Accepted Manuscript

Evaluation of entomopathogenic fungi for the control of Tetranychus urticae

(Acari: Tetranychidae) and the effect of Metarhizium brunneum on the predatory
mites (Acari: Phytoseiidae)

YagmurOyku Dogan, Selcuk Hazir, Ayhan Yildiz, Tariq M. Butt, Ibrahim

Cakmak

PII: S1049-9644(17)30096-8
DOI: http://dx.doi.org/10.1016/j.biocontrol.2017.05.001
Reference: YBCON 3585

To appear in: Biological Control

Received Date: 25 December 2016

Revised Date: 1 May 2017
Accepted Date: 7 May 2017

Please cite this article as: Dogan, Y., Hazir, S., Yildiz, A., Butt, T.M., Cakmak, I., Evaluation of entomopathogenic
fungi for the control of Tetranychus urticae (Acari: Tetranychidae) and the effect of Metarhizium brunneum on the
predatory mites (Acari: Phytoseiidae), Biological Control (2017), doi: http://dx.doi.org/10.1016/j.biocontrol.
2017.05.001

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 1 Evaluation of entomopathogenic fungi for the control of Tetranychus urticae (Acari:
2 Tetranychidae) and the effect of Metarhizium brunneum on the predatory mites (Acari:
3 Phytoseiidae)
4
5 YagmurOyku Dogan1, Selcuk Hazir2, Ayhan Yildiz1, Tariq M. Butt3 and Ibrahim Cakmak1*
6
1
7 Adnan Menderes University, Faculty of Agriculture, Department of Plant Protection, 09010 Aydin,
8 Turkey
2
9 Adnan Menderes University, Faculty of Arts and Sciences, Department of Biology, 09010 Aydin,
10 Turkey
3
11 Department of Biosciences, Swansea University, Singleton Park, Swansea SA2 8PP, UK
12
13
14 *Corresponding author: icakmak@adu.edu.tr, Tel: +90 256 772 70 23 Fax: +90 256 772 72 33
15
16

Page 1 of 26
17 Abstract

18 The efficacy of the entomopathogenic fungi (EPF), Metarhizium brunneum (strains ARSEF

19 4556 and V275), Metarhizium flavoviride UPH-0288, Lecanicillium lecanii UPH-0241, and

20 Beauveria bassiana UPH-1103 against the different developmental stages of the two spotted

21 spider mite (TSSM), Tetranychus urticae, was investigated at 1 x 107 conidia/ml

22 concentration in detached leaf Petri dish and whole plant pot assays. One of the most

23 virulent strains, M. brunneum V275 (= Novozymes Met52/F52), was screened using a range

24 of doses against adult female predatory mites, Phytoseiulus persimilis and Neoseiulus

25 californicus. All the EPF tested killed 11.8-14.3% and 12.8-17% of the TSSM eggs in Petri dish

26 and pot assays, respectively. They also caused 57.3-90.3% and 29.5-67.5%, mortality of the

27 mobile stages in the Petri dish and pots trials, respectively. TSSM adults were generally more

28 susceptible to the EPF than the juveniles. Adult mortality was >80% for all strains except M.

29 flavoviride which caused 67% mortality. Predatory female mites were also susceptible to M.

30 brunneum V275 with mortality being dependent on the dose and predator species; thus

31 mortality at 1x106, 1x107 and 1x108 conidia/ml was 57.5, 80.5, and 99.5% for P. persimilis

32 and 51.5, 75.0, and 90.5% for N. californicus, respectively. However, subsequent biological

33 stages (eggs and juveniles) were not affected by the fungal pathogen, and the number of

34 new generation mobile stages increased 3-9 fold compared with the initial number of

35 released predatory mites. The current study thus shows that M. brunneum V275 application

36 with the releases of predatory mites have the potential to suppress T. urticae

37

38
39 Keywords: Tetranychus urticae, spider mites, entomopathogenic fungi, Metarhizium
40 brunneum, Phytoseiulus persimilis, Neoseiulus californicus
41

Page 2 of 26
42 Introduction

43 The two-spotted spider mite (TSSM), Tetranychusurticae Koch, is a major pest of a wide

44 range of outdoor and protected crops worldwide. It is estimated that it attacks over 1000

45 different plant species including economically important food crops and ornamentals (van

46 Leeuwen et al., 2010, 2015). TSSM feeding damage can result in chlorosis, leaf and fruit

47 deformation and stunted plant growth leading to reduced yields or poor marketability of the

48 produce (Lahai et al., 2003). Acaricides are still widely used to control TSSM, however, many

49 of these chemicals are failing because the pest develops resistance rapidly due to its short

50 generation time, high fecundity and arrhenotokous parthenogenesis (Cakmak et al., 2003,

51 2005). Acaricide resistance is creating considerable pressure on farmers who are increasingly

52 seeking alternative methods of control with many using macro and microbial biocontrol

53 agents (Dermauw et al., 2013).

54

55 Several predatory mites, such as Phytoseiulus persimilis and Neoseiulus californicus (Acari:

56 Phytoseiidae), and entomopathogenic fungi (EPF), such as Metarhizium anisopliae,

57 Beauveria bassiana and Lecanicillium lecanii, are often used to control TSSM and other

58 spider mites (Chandler et al., 2005; Maniania et al., 2008; Cakmak et al., 2009). The

59 predatory mites feed on the different developmental stages of the TSSM. Control can vary

60 depending on a range of factors such as predator-prey ratio, host plant and climate

61 (Escudero and Ferragut, 2005). EPF conidia germinate on and penetrate the cuticle then

62 rapidly colonize the haemocoel before emerging and sporulating on the mite cadaver. EPF

63 efficacy in controlling TSSM depends on the strain, dose, formulation, environmental factors

64 and compatibility with pesticides (Irigaray et al., 2003; Bugeme et al., 2009, 2014; Gatarayiha

65 et al., 2010a, b; Ullah and Lim, 2015; Afifi et al., 2015).

Page 3 of 26
66

67 To enhance control, attention is focusing on the development of integrated pest

68 management (IPM) programmes where combinations of several biological control agents are

69 used to suppress pest populations (Premachandra et al., 2003). The potential exists for using

70 combinations of EPF and predatory mites for maintaining TSSM populations below the

71 economically damaging levels, including acaricide resistant populations. Although EPF have

72 been used together with predatory mites to control spidermites, however, results are often

73 variable (Vergel et al., 2011). More studies on this topic may generate information which

74 could help improve pest control, reassuring the grower that the combined use of predators-

75 pathogens is robust, economical and efficacious. The risks and benefits of the simultaneous

76 deployment of EPF and predatory mites is one area that has not been fully evaluated. The

77 biggest risk being the potential of EPF to debilitate or kill the beneficial predatory mite.

78 Limited studies have been conducted to determine the susceptibility of predatory mites to

79 EPF or their combined use in spider mite control. Strains of B. bassiana were found to have

80 no negative effect on the thrips predator Neoseiulus (Amblyseius) cucumeris (Jacobson et al.,

81 2001) and Neoseiulus barkeri (Wu et al., 2014). Some researchers report that predatory

82 mites are less susceptible to EPF than the pest with some predators actively avoiding EPF-

83 infected mites (Seiedy et al., 2013; Wu et al., 2016). Some strains of EPF do, however,

84 appear to affect survival, longevity or fecundity of some predatory mites (Seiedy et al.,

85 2012).

86

87 The aims of this study were to determine the efficacy of M.brunneum, M.flavoviride, L.lecanii

88 and B.bassiana against different developmental stages of T. urticae and then test the most

89 virulent strain against the beneficial predatory mites, P.persimilis and N.californicus. The

Page 4 of 26
 90 overall goal was to determine if virulent strains of EPF could be used with predatory mites

91 for suppressing TSSM populations.

92

93

94 Material and Methods

95 Rearing of Tetranychus urticae, Phytoseiulus persimilis and Neoseiulus californicus

96 TSSM collected from strawberry fields in Aydin, Turkey were maintained on Phaseolus

97 vulgaris cv. Barbunia grown in 0.5 litre pots at 252oC, 6010% Rh, and 16 h light

98 photoperiod. Plants were inoculated with TSSM from the 5-6 leaf stage onwards. The rearing

99 of T. urticae was conducted as described by Cakmak et al. (2005, 2009). The predatory mites,

100 P.persimilis and N.californicus, were collected from bean plants in Hatay and strawberry

101 fields in Aydin, Turkey, respectively. The predatory mites were reared at 251oC on detached

102 bean leaves infested with T. urticae. Three bean leaves infested with T. urticae were added

103 to the predator cultures twice weekly.

104

105 Entomopathogenic fungi: strains and preparation of inoculum

106 Metarhizium brunneum strains ARSEF 4556 and V275 (= Met52, F52, BIPESCO 5) were

107 obtained from Swansea University, UK while Metarhizium flavoviride UPH-0288,

108 Lecanicillium lecanii UPH-0241, and Beauveria bassiana UPH-1103 strains were obtained

109 from Siedlce University, Poland. All EPF strains were grown on Sabouraud Dextrose Agar

110 (SDA) medium at 25C for 7-10 days. Conidia were harvested from sporulating cultures with

111 the aid of a spatula, washed with sterile distilled water and filtered through 4 layers of gauze

112 to remove any hyphae. Conidia were suspended in 0.03% (v/v) aqueous Tween 80 to the

113 desired concentration. Three doses were used in assays: 1 x 106, 1 x 107 and 1 x 108

Page 5 of 26
114 conidia/ml. Conidia germination rates were assessed as outlined by Ansari and Butt (2011)

115 and always exceeded 90%.

116

117

118 Efficacy of EPF against T. urticae

119 EPF were initially assayed against TSSM in Petri dishes, then pot trials. For Petri dish assays, a

120 bean leaf was placed on top of moist cotton wool with the abaxial (underside) surface facing

121 upwards. The petiole was immersed into the cotton to ensure the leaf remained hydrated.

122 Each of the five Petri dishes was placed in a transparent plastic box (33.5x46x8.5 cm) and

123 covered with another box to maintain high humidity. For pot trials, one of the cotyledon

124 leaves was removed from the plant and the remained leaf was inoculated with synchronized

125 stages as outlined below (there was only one leaf in per pot). A transparent plastic cloche

126 (12x10.5x12 cm) was placed over each pot to maintain high humidity. The humidity was

127 determined as 855% Rh in Petri dish experiments and 805% Rh in pot experiments (Hobo

128 U12-013, Onset Computer Corporation, Massachusetts, USA).

129
130 To obtain synchronized developmental stages [eggs, larvae, nymphs (mix of protonymphs

131 and deutonymphs), female adults] 25 gravid female TSSM were placed on detached bean

132 leaves then removed 24hrs later. Twenty individuals of the required developmental stage

133 were subsequently transferred to leaves in the Petri dish assays and whole plant trials.

134
135 The EPF strains were evaluated by spraying TSSM infested leaves with 2.5ml of a spore

136 suspension (1x107 conidia/ml) using a hand sprayer. In Petri dish assay, spore suspension

137 was applied to the surface of abaxial side, whereas both sides of leaf were sprayed (2.5ml for

138 each side) in pot experiment. Control leaves were treated with 0.3% aqueous Tween carrier

Page 6 of 26
139 only. The number of living and dead individuals was recorded 7 days post inoculation (pi).

140 Dead mites were transferred to a 10 cm diam. Petri dish lined with double filter paper

141 moistened with 1 ml sterile distilled water to encourage external sporulation. Assays were

142 conducted at 251C, 70 5% relative humidity and a light: dark cycle of 16:8 h in climate

143 room (PG34-3 Digitech Ltd., Ankara, Turkey). For both Petri dish and pot studies, there were

144 five replicates per treatment with the whole study being repeated four times.

145

146 Efficacy of M. brunneum V275 against predatory mites

147 Since both strains of M. brunneum V275 and 4556 proved to be highly virulent against TSSM,

148 strain V275 was assayed against the predatory mites, P. persimilis and N. californicus.

149 Moistened cotton (10 cm diameter) was placed in a 15 cm diameter Petri dish and then a

150 bean leaf was placed as described above. The gap (2.5 cm diameter) between moistened

151 cotton and the Petri dish was created and the gap was filled with water to prevent the

152 escape of the predatory mites. Each of the five Petri dishes was placed in a transparent

153 plastic box (33.5x46x8.5 cm) and covered with another box to maintain high humidity. The

154 humidity was determined as 855% Rh (Hobo U12-013, Onset Computer Corporation,

155 Massachusetts, USA).

156 The leaf was inoculated with mixed populations (eggs, juveniles and adults) of 300 TSSM by

157 means of brushing the infested leaves and 24 hrs later 10 female predatory mites were

158 transferred to the bean leaf. The leaf was then sprayed with 2.5 ml of a conidial suspension

159 (1x106, 1x107 or 1x108 conidia/ml) of M. brunneum V275 applied using a hand sprayer.

160 Control treatments were sprayed with sterile aqueous 0.03% v/v Tween 80 only. Another

161 mixed populations of 300 TSSM were provided as food every 2 days to ensure this was not a

162 limiting factor as regards predator development. Eggs deposited by predatory mites were

Page 7 of 26
163 removed by fine brush (000) and counted daily. The treated Petri dishes were monitored

164 everyday and the number of live and dead individuals was recorded 6 days pi. In another

165 experiment, the susceptibility of juvenile stages of predatory mites was assessed. The same

166 experimental design as explained above was established. But this time, the eggs of predatory

167 mites were not removed from the arena and the treated Petri dishes were monitored

168 everyday whether laid eggs and emerged juveniles of predatory mites were affected by

169 initially sprayed EPF. The number of all alive mobile stages was counted 6 days pi. Assays

170 were conducted at 251C, 70 5% relative humidity and a light: dark cycle of 16:8 h in

171 climate room (PG34-3 Digitech Ltd., Ankara, Turkey). There were five replicates per

172 treatment with the whole study being repeated four times in one week intervals.

173
174 Statistical Analyses
175 Data were analyzed using a general linear model in Equation 1 as for a randomized complete
176 block design:
177
178 = + + + () + [1]
179
180 where is the kth observed value from the response variable (, , or ) for ith fungus
181 and jth block (week); is the overall mean; is the effect of ith fungus (i=1, 2, ..6); is the
182 effect of jth block (week) (j=1, 2, , 4); () is the interaction between fungus and block
183 (week) and is the residual random error distributed normal with mean zero and variance
184 . Statistical analysis was performed using the General Linear Model procedure. After
185 significant effects were identified, differences between means were considered significant at
186 0.05 based on Tukeys Honestly Significant Difference (Tukey HSD) test. Arcsine
187 transformation was performed on mite mortality before statistical analyses. Data on the
188 effect of M. brunneum at different concentrations were analyzed separately between P.
189 persimilis and N. californicus with Students t-test (SPSS, 2011).
190

Page 8 of 26
191 Results

192 The virulence of EPF was based on mortality. It was observed that EPF sporulated on the

193 different stages of TSSM and M. brunneum V275 sporulated on the predatory mites.

194 Susceptibility of T. urticae to entomopathogenic fungi

195 TSSM exhibited differential susceptibility to EPF with adult mites being most susceptible

196 followed by nymphs and larvae with the eggs being the least susceptible (Table 1). Ovicidal

197 activity in the Petri dish assays ranged between 11.8 2.3 to 14.3 2.4% with no statistical

198 difference between the EPF strains (F=3.81; df=5,113; P>0.01, Table 1). Larvae were more

199 susceptible, with M. brunneum V275, B. bassiana, L. lecanii, and M. brunneum 4556,

200 respectively (Table 1). No significant differences were noted between the EPF (P> 0.05)

201 except M. flavoviride (F=129.95; df=5,113; P<0.001; Table 1). Nymphs were highly

202 susceptible to M. brunneum 4556 and V275 and M. flavoviride (Table 1). Nymphal mortality

203 was statistically lower for B. bassiana (62.31.9%) and L. lecanii (60.02.2%) than M.

204 brunneum strains (F=133.59;df=5,113; P<0.001). All EPF tested caused over 80% adult

205 mortality except for M. flavoviride which caused 67% mortality (Table 1). TSSM control

206 mortality for all treatments was below 4% (Table 1).

207
208 Ovicidal activity was also observed in pot experiments, with L. lecanii, M. brunneum V275

209 and 4556, B. bassiana and M. flavoviride causing 17.01.1, 15.81.0, 14.80.8, 14.80.8 and

210 12.81.1% mortality, respectively (Table 1). There were clear statistical differences between

211 the treatments and control (F=32.83; df=5,113; P<0.001). All the emergent nymphs and

212 larvae collected from EPF treated eggs 7 days pi appeared to be healthy with no symptoms

213 of fungal infection (Fig. 1A). The number of larvae and nymphs was directly proportionate to

Page 9 of 26
214 egg hatchability rate. The number of larvae and nymph was statistically different between

215 control and fungal applications (F=33.18; df=5,113; P<0,001) (Fig.1A).

216

217 Larval mortality caused by B. bassiana, L. lecanii, M. brunneum V275 and 4556 and M.

218 flavoviride ranged between 29.51.1 and 33.31.4% with no major statistical difference

219 between fungal treatments (F=74.76; df=5,113; P<0.001; Table 1). Larvae that survived

220 fungal infection, 7 days pi, ultimately developed into nymphs and adults with no decline in

221 fecundity (Fig. 1B). Statistical differences in the number of nymphs plus adults and eggs were

222 observed between the EPF treatments and control (P<0.001).

223

224 The highest nymphal mortality recorded in pot assays was for M. brunneum 4556

225 (47.01.3%) followed by M. brunneum V275 (40.51.0%), M. flavoviride (36.51.2%), L.

226 lecanii (31.51.1%) and B. bassiana (27.81.1%) with significant differences between strains

227 (Table 1; F=192.12; df=5,113; P<0.001). The lowest number of live adults (F=204.45;

228 df=5,113; P<0.001) and eggs produced by these adults (F=372.39; df=5,113; P<0.001) were

229 observed in the M. brunneum 4556 treatment which corresponded with the highest

230 nymphal mortality (Fig. 1C). The highest number of live adults and eggs was observed in the

231 control (Fig. 1C).

232

233 Adult TSSM were susceptible to all the EPF strains with M. brunneum V275 (67.51.9%) and

234 4556 (61.32.5%) being the most virulent (Table 1). Adult mortality rates were significantly

235 higher in the EPF treatments than the control (F=162.25; df=5,113; P<0.001). The lowest

236 number of live adults (F=202.38; df=5,113; P<0.001) and larvae plus nymphs (F=93.85;

237 df=5,113; P<0.001) was found in the M. brunneum 4556 and V275 treatments where the

Page 10 of 26
238 highest nymphal mortality were observed (Fig. 1D). The highest number of live adults, eggs,

239 larvae, and nymphs was in the control.

240
241
242 Susceptibility of predatory mites to Metarhizium brunneum V275

243 Adult female P. persimilis and N. californicus were susceptible to M. brunneum V275 with

244 their respective mortalities at 1 x 106, 1 x 107 and 1 x 108 conidia/ml being 57.53.5,

245 80.53.4, 99.50.5% and 51.53.3, 75.03.2, 90.52.0% (Table 2). Mortality was dose

246 dependent for both P. persimilis (F=215.55; df=3,75; P<0.001) and N. californicus (F=160.50;

247 df=3,75; P<0.001). Although mortality was similar for both predators at 1 x 106 conidia/ml (t=

248 1.305; df=1,37; P>0.05) and 1x107 conidia/ml (t= 1.313; df=1,37; P>0.05), it was significantly

249 higher for P. persimilis at 1x108 conidia/ml (t= 4.915; df=1,37; P<0.05; Table 2).

250

251 Female predatory mites started to die 3 days pi with M. brunneum V275 but were still able

252 to lay eggs before dying (Table 3). As the mortality of predatory mites was highest at 1 x 108

253 conidia/ml, oviposition was lowest at this conidial concentration (Table 3). Subsequent

254 biological stages (eggs and juveniles) appeared to be unaffected by M. brunneum. The

255 number of new generation mobile stages of predatory mites was 90.88.3, 60.23.2,

256 31.22.6 for P. persimilis and 70.22.0, 51.43.2, 32.41.7 for N. californicus at 1 x 106, 1 x

257 107 or 1 x 108 conidia/ml, respectively. This amount was 3-9 fold compared with the number

258 of predatory mites released initially.

259

260 Discussion

261 This study shows that TSSM are susceptible to various species of EPF with the adults being

Page 11 of 26
262 the most susceptible followed by the juveniles (nymphs, larvae) and with the eggs being the

263 least susceptible. Susceptibility of the TSSM appeared to be dependent upon the fungal

264 species/strain, dose, and assay conditions. The higher control obtained in Petri dishes may

265 be partially due to high humidity which favors EPF infection and sporulation (Butt and

266 Goettel, 2000). Whole plants exhibit dynamics which could interfere with EPF efficacy. For

267 example, leaf expansion and fungistatic plant volatiles released from intact plants have been

268 shown to reduce the efficacy of M. anisopliae in the control of mustard beetle (Inyanget al.,

269 1999). It is also possible that on detached leaves, the TSSM is nutritionally stressed which

270 increases its susceptibility to fungal infection. Stress has been shown to increase insect

271 susceptibility EPF irrespective of whether the stress is chemical or biotic (Butt et al., 2016).

272 The host plant is known to influence susceptibility of insects to a range of microbial

273 pathogens (Cory and Hoover, 2006; Butt et al., 2016; Hazir et al., 2016).

274

275 The differential susceptibility of adults, juveniles and eggs of TSSM to EPF has also been

276 reported by others. Furthermore, this phenomenon has also been observed in other

277 arthropods. Various mechanisms have been reviewed to explain why the different

278 developmental stages differ in their susceptibility to EPF with much emphasis being given to

279 the physical-chemical properties of the arthropod cuticle and ecdysis (Butt et al., 2016).

280 Bugeme et al. (2014) noted that time interval between consecutive moulting stages in larvae

281 and protonymphs of the TSSM was short which meant that spores would be shed with the

282 old cuticle before they had the chance to penetrate the underlying new cuticle. Some

283 researchers have suggested that the immatures are less susceptible than mature stages to

284 EPF because of their small size and limited movement, which reduce contact with conidia

285 (Oduor, 1995). Bugeme et al. (2014) reported that T. urticae adults and deutonymphs were

Page 12 of 26
286 more susceptible to M. anisopliae and B. bassiana than larvae and eggs. Whereas some

287 researchers have found TSSM eggs to be highly susceptible to EPF infection others found

288 them to be more tolerant as reported here. For example, exposing TSSM eggs to high doses

289 of B. bassiana resulted in 70% mortality with eggs of different ages being equally susceptible

290 (Irigaray et al., 2003). This suggests that some EPF strains exhibit greater ovicidal activity

291 than others. Indeed, Shi and Feng (2004) screened 10 isolates of B. bassiana, M. anisopliae

292 and Paecilomyces fumosoroseus and found only 4 to be highly efficacious in killing T.

293 cinnabarinus eggs but only at high doses., At lower doses all ten isolates caused significantly

294 lower egg mortality (Shi and Feng, 2004). Erler et al. (2013) compared M. anisopliae F52 (=

295 M. brunneum V275) with a strain of B. bassiana and found the latter to be more effective

296 against T. cinnabarinus eggs. Other researchers have also reported egg mortality to be dose

297 dependent (Irigaray et al., 2003; Shi and Feng, 2004; Erler et al., 2013; Bugeme et al., 2014).

298 The low egg mortality reported here is probably due to the strain having low ovicidal activity.

299
300 Controlling the adult TSSM, especially females (to prevent oviposition), is essential for

301 suppressing TSSM populations. The current study shows that the most virulent strains (M.

302 brunneum V275, ARSEF 4556) caused > 60% adult mortality in whole plant studies and >80%

303 in Petri dish assays. The control was similar to that reported by other researchers. However,

304 Wu et al. (2016) reported that the virulence of B. bassiana strains at 1x107 m/L conidia

305 ranged between 37-49% against T. urticae adults in petri dishes. Fungal virulence is not just

306 dependent upon the fungal strain, but also the formulation, dose and frequency of

307 application. For example, >80% mortality of adult female T. evansi and T. cinnabarinus was

308 obtained with virulent strains of B. bassiana (Wekesa et al., 2005; Shi et al., 2008a). Field

309 studies show that effective spider mite control can be achieved through high application

Page 13 of 26
310 rates (dose and/frequency) and use of appropriate carriers. Emulsifiable formulations appear

311 to give the best control as reported for B. bassiana against the citrus red mite, Panonychus

312 citri (Shi and Feng, 2006), European red mite, Panonychus ulmi on apple (Marcic et al., 2012),

313 and cotton spider mites, T. truncates and T. turkestani (Shi et al., 2008b). Similar results were

314 obtained with oil formulations of M. anisopliae and B. bassiana against T. cinnabarinus on

315 okra (Naik and Shekharappa, 2009). Bugeme et al. (2015) showed that formulated M.

316 anisopliae (ICIPE78) used at 1x108 conidia/ml was as effective as the abamectin insecticide in

317 reducing T .urticae densities on protected and field crops. This dose was 10 fold higher than

318 that used in the current study so it is feasible that M. brunneum 4556 and V275 could have

319 given even greater control of TSSM if used at the higher dose and/or applied several times at

320 set intervals.

321

322 Adult TSSM were more susceptible to M. brunneum V275 than the predatory mites P.

323 persimilis and N. californicus when exposed to the same spore concentration. Interestingly,

324 N. californicus was less susceptible to V275 than P. persimilis at the highest dose. No

325 obvious oviposition or behavioral changes were observed in infected predatory mites or

326 TSSM at least shortly before death. Furthermore, freshly laid eggs and subsequent

327 developmental stages were also unaffected presumably due to poor exposure to conidia but

328 the possibility of greater resistance cannot be ruled out. Higher application rates of M.

329 brunneum may kill more TSSM but it could reduce the number of predators significantly. The

330 susceptibility of P. persimilis and N. californicus to EPF appears to be dependent upon the

331 strain and dose. For example, Vergel et al. (2011) found that B. bassiana and P.

332 fumosoroseus at 1.25x107conidia/ml caused 43% and 14% female mortality of P. persimilis

333 and 31% and 16% of N. californicus, respectively. In contrast, Wu et al. (2014) found that a

Page 14 of 26
334 thrips pathogenic strain of B. bassiana was not harmful to neither adult nor larval stages of

335 the predatory mite, Neoseiulus barkeri. More recently, ultrastructural studies have revealed

336 that B. bassiana conidia adhered to the cuticle of predatory mites but failed to infect

337 because the conidia were either shed or shriveled whereas they germinated and penetrated

338 the cuticle of T. urticae (Wu et al., 2016). EPF strains do differ in their host specificity with

339 fatty acids in the epicuticular waxes often influencing spore adhesion, germination and/or

340 penetration of the host cuticle (Butt et al., 2016).

341

342 The current study shows that female predatory mites will freely lay eggs right up till the time

343 they are killed by M. brunneum V275. Furthermore, none of their progeny (eggs, juveniles,

344 adults) become infected. These factors combined with the fact that the development time of

345 the predatory mites is significantly faster than that of Tetranychus species (Cakmak and

346 Demiral, 2007; Kazak, 2008; Kustutan and Cakmak, 2009) may account for the significant

347 increase in the predator population (up to 9 fold higher than initial number released).

348 Although not observed in our study, it is possible that the predatory mites are able to detect

349 the presence of M. brunneum V275 and avoid it. Several workers show that predators avoid

350 EPF treated surfaces or infected hosts (Butt et al., 2016). For example, the predators P.

351 persimilis, Amblyseius swirskii and Anthocoris nemorum avoid their respective prey

352 (Microlophium carnosum, Trialeurodes vaporariorum, T. urticae) if they are infected with B.

353 bassiana (Meyling and Pell, 2006; Seiedy, 2014, 2015).

354

355 In conclusion, this study shows that EPF are effective in killing TSSM but pose a risk, albeit a

356 comparatively smaller one, to beneficial predatory mites. When used together, these two

357 biological control agents show much promise in suppressing the TSSM population, however,

Page 15 of 26
358 their application rate and timing needs to be optimized if any potential synergies are to be

359 fully exploited. It should also be remembered that many of the crops attacked by TSSM are

360 also susceptible to other pests notably aphids, thrips, and whitefly. Selecting strains of EPF

361 that kill these pests and not beneficial arthropod predators and parasitoids will be vitally

362 important in the development of robust, efficacious, cost effective pest control programmes.

363

364 Acknowledgements

365 The authors thank Merve Gkay, Mine Pelin Ylmaz, Ceren Erolu, and Hlya Dizlek for their

366 assistance in the experiments, Dr. Cezary Tkaczuk (Siedlce University, Poland) for providing

367 M. flavoviride, L. lecanii and B. bassiana, Kadir Kizilkaya (Adnan Menderes University,

368 Turkey) for statistical analyses, and Dr. Harry K. Kaya (University of California, Davis) for

369 editing an earlier version of the manuscript. We also thank the anonymous referees for their

370 critical reviews and suggestions to improve the quality of the manuscript. This study was

371 supported by Adnan Menderes University Research Foundation (Grant no: ZRF-14024).

372

373

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539 0090-x
540
541
542

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543

544 Table 1. Susceptibility of different developmental stages of Tetranychus urticae exposed to 1

545 x 107 conidia/ml of five strains of entomopathogenic fungi in Petri dish and pot experiments.
Mortality rate (%SE)
Egg Larva Nymph Adult
Petri dish experiment
Beauveria bassiana 11.82.3 aC 63.52.2 aB 62.31.9 bB 80.33.8 bA
Lecanicillium lecanii 12.32.6 aC 64.02.2 aB 60.02.2 bB 90.31.9 aA
Metarhizium brunneum V275 13.32.4 aC 66.51.9 aB 71.51.8 aB 86.32.3 abA
Metarhizium brunneum 4556 13.52.3 aD 61.52.2 abC 73.01.8 aB 84.33.0 abA
Metarhizium flavoviride 14.32.4 aC 57.32.2 bB 66.52.2 abA 67.33.2 cA
Control 2.00.8 bA 3.50.9 cA 4.01.2 cA 3.80.8 dA
Pot experiment
Beauveria bassiana 14.80.8 abC 30.01.4 aB 27.81.1 cB 45.81.4 cA
Lecanicillium lecanii 17.01.1 aC 31.02.0 aB 31.51.1 cB 50.02.0 cA
Metarhizium brunneum V275 15.81.0 abD 33.31.4 aC 40.51.0 bB 67.51.9 aA
Metarhizium brunneum 4556 14.80.8 abD 30.01.3 aC 47.01.3 aB 61.32.5 abA
Metarhizium flavoviride 12.81.1 bD 29.51.1 aC 36.51.2 bB 57.81.0 bA
Control 2.50.6 cA 3.30.8 bA 3.00.8 dA 3.30.9 dA
546 Means within a row followed by the different upper-case letters and within a column followed by the different
547 lower-case letters are significantly different (P<0.05, Tukey test).
o
548 Mortality was recorded at seven days post-infection at 251 C.
549

550

Page 22 of 26
551 Table 2. Mortality rate of Metarhizium brunneum V275 on females of Phytoseiulus persimilis
552 and Neoseiulus californicus in Petri dish experiment.
Mortality rate (%SE)
Fungal concentrations (conidia/ml)
Control F ratio
106 107 108
P. persimilis 3.51.1 d 57.53.5 c 80.53.4 b 99.50.5 aA 215.55
N. californicus 2.51.0 d 51.53.3 c 75.03.2 b 90.52.0 aB 160.50
t-test value 0.677 1.305 1.313 4.915
P>0.05 P>0.05 P>0.05 P<0.05
553 Means within a row followed by the different lower-case letters are significantly different (P<0.05, Tukey test).
554 Means within a column followed by the different upper-case letters are significantly different (P<0.05, Student
555 t-test).
556 Mortality was recorded at 6 days post-infection at 251oC.
557

Page 23 of 26
558 Table 3. The total number of eggs deposited by the predatory mites after Metarhizium
559 brunneum V275 application in Petri dish experiment.
Fungal concentrations (conidia/ml)
Control F ratio
106 107 108
*
Phytoseiulus persimilis 168.99.5 a 147.910.3 a 93.08.1 b 42.36.6 c 65.04
Neoseiulus californicus 88.23.9 a 79.02.9 ab 68.52.9 b 51.73.6 c 29.92
560 Means within a row followed by the different lower-case letters are significantly different (P<0.01, Tukey test).
o
561 The total number of deposited eggs was recorded at 6 days post-infection at 251 C. 10 female predatory
562 mites were used for each treatment.
563

Page 24 of 26
564

565 Figure Legend

566 Figure 1. The mean number of survival stages of Tetranychus urticae after spraying
567 entomopathogenic fungi on (A) eggs, (B) larvae, (C) nymphs, (D) adult stages in pot
568 experiments. Initial population of the each stage was 20 individuals.
569

Page 25 of 26
570 - Tetranychus urticae adults were more susceptible to the Entomopathogenic fungi (EPF)
571 than the juvenile stages.

572 - The EPF exhibited weak ovicidal activity.

573 - Metarhizium brunneum V275 was also pathogenic to the predatory mites, Phytoseiulus
574 persimilis and Neoseiulus californicus.

575 - These predatory mites were much less susceptible than the pest mite species.

576 - EPF application with the releases of predatory mites have the potential to suppress T.
577 urticae.
578
579

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