. Strains were re-isolated from the soil mixture by using each of the three isolation methods and genotypes were determined by microsatellite analysis. All six genotypes have been re-isolated with the GB method and five genotypes have been re-isolated with either the MB and the SM method. In most cases the observed abundance of the genotypes corresponded with the expected abundance calculated based on equal re-isolation rates for each isolate with each method. A broader spectrum of genotypes will need to be tested in order to confirm the findings and allow statistically valid generalizations. In this study we have described and evaluated a strategy to compare and validate isolation techniques for entomopathogenic soil fungi. Keywords: isolation techniques, genotype selection, virulence, monitoring Introduction The fungus Beauveria brongniartii (Sacc.) Petch is a well established and commercially available biocontrol agent (BCA) to control the larvae of Melolontha melolontha in grasslands and orchards (Keller, 1992; Zelger, 1996). Three techniques are currently available for monitoring and isolation of B. brongniartii from soil samples. The Melolontha bait (MB) or Galleria bait (GB) methods where B. brongniartii is selected from soil samples by baiting either with M. melolontha or Galleria mellonella larvae (Keller et al., 1997; Kessler et al., 2003) and the selective medium (SM) method where soil suspensions are plated on a selective medium (Kessler et al., 2003; Strasser et al., 1996). Although all three techniques are widely applied for pre- and post-treatment monitoring and isolation of new biocontrol strains they have never been compared regarding their selectivity i.e. whether they select for certain B. brongniartii genotypes or whether they allow for equal growth of different strains. 25
26 Particularly, differences in the selective ability of MB and GB methods, which are based on selection for a virulent phenotype, compared to the SM method would be reasonable. Availability of validated selection and isolation techniques is important for reliable monitoring of applied biocontrol agents in the field, selection of new biocontrol strains or investigations of natural population structures and it is crucial to compare selective behavior of different techniques. Sensitive genetic identification tools are an important requirement to perform such comparisons and for B. brongniartii appropriate techniques recently have become available with the development of microsatellite markers (Enkerli et al., 2001). This genetic tool has been applied to investigate genetic diversity in a natural population of B. brongniartii in Switzerland (Enkerli, Keller, and Widmer, unpublished). Preliminary results of this study have suggested that certain genotypes might preferentially be selected by either of the three isolation techniques. The aim of the present study was to investigate whether the three isolation techniques select for certain isolates of B. brongniartii or whether they allow to equally grow different B. brongniartii strains. First, we tested whether genetically different isolates that have been selected with only one of the three isolation techniques differ in their virulence towards M. melolontha. Second, we mixed six genetically different isolates of which always two were isolated with one technique into a soil samples. Subsequently, strains were re-isolated with all three techniques to test whether different techniques select preferentially for isolates with a certain genotype or whether each genotype can be re-isolated at the same rate. Materials and methods All fungal isolates originate from a grassland plot of 1ha at Jenaz (eastern Switzerland). The plot was sampled continuously once to twice per year since 1999 to monitor B. brongniartii density and population structure. Isolates were obtained by applying three techniques: (i) Isolation from collected M. melolontha larvae, (ii) baiting with G. mellonella or (iii) plaiting soil suspensions on selective medium and subsequent isolation of single B. brongniartii colonies. For isolation from M. melolontha, larvae were collected from soil, placed individually into plastic cups (4.5 cm ∅, 6 cm high) filled with damped peat and incubated in constant darkness at 22˚C. Subsequently, single isolates were obtained from sporulating mycelium of diseased cadavers. Galleria baiting (GB) as well as isolation from selective medium (SM) were performed as described by Kessler et al. (2003). One hundred and fiftyone B. brongniartii isolates were collected between 1999 and 2001 and genotyped based on 6 microsatellite markers as described by Enkerli et al. (2004). For each isolation technique 10 isolates were selected, which displayed a genotype that was only detected in isolates obtained with a particular technique. This resulted in a collection of 30 isolates with unique genotypes. Virulence of the B. brongniartii strains was determined by dipping 30 M. melolontha larvae per strain in a suspension containing blastospores (10 7 /ml) for 4 seconds. Excess water was removed with a paper towel and the larvae were placed individually into plastic cups (4.5 cm ∅, 6 cm high) filled with damped peat. The larvae were incubated in constant darkness at 22˚C and fed with sliced carrots. Mortality was recorded daily starting five days after inoculation. Dead insects, with typical signs for fungal infection were moved to a separate moist chamber and incubated until sporulation. Conidia were identified under the microscope. Calculation of average survival time of M. melolontha larvae for each isolate and significance analyses (Kruskal-Wallis H test) were performed with the software SSPS V.10.1. Two isolates per isolation technique, all with comparable growth and virulence characteristics, were selected for the re-isolation experiment (Table 1.) and grown on complete medium (CM) plates (Riba & Ravelojoana, 1984). For each isolate 10 plates were
Page 1 and 2: IOBC / WPRS Working group „Insect
Page 3: Preface The Working Group “Integr
Page 6 and 7: iv ERICSSON Jerry University of Bri
Page 8 and 9: vi PÁZMÁNDI Christian Centre for
Page 10 and 11: viii
Page 12 and 13: x Metarhizium anisopliae for white
Page 14 and 15: xii
Page 16 and 17: Insect Pathogens and Insect Parasit
Page 18 and 19: stands (Levine et al. 2002) to feed
Page 20 and 21: In conclusion, the chemical control
Page 22: Kuhlmann, U. & W.A.C.M. Burgt 1998:
Page 25 and 26: 10 Austria, Belgium, Czech Republic
Page 27 and 28: 12 The control strategies used agai
Page 29 and 30: 14 1) Case study: Soil application
Page 31 and 32: 16 2) Case study: Genetic diversity
Page 33 and 34: 18 Bidochka, M.J., Menzies, F.V. &
Page 38 and 39: Results and discussion The first co
Page 42 and 43: grown in the dark for 3 weeks at 22
Page 44: genotypes (A, B, D, and F) were re-
Page 47 and 48: 32 Since 1974 the financial losses
Page 49 and 50: 34 there is no more a strict three
Page 54 and 55: Numbers of captured beetles were an
Page 56 and 57: Mean captues +/- SE per trap & day
Page 58 and 59: dilution for plating (see above). F
Page 60: 45 “New” white grubs
Page 63 and 64: 48 The goal of this study was to de
Page 65 and 66: 50 In spring 2003 the density of M.
Page 67 and 68: 52 (Germany). The first treatment w
Page 69 and 70: 54 Application timing of H. bacteri
Page 74 and 75: Results and discussion Collection o
Page 76: Discussion During this study the en
Page 79 and 80: 64 Materials and methods Source of
Page 81 and 82: 66 100 80 60 40 20 0 100 80 60 40 2
Page 83 and 84: 68 During this study, selectivity a
Page 86: 71 Wireworms
Page 89 and 90: 74 Solanum tuberosum L. (Solanaceae
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76 % CATEGORIZATION OF MORBIDITY FI
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78 of adults caught can reliably pr
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Insect Pathogens and Insect Parasit
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Results and discussion Before spray
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References Parker, W.E. & Howard, J
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88 insecticide residues. Nonetheles
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90 References Cockbill, G.F., Hende
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92 Results and discussion a. Taxono
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94 published; some information has
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96 C2a: no sensitive crops in the s
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98 Furlan, L., Tóth, M., Ujvary, I
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100 Roberts, A.W.R. 1928: On the li
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102 Results and discussion Crop rot
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104 % damage by wireworms 100 80 60
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106 noticed that grass-clover in cr
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108 % damage by wireworms 60 50 40
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110 Materials and methods Soil Soil
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112 Emigration - deterrence of wire
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114 Table 1. The effect of broadcas
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116
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118 presence and abundance of wirew
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120 Furthermore, A. obscurus larvae
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122 Horton, D.R. & Landolt, P.J. 20
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124 Bait trapping 48 bait traps wer
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126 Acknowledgements We thank Anton
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128 history can be obtained (Scheu
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130 lower left hand corner of fig.
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132 Eggers, T. & Jones, T.H. 2000:
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134 pheromone combinations for each
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136 Fig. 2. Click beetle spp. captu
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138 Fig 4. Click beetle spp. captur
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140 Fig 6. Click beetle spp. captur
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142 Siirde, K., Lääts, K., Erm, A
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Fig. 2. Comparison of several trap
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Fig. 5. Catches of WCR in VARs+ tra
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Table 1. Performance characteristic
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Miscellaneous
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158 Material and methods Field tria
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160 Figure 2: Abundances of Collemb
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162
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164 but also when used against the
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166 based EPN product in this exper
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168
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170 branches. Beetles can walk up t
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172 1999/ 2000, I 1999/ 2000, C 200
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174
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176 Experimental design During the
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178 Acknowledgements We thank Stefa
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180 Spore suspension Conidia of M.
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182 Data about the stability of spo
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184 dence of host presence and appl
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186 Material and methods All habita
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188 Hajek, A.E. & Leger, R. J. St.
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190 Risk Assessment of Fungal Biolo
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192 It is scheduled that our future
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IOBC/wprs Bulletin Vol. 28(2) 2005

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