Microsclerotia (MS) production by two isolates of Lecanicillium lecanii on various culture media is described, and the efficacy of MS against western flower thrips is evaluated. High concentrations of MS (2.9-3.1×10⁵•mL⁻¹) were produced in media with C: N ratios of 7.4:1 and 10.3:1 by isolate SN21. Bioassays using soil-incorporated MS resulted in significant infection and mortality of thrips.Keywords: Frankliniella occidentalis, Lecanicillium lecanii, fermentation, integrated biological pest control, microscleroti

Lei, Zhongren

Li, Yinping

Reitz, Stuart

Wang, Haihong

Xu, Xuenong

Undetermined

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For submission to:  1 Biocontrol science and technology 2  3  4  5  6  7  8  9  10 Running title: Microsclerotia of Lecanicillium lecanii 11  12  13 Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii 14 (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of 15 Frankliniella occidentalis (Thysanoptera: Thripidae) 16  17  18 HAIHONG WANG1, ZHONGREN LEI1,3, STUART R REITZ2 ,YINPING LI1 XUENONG XU1 19  20  21  22  23  24  25  26 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant 27 Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China 28 2 Malheur County Extension, Oregon State University, 710 SW 5th Avenue, Ontario, Oregon 29 97914, USA 30 3 Corresponding author, e-mail: leizhr@sina.com 31  1 Microsclerotia (MS) production by two isolates of Lecanicillium lecanii on various culture 32 media is described, and the efficacy of MS against western flower thrips is evaluated. High 33 concentrations of MS (2.9-3.1×105•mL-1) were produced in media with C: N ratios of 7.4:1 and 34 10.3:1 by isolate SN21. Bioassays using soil-incorporated MS resulted in significant infection 35 and mortality of thrips. 36  37 Key words: Lecanicillium lecanii, Frankliniella occidentalis, microsclerotia, Fermentation 38 integrated biological pest control 39 Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is a 40 pest of global significance on a wide range of economically important crops (Kirk and Terry, 41 2003; Reitz, 2009). Many isolates of Lecanicillium spp. (previously known as Verticillium lecanii) 42 have been applied as biopesticides for control of F. occidentalis (Faria and Wraight, 2007; 43 Ansari et al., 2008). This study describes the formation of microsclerotia (MS) by two isolates 44 of Lecanicillium lecanii (Zimm.) Zare & W. Gams, and investigates their activity against F. 45 occidentalis in order to provide a basis for development of a biological control technique using 46 L. lecanii MS. 47 A colony of western flower thrips was maintained as described by Ansari et al. (2008). 48 Briefly, about 40-50 adult thrips were introduced into 0.5 L ventilated glass jars containing 3-4 49 pieces of green bean (Phaseolus vulgaris L.) pods at 26 ± 1°C, 60–70% RH and L14:D10 50 photoperiod. After 12 hours, egg-infested beans were transferred to fresh glass jars.  First 51 instars hatched 3 days later and were transferred to fresh glass jars with fresh green bean pods, 52 using a fine camel hair brush. After 3 days, second instars (L2) were collected for experimental 53 use. 54 The origin and source of the two fungal isolates are as follows: (1) NA1, derived from 55 Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) collected in Nongan, Jilin (2010); (2) 56 SN21, derived from Thrips tabaci Lindeman (Thysanoptera: Thripidae) collected in Shenyang, 57 Liaoning (2011). Stocks of isolates NA1 and SN21 were identified as Lecanicillium lecanii sensu 58 stricto (Kouvelis et al., 2008) and deposited in the Agricultural Culture Collection of China at 59 the Chinese Academy of Agricultural Sciences (Beijing, China) under accession numbers 30840 60 and 30841. Both isolates have been under evaluation by our research group as 61  2 mycoinsecticides for control of western flower thrips (unpublished data). Each isolate was 62 cultured on agar media and stored in 10% glycerol at -80°C until use.  63 Liquid culturing, measuring and drying of MS were carried out, as described by Shearer 64 and Jackson (2006) and Jackson and Jaronski (2009), with minor modifications. Briefly, in all 65 liquid culture experiments, an initial concentration of 5×106 conidia•ml-1 of L. lecanii was 66 incubated in medium composed of a nitrogen source (either soybean powder or casamino 67 acids, 10-50 g•L-1), carbon source (glucose, 10-72 g•L-1) and basal salt solution (Jackson and 68 Jaronski, 2009). Cultures were incubated at 25±1°C on rotary shaker incubators (SPH-103B, 69 SHIPING lab equipment Co. Ltd., Shanghai, China) at 150 rpm. Carbon concentration and C: N 70 ratio calculations were based on 40% carbon in glucose, and on 10% carbon and 11% nitrogen 71 in soybean powder (Zabriske et al., 1982) (Table 1).  Three replicate flasks for each isolate 72 and media treatment were used, and the entire experiment was repeated twice at different 73 times.  74 At seven days post-inoculation, all the MS (>50 µm in diameter) in 50 µl of culture broth 75 from each flask were counted using a microscope (Olympus BX41, Olympus Optical Co., Ltd., 76 Tokyo, JAPAN). Fifteen grams of diatomaceous earth (DE) were then added to 300 ml culture 77 broth. The MS-DE mixture was filtered in a circulating water pump (SHZ-Ⅲ, Yarong 78 Biochemical Instrument Factory. Shanghai, China), dried (moisture content ≤5%) in a vacuum 79 dryer (LGJ-10D, Four Rings Scientific Instrument Co., Ltd. Beijing, China), vacuum packed in 80 aluminum foil bags with a vacuum packer (DZ-260, Beijing Jiaode Packaging Machinery Co., 81 Ltd. ), and stored at 4°C for later experimental use.  The liquid culturing media that produced 82 the most MS for each isolate were used in the following experiments to determine conidial 83 production and the efficacy against western flower thrips of the isolates. 84 Conidial production was determined for air-dried, MS–DE preparations according to the 85 methods of Jackson and Jaronski (2009). Twenty-five mg of MS–DE formulation was sprinkled 86 onto the surface of a water agar plate, and plates were incubated for eight days at 25°C. Each 87 water agar plate was then flooded with 5 ml of sterile water and the conidia were dislodged 88 from the MS–DE granules using a sterile loop. The concentration of conidia was measured 89 using a hemocytometer under a microscope. To determine the number of conidia that each L. 90  3 lecanii isolate produced per gram of dried MS–DE preparation, the number of conidia harvested 91 per plate was divided by the weight of the dried MS–DE preparation added to each water agar 92 plate (0.025 g). 93 The entomopathogenic efficacies of the MS–DE mixtures of the L. lecanii isolates were 94 assessed according to the methods of Ansari et al. (2008), with minor modifications. Briefly, in 95 250-ml plastic pots, dried MS–DE mixtures that had been stored for either 1 or 90 days were 96 uniformly mixed into plant growth media, composed of vermiculite, clay loam, and nutrition 97 soil in a 2:1:1 proportion, to yield a final concentration of 100 μg MS–DE•100 g-1 media. One 98 ventilation hole (6 cm diameter) was made in the lid of each pot and covered with nylon gauze 99 (64 µm pore size). A 5×4 cm section of a yellow sticky trap with thrips attractant added (Lei et 100 al., Chinese patent no. 200910090398.1) was attached to the inner part of the lid to trap adult 101 thrips. Ample water was added to pots (effluent water was observed from the bottom of pots) 102 every 6 days. Controls consisted of growing media, water and no MS-DE.  103 One week after the first watering of the MS-mixed medium, a small piece of bean pod (2 104 cm length) was placed on the media surface in each pot as a source of food for thrips, and 105 twenty L2 thrips were added to each pot. Pots were kept in a constant temperature room 106 (25±1°C, 60–70%R.H., and L14:D10 photoperiod). Experiments were replicated five times for 107 each isolate and the whole experiment was conducted twice.  108 Emerging adults were attracted to and then adhered to the sticky traps. On the eighth day 109 after the first thrips adult emergence, sticky traps were removed from pots, and adults trapped 110 on sticky cards were incubated separately at 25°C in Petri dishes lined with moist filter paper. 111 At ten days post-incubation, thrips were examined microscopically, and the numbers of 112 fungi-infested and healthy adults were recorded. 113 For each study (MS production and efficacy), data from the repeated experiments were 114 pooled for statistical analysis.  In the efficacy study, mortality data were normalized using the 115 arcsine square root transformation before further analysis.  SPSS (version 11.0) software was 116 used for ANOVA followed by Duncan’s multiple range tests to detect statistical differences 117 among treatments. Significance was set at the 5% probability value. 118 Both isolates produced fewer and less uniform MS with casamino acids as the nitrogen source 119 than with soybean powder as the nitrogen source (data not shown).  With soybean powder 120  4 as the nitrogen source, the yield of L. lecanii MS varied among culture media for each isolate 121 (Table 1). For isolate NA1, the highest MS concentrations were produced in both carbon-poor 122 (5 g•L-1) media with C: N ratios of 4.5:1 and carbon-rich (25 g•L-1) media with C: N ratios 123 ranging from 4.5:1 to 10.3:1; lower MS concentrations were produced in other culture media 124 (F=13.29; df=5, 12; P<0.001). For isolate SN21, the highest MS concentrations were produced 125 in carbon-rich (29.2 and 31.6 g•L-1) liquid media with C: N ratios of 7.4:1 and 10.3:1 126 respectively, and significantly lower MS concentrations were produced in other culture media 127 (F=17.412; df= 5, 12; P<0.001). Conidia production by air-dried, MS–DE preparations of L. 128 lecanii was 87.3×105 conidia•g-1 for isolate NA1 and 87.3×105 and 142.6×105 conidia•g-1 for 129 isolate SN21. 130 MS-DE treated soils produced significantly greater mean mortalities of western flower 131 thrips than control soils, but there were no significant differences in mortality between the 132 isolates (storage for 1 day: 98%, 100% and 0% for NA1, SN21 and control, respectively; 133 F=4574.129, df=2,6, P<0.001; storage for 90 days: 72%, 84% and 0% for NA1, SN21, and control, 134 respectively; F=95.588, df=2,6, P<0.001).    135     Our results indicate that MS production by L. lecanii isolates NA1 and SN21 is affected by 136 liquid culture conditions and that MS of both isolates significantly reduce F. occidentalis survival 137 through the production of infective conidia. In our study, the use of casamino acids was not 138 conducive to MS formation by L. lecanii, which is in contrast to results of studies of MS 139 formation by other fungal species, including Colletotrichum truncatum (Schwein.) Andrus & 140 W.D. Moore (Jackson and Schisler, 1995), Mycoleptodiscus terrestris (Gerd.) Ostaz. (Shearer 141 and Jackson, 2006), and Metarhizium anisopliae (Metchnikoff) Sorokin (Jackson and Jaronski, 142 2009). Furthermore, the highest MS concentrations by the two isolates in our study, NA1 and 143 SN21, were achieved with different carbon content and C: N ratios in the liquid media. Thus, it 144 is apparent that environmental conditions required for formation of MS varies among fungal 145 species and even among isolates of a particular species. The MS of both isolates proved to be 146 highly virulent to western flower thrips, even after 90 days of storage. Both isolates produced 147 greater mortality than observed in previous studies (Vestergaard et al. 1995; Gouli et al. 2009), 148 although differences in thrips species and development stages used in experiments cannot be 149 discounted.  150  5  151 Acknowledgements 152 The authors would like to thank Xue li, Institute of Plant protection, CAAS, for her molecular 153 characterization of L. lecanii. This research project was supported by the National Special Fund 154 for the Commonwealth Agricultural Research (200903032) and China Agriculture Research 155 System (CARS-25-B-07).  156  157 References 158 Ansari, M.A; Brownbridge, M., Shah, F. A., Butt, T.M. (2008), Efficacy of entomopathogenic 159 fungi against soil-dwelling life stages of western flower thrips, Frankliniella occidentalis, in 160 plant-growing media, Entomologia Experimentalis et Applicata.,127, 80–87. 161 Faria, M.R., Wraight, S.P. (2007), Mycoinsecticides and Mycoacaricides: a comprehensive list 162 with worldwide coverage and international classification of formulation types, Biological 163 Control, 43, 237–256.  164 Gouli, V. V., Gouli, S. Y., Skinner, M., Shternshis, M. V. (2009), Effect of the entomopathogenic 165 fungi on mortality and injury level of western flower thrips, Frankliniella occidentalis, 166 Archives of Phytopathology and Plant Protection, 42, 118-123 167 Jackson, M.A., Jaronski, S.T. (2009). Production of microsclerotia of the fungal entomopathogen 168 Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting 169 insects, Mycological Research, 113, 842–850. 170 Jackson, M.A., Schisler, A.D. (1995). Liquid culture production of microsclerotia of 171 Colletotrichum truncatum for use as bioherbicidal propagules. Mycological Research, 99, 172 879-884. 173 Kirk, W.D.J., Terry, L.I. (2003), The spread of the western flower thrips Frankliniella occidentalis 174 (Pergande), Agricultural and Forest Entomology, 5, 301–310. 175 Kouvelis, V. N., Silalkouma, A., Typas, M. A. (2008), Mitochondrial gene sequences alone or 176 combined with ITS region sequences provide firm molecular criteria for the classification of 177 Lecanicillium species. Mycological Research, 112, 829-844. 178 Lei, Z. H., Liang, X. H., Wang, H. H., Wen, J. Z. (2009), Preparation and application of a kind of 179 insect-attracted yellow plate, Chinese patent no. 200910090398.1 180  6 Reitz, S. R. (2009), Biology and ecology of the western flower thrips (Thysanoptera: Thripidae): 181 The making of a pest. Florida Entomology, 92, 7-13. 182 Shearer, J.F., Jackson, M.A. (2006), Liquid culturing of microsclerotia of Mycoleptodiscus 183 terrestris, a potential biological control agent for the management of Hydrilla, Biological 184 Control, 38, 298–306. 185 Vestergaard, S., Gillespie, A.T., Butt, T.M., Schreiter, G., Eilenberg, J. (1995). Pathogenicity of the 186 hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to the western flower 187 thrips, Frankliniella occidentalis. Biocontrol Science and Technology, 5, 185-192.  188 Zabriske, D.W., Armiger, W.B., Phillips, D.H., Albano, P.A. (1982). Trader’s guide to fermentation 189 media formulation. Trader’s Protein. Memphis, Tennessee. USA  190 191  7 Table 1. Comparison of MS production by isolates of L. lecanii in various liquid culture mediaa 192  193 Isolate Culture Media Microsclerotia  (mean MS× 104•mL -1)  C (g•L-1) C:N Glucose (g•L-1) Soybean powder (g•L-1) NA1 5 5.8 6.4 25 29.2 31.6 4.5:1 7.4:1 10.3:1 4.5:1 7.4:1 10.3:1 10 12.8 14.4 50 64 72 10 7.2 5.6 50 36 28 16.9a 6.8bc 5.8c 12.6ab 10.8abc 14.6a SN21 5 5.8 6.4 25 29.2 31.6 4.5:1 7.4:1 10.3:1 4.5:1 7.4:1 10.3:1 10 12.8 14.4 50 64 72 10 7.2 5.6 50 36 28 6.4c 12.0b 8.1bc 9.3bc 29.3a 31.2a a For each isolate, mean values followed by the different letters are significantly different using 194 Duncan’s multiple range test (α = 0.05). 195  196  197      198  8 

Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of Frankliniella occidentalis (Thysanoptera: Thripidae)

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Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of Frankliniella occidentalis (Thysanoptera: Thripidae)

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