Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the field

Fernanda de Cássia Neves Esteca

In a 3-year, replicated on-farm study, we compared the incidence of root fungi in a certified organic and a conventional strawberry production system in coastal central California. The organic production system received yearly inputs of composted manures and organic fertilizers, and no pesticides were used. The conventional pro- duction system received preplant fumigation with methyl bromide/chloropicrin, fungicide dip at plant-out, syn- thetic fertilizers, and periodic insecticide and acaricide sprays during the harvest season. Whole plants were dug up and root segments incubated on selective media. Fusarium, Pythium, Rhizoctonia, Cylindrocarpon, Trichod- erma, and Verticillium were isolated from the strawberry roots. Root growth and fruit yield was lower in the or- ganic system, although epidemic populations of potential root pathogens were not observed. Fungal communities in the two production systems differed more after 2 years of organic management. The organic system was char- acterized by increases in the population of Cylindrocarpon. We discuss the role of organic matter management in the biological control of soil-borne plant pathogens.

Strawberry fields receive several pesticides spraying and residues in fruits are recurrent. Development and application of methods that avoid applying pesticides mostly during fructification period are imminent needs. Comparison of population of two-sppoted spider mite (TSSM) Tetranychus urticae Koch. in organic and mineral fertilized plants (as used by growers) and treated with alternative products were done. Plants were grown in vessels in a greenhouse infested with local population of TSSM. Extracts of Tephrosia voggeli (Hook), neem oil (Azadirachta indica A. Juss.), and lime sulfur were applied three times against TSSM in ten days intervals. Assessments were done three times after each spraying. Interaction between fertilization and spraying alternatives was also assessed but was not significant. TSSM population was about 44% lesser in organic fertilized plants than those in inorganic fertilized. T. voggeli extracts reduced in large amounts TSSM; neem oil reduced in some assessm...

Considering the economic importance of strawberry in Brazil, with a clear migration trend from the soil to innovative systems on substrates, it is fundamental to evaluate the available cultivars in the market regarding tolerance to pathogens with potential for infestation. The reaction of eight commercial strawberry cultivars (Festival, Camino Real, Camarosa, Oso Grande, Monterey, San Andreas, Aromas, and Albion) to Meloidogyne and Pratylenchus species was evaluated. Strawberry cultivars were maintained in individual pots with sterilized soil and inoculated with 5,000 eggs + second stage juveniles of Meloidogyne arenaria, M. incognita, M. javanica or M. hapla or 1,000 specimens of Pratylenchus zea or P. brachyurus per plant. The experiment was carried out in a completely randomized design with six replications using tomato ‘Rutgers’ and sorghum ‘506’ plants as control to root-knot and lesion nematodes, respectively. Ninety days after inoculation, the nematode reproduction factor (RF...

Metadata of the article that will be visualized in OnlineFirst ArticleTitle Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the field Article Sub-Title Article CopyRight Springer-Verlag GmbH Germany, part of Springer Nature (This will be the copyright line in the final PDF) Journal Name Journal of Pest Science Corresponding Author Family Name Delalibera Particle Given Name Italo Suffix Jr. Division Department of Entomology and Acarology Organization “Luiz de Queiroz” College of Agriculture/University of São Paulo (ESALQ/ USP) Address Piracicaba, São Paulo, 13418-900, Brazil Phone +55 (19) 3429-4199 Fax Email delalibera@usp.br URL Author ORCID http://orcid.org/0000-0001-9770-9216 Family Name Canassa Particle Given Name Fernanda Suffix Division Department of Entomology and Acarology Organization “Luiz de Queiroz” College of Agriculture/University of São Paulo (ESALQ/ USP) Address Piracicaba, São Paulo, 13418-900, Brazil Division Department of Plant and Environmental Sciences Organization University of Copenhagen Address Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark Phone Fax Email fernanda.canassa@usp.br URL Author ORCID http://orcid.org/0000-0001-9186-9278 Family Name Esteca Particle Given Name Fernanda C. N. Suffix Division Department of Entomology and Acarology Organization “Luiz de Queiroz” College of Agriculture/University of São Paulo (ESALQ/ USP) Address Piracicaba, São Paulo, 13418-900, Brazil Phone Fax Email fernanda.esteca@usp.br URL Author ORCID http://orcid.org/0000-0001-8043-2433 Family Name Moral Particle Given Name Rafael A. Suffix Division Department of Mathematics and Statistics Organization Maynooth University Address Maynooth, Co. Kildare, Ireland Phone Fax Email rafael.deandrademoral@mu.ie URL Author ORCID http://orcid.org/0000-0002-0875-3563 Family Name Meyling Particle Given Name Nicolai V. Suffix Division Department of Plant and Environmental Sciences Organization University of Copenhagen Address Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark Division Biotechnology and Plant Health Division Organization Norwegian Institute of Bioeconomy (NIBIO) Address NO-1431, P.O. Box 115, Ås, Norway Phone Fax Email nvm@plen.ku.dk URL Author ORCID http://orcid.org/0000-0003-3025-4370 Family Name Klingen Particle Given Name Ingeborg Suffix Division Biotechnology and Plant Health Division Organization Norwegian Institute of Bioeconomy (NIBIO) Address NO-1431, P.O. Box 115, Ås, Norway Phone Fax Email ingeborg.klingen@nibio.no URL Schedule ORCID http://orcid.org/0000-0002-8230-5857 Received 14 March 2019 Revised 28 June 2019 Accepted 5 August 2019 Abstract The effect of inoculation of strawberry roots by two entomopathogenic fungal isolates, Metarhizium robertsii (ESALQ 1622) and Beauveria bassiana (ESALQ 3375), on naturally occurring arthropod pests and plant diseases was investigated in four commercial strawberry fields during two growing seasons in Brazil. Three locations represented open-field production while strawberries were grown in low tunnels at the fourth location. Population responses of predatory mites to the fungal treatments were also assessed. Plants inoculated by the fungal isolates resulted in significantly fewer Tetranychus urticae adults compared to control plants at all four locations. The mean cumulative numbers ± SE of T. urticae per leaflet were: M. robertsii (225.6 ± 59.32), B. bassiana (206.5 ± 51.48) and control (534.1 ± 115.55) at the three open-field locations, while at the location with tunnels numbers were: M. robertsii (79.7 ± 10.02), B. bassiana (107.7 ± 26.85) and control (207.4 ± 49.90). Plants treated with B. bassiana had 50% fewer leaves damaged by Coleoptera, while there were no effects on numbers of whiteflies and thrips. Further, lower proportions of leaflets with symptoms of the foliar plant pathogenic fungi Mycosphaerella fragariae and Pestalotia longisetula were observed in the M. robertsii (4.6% and 1.3%)- and B. bassiana (6.1% and 1.3%)-treated plots compared to control plots (9.8% and 3.7%). No effect was seen on numbers of naturally occurring predatory mites. Our results suggest that both isolates tested may be used as root inoculants of strawberries to protect against foliar pests, particularly spider mites, and also against foliar plant pathogenic fungi without harming naturally occurring and beneficial predatory mites. Keywords (separated by '-') Endophytic entomopathogenic fungi - Microbial control - Plant–microbe interactions - Tetranychus urticae - Integrated pest management (IPM) Footnote Information Communicated by E. Quesada-Moraga. Journal of Pest Science https://doi.org/10.1007/s10340-019-01147-z 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 AQ1 26 27 · Fernanda C. N. Esteca1 · Rafael A. Moral4 · Nicolai V. Meyling2,3 Received: 14 March 2019 / Revised: 28 June 2019 / Accepted: 5 August 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 · Ingeborg Klingen3 PR O O F 8 9 Fernanda Canassa1,2 Italo Delalibera Jr.1 · Abstract The efect of inoculation of strawberry roots by two entomopathogenic fungal isolates, Metarhizium robertsii (ESALQ 1622) and Beauveria bassiana (ESALQ 3375), on naturally occurring arthropod pests and plant diseases was investigated in four commercial strawberry ields during two growing seasons in Brazil. Three locations represented open-ield production while strawberries were grown in low tunnels at the fourth location. Population responses of predatory mites to the fungal treatments were also assessed. Plants inoculated by the fungal isolates resulted in signiicantly fewer Tetranychus urticae adults compared to control plants at all four locations. The mean cumulative numbers ± SE of T. urticae per lealet were: M. robertsii (225.6 ± 59.32), B. bassiana (206.5 ± 51.48) and control (534.1 ± 115.55) at the three open-ield locations, while at the location with tunnels numbers were: M. robertsii (79.7 ± 10.02), B. bassiana (107.7 ± 26.85) and control (207.4 ± 49.90). Plants treated with B. bassiana had 50% fewer leaves damaged by Coleoptera, while there were no efects on numbers of whitelies and thrips. Further, lower proportions of lealets with symptoms of the foliar plant pathogenic fungi Mycosphaerella fragariae and Pestalotia longisetula were observed in the M. robertsii (4.6% and 1.3%)- and B. bassiana (6.1% and 1.3%)-treated plots compared to control plots (9.8% and 3.7%). No efect was seen on numbers of naturally occurring predatory mites. Our results suggest that both isolates tested may be used as root inoculants of strawberries to protect against foliar pests, particularly spider mites, and also against foliar plant pathogenic fungi without harming naturally occurring and beneicial predatory mites. Keywords Endophytic entomopathogenic fungi · Microbial control · Plant–microbe interactions · Tetranychus urticae · Integrated pest management (IPM) A1 Communicated by E. Quesada-Moraga. A2 A3 * Italo Delalibera Jr. delalibera@usp.br U Author Proof 7 D 6 TE 5 EC 4 R 3 Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the ield R 2 ORIGINAL PAPER NC O 1 A4 A5 Fernanda Canassa fernanda.canassa@usp.br A6 A7 Fernanda C. N. Esteca fernanda.esteca@usp.br A8 A9 Rafael A. Moral rafael.deandrademoral@mu.ie A10 A11 Nicolai V. Meyling nvm@plen.ku.dk A12 A13 Ingeborg Klingen ingeborg.klingen@nibio.no 1 Department of Entomology and Acarology, “Luiz de Queiroz” College of Agriculture/University of São Paulo (ESALQ/USP), Piracicaba, São Paulo 13418-900, Brazil A14 A15 A16 2 Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark A17 A18 A19 3 Biotechnology and Plant Health Division, Norwegian Institute of Bioeconomy (NIBIO), NO-1431, P.O. Box 115, Ås, Norway A20 A21 A22 4 Department of Mathematics and Statistics, Maynooth University, Maynooth, Co. Kildare, Ireland A23 A24 13 Vol.:(0123456789) Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 Journal of Pest Science AQ2 36 37 Author Proof 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 AQ3 62 63 64 65 66 67 68 69 70 71 72 73 Introduction Strawberry is an important fruit throughout the world, and in 2016, approximately 9.2 million tons of fruits were produced worldwide, with a yield of 22.690 kg ha−1 (FAOSTAT 2018). Cultivated strawber r y, Fragaria × ananassa (Duch; Rosales: Rosacea), is attacked by a large complex of arthropod pests and plant diseases that may reduce the yield (Solomon et al. 2001). The twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is an important pest of many crops throughout the world (Greco et al. 2005), including strawberries (Raworth 1986; Easterbrook et al. 2001; Solomon et al. 2001). Tetranychus urticae feed mainly on the underside of leaves, and this feeding may lead to reduced photosynthesis and increased transpiration as well as injection of phytotoxic substances when feeding on mesophyll and parenchyma plant cells (Sances et al. 1979, 1982; Attia et al. 2013). The feeding damage therefore decreases foliar and loral development causing reductions in quality and quantity of fruits (Rhodes et al. 2006). Other important pest of strawberries worldwide includes the western lower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), which causes damage by the feeding of nymphs and adults resulting in lower abortion, fruit bronzing and malformation, and consequently yield loss (Solomon et al. 2001; Coll et al. 2007). Strawberries are also attacked by aphids of diferent species such as Chaetosiphon fragaefolli Cockerell, Aphis forbesi Weed, A. gossypii Glover and Mizus persicae Sulzer (Hemiptera: Aphididae) (Solomon et al. 2001; Bernardi et al. 2015; Dara 2016). The whitely Trialeurodes PR O O F 35 D 34 vaporariorum (Westwood) (Hemiptera: Aleyrodidae) is also a signiicant pest of strawberry crop in many regions (Solomon et al. 2001; Bernardi et al. 2015; Dara 2016). Moreover, Neopamera bilobata Say (Hemiptera: Rhyparochromidae) and the spotted wing fruit ly Drosophila suzukii Matsumura (Diptera: Drosophilidae) have recently invaded and caused economic losses in the production of many strawberry ields in Brazil (Kuhn et al. 2014; Andreazza et al. 2016). High incidence of plant pathogens, especially fungal pathogens, is another challenge faced by strawberry farmers in all producing countries and causes problems throughout the crop cycle, from the newly planted seedlings to the inal fruit-producing stage (Garrido et al. 2011). The main pest control strategy in strawberries throughout the world is the use of synthetic chemical pesticides (Solomon et al. 2001; Garrido et al. 2011). Dependency of these chemicals for pest control in strawberries is associated with undesirable efects on environment and human health (e.g., Attia et al. 2013; Barzman et al. 2015; Czaja et al. 2015). Outbreaks of T. urticae are often observed following continuous pesticide treatments (Klingen and Westrum 2007; Van Leeuwen et al. 2009, 2010) due to the emergence of pest resistance to the particular pesticides and destruction of the pests’ natural enemies (Solomon et al. 2001; Sato et al. 2005). The use of invertebrate predators, parasitoids and microbial control agents in biological control is considered a sustainable alternative to synthetic chemical pesticides for control of arthropod pests (Garcia et al. 1988; Eilenberg et al. 2001). Except the application of predatory phytoseiid mites to control T. urticae, biological control is not widely used in strawberry production, and more development of macro- and microbial control agents and application strategies is therefore necessary (Solomon et al. 2001; Attia et al. 2013). Entomopathogenic fungi within the order Hypocreales are used in microbial control, and many species are known to have a quite wide host range (Goettel et al. 1990; Rehner 2005). The species Beauveria bassiana (BalsamoCrivelli) Vuillemin (Cordycipitaceae) and several species of Metarhizium (Clavicipitaceae) have been considered promising microbial control agents in strawberries (Sabbahi et al. 2008; Castro et al. 2018) and may be implemented in programs for integrated pest management (IPM) (Hajek and Delalibera 2010). There are, however, constraints in the use of entomopathogenic fungi as microbial control agents, such as non-consistent efects against pests, short survival time of the fungal propagules in the environment, quality of commercial products, shelf life and costs (Lacey et al. 2015). These aspects are inluenced by abiotic factors such as temperature, light intensity and quality, humidity and rainfall (Meyling and Eilenberg 2007; Castro et al. 2013) and by biotic factors such as multitrophic interactions with plants, TE 33 EC 32 R 31 inoculated with entomopathogenic fungi as microbial control agents under natural ield conditions. • The irst report of reduced Tetranychus urticae numbers on strawberry plants receiving root inoculation with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana under commercial cultivation regimes. • Reduction in foliar plant pathogenic fungi and no harmful efects on naturally occurring predatory mites were also observed. • This represents a new tool and an innovative biocontrol strategy that may be implemented in IPM and organic strawberry production. R 30 • Few studies have investigated the potential of plant NC O 29 Key message U 28 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 Journal of Pest Science 135 136 137 138 Author Proof 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 PR O O F 134 Article No : 1147 Pages : 14 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 Fungal isolates 200 Based on earlier eicacy studies (F. Canassa, unpubl.), two entomopathogenic fungal isolates M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375, identiied to species level by molecular techniques according to Rezende et al. (2015) and Rehner and Buckley (2005), were selected. Isolates were kept at − 80 °C in the entomopathogen collection “Prof. Sérgio Batista Alves” in the “Laboratory of Pathology and Microbial Control of Insects” at Escola Superior de Agricultura “Luiz de Queiroz” at University of São Paulo (ESALQ/USP), Piracicaba, São Paulo, Brazil. The M. robertsii ESALQ 1622 isolate originated from soil of a corn ield in Sinop City (11°51 47 S; 55°29 01 W), Mato Grosso State, Brazil, and the B. bassiana ESALQ 3375 isolate was obtained from soil of a strawberry ield in Senador Amaral City (22°33 12 S; 46°13 41 W), Minas Gerais State, Brazil. Experimental setup The experiments were conducted in four diferent commercial strawberry ields (Fig. 1). The roots of the strawberry seedlings were immersed in one of the following treatments before planting: A) M. robertsii ESALQ 1622 in water + 0.05% Tween 80; B) B. bassiana ESALQ 3375 in water + 0.05% Tween 80; C) Water + 0.05% Tween 80 (control). A randomized block design was used in all four ield experiments. Three experiments were conducted in Atibaia City, São Paulo State, Brazil, from March to September 2018 MS Code : 1147 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 13 Journal : Large 10340 180 Materials and methods D 133 TE 132 pseudobassiana Rehner & Humber applied as granulates close to strawberry roots was conirmed in studies in Norway (Klingen et al. 2015). However, none of these studies evaluated the potential of these fungi for improving plant productivity or controlling pests aboveground in strawberry. The aim of the present study was therefore to evaluate the potential of two selected isolates of entomopathogenic fungi as root inoculants of strawberry plants for aboveground pest management under ield conditions in Brazil. The fungal species used were M. robertsii and B. bassiana, and the origin of the isolates was Brazil. They were selected based on the ability to reduce T. urticae numbers on strawberry (F. Canassa, unpubl.) and on common beans P. vulgaris (Canassa et al. 2019), in greenhouse experiments. The efects on natural predatory mite populations were also assessed to evaluate the efect of the fungal inoculation strategy on natural enemies of T. urticae in the strawberry foliage. Further, prevalence of insect pests and important strawberry foliar pathogens was also monitored. EC 131 R 130 R 129 invertebrates, other microorganisms and plant pathogens (Klingen and Haukeland 2006; Meyling and Eilenberg 2007; Meyling and Hajek 2010). In order to optimize pest control by entomopathogenic fungi, it is important to understand how these factors and their interactions afect the eicacy of the microbial control agent in question. Recent studies have reported that entomopathogenic fungi in the Hypocreales, mainly Metarhizium spp. and Beauveria spp., may also interact with plants as endophytes (Vega 2008, 2018; Vega et al. 2009; Greenield et al. 2016). Endophytic fungi are able to colonize the internal tissues of a host plant and cause no apparent negative efect on the plant (Carroll 1988; Stone et al. 2004; Vega 2008). This relationship between entomopathogenic fungi and their host plant may protect the plant against arthropod pests and plant diseases (Bing and Lewis 1991; Ownley et al. 2010; Jaber and Ownley 2018). Furthermore, endophytic fungi are protected inside the plant tissues from the efect of ambient abiotic factors (Vega 2008, 2018) and the challenge of short survival time of fungal propagule in the environment due to abiotic factors may therefore be reduced. The mechanisms responsible for any plant protection capacity of plant-associated entomopathogenic fungi against arthropod pests and plant pathogens remain uncertain (Vidal and Jaber 2015; McKinnon et al. 2017). Most of the published studies on entomopathogenic fungi as plant inoculants were carried out under controlled experimental conditions, and so far, only few studies have investigated the pest control potential of entomopathogenic fungi as inoculants of plants under ield conditions while no ield studies have evaluated efects against plant pathogens (Jaber and Ownley 2018). Field studies have been carried out with inoculation of common beans, Phaseolus vulgaris L. (Fabales: Fabaceae) with B. bassiana against Liriomyza leafminers (Diptera: Agromyzidae) (Gathage et al. 2016); of Sorghum bicolor L. (Moench) (Poales: Poaceae) with B. bassiana, Metarhizium robertsii Bisch., Rehner & Humber, and Isaria fumosorosea (Wize) Brown & Smith (Cordycipitaceae) (Mantzoukas et al. 2015); and of cotton Gossypium spp. (Malvales: Malvaceae) with B. bassiana against Aphis gossypii Glover (Homoptera: Aphididae) (Castillo-Lopez et al. 2014). These recent ield studies report signiicant efects against foliar arthropod pests under ield conditions, suggesting that implementation of entomopathogenic fungi as plant inoculants into outdoor IPM programs has a major potential (Lacey et al. 2015; Jaber and Ownley 2018). Few ield studies have been conducted on strawberry. One study was conducted on soil drench granulate or root dipping application of Met52® Metarhizium brunneum [reported as M. anisopliae (Metsch.) Sorokin] to strawberry against the soil living larvae of the black vine weevil Otiorhyncus sulcatus in a temperate region (UK), and it was suggested to be a potential strategy (Ansari and Butt 2013). Further, the persistence of locally adapted isolates of M. brunneum Petch and Beauveria NC O 128 U 127 Dispatch : 10-8-2019 217 218 219 220 221 222 223 224 225 226 Journal of Pest Science 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 EC 231 R 230 R 229 in three separate open commercial strawberry ields with black plastic mulching and drip irrigation. (Open-field locations 1, 2, 3 are shown in Fig. 1.) At all three locations, an experimental strawberry block was 60 m long (20 m for each treatment), 1.1 m wide and contained 600 plants (200 plants for each treatment). Experiments at location 1 (23°04 14.32 S; 46°40 58.2 W) and location 2 (23°04 33.5 S; 46°40 30.1 W) had 6 blocks (= strawberry beds), where the three treatments A), B) and C) were randomized inside each block, totaling 3.600 plants, while at location 3 (23°08 00.7 S; 46°37 04.5 W) there were 4 blocks (= strawberry beds), where the three treatments (A), (B) and (C) were also randomized inside each block, totaling 2.400 plants. Strawberry cultivars of locations 1, 2 and 3 were Camarosa (University of California, 1993), Camino real (University of California, 2001) and Oso grande (University of California, 1989), respectively. At these three locations, bare root strawberry plants (Fragaria × ananassa) were planted at the 4-leaf stage in three rows per bed with a distance of 0.27 cm between rows. The experiment at location 4 was conducted in Senador Amaral City (22°33 12.1 S; 46°13 41.8 W), Minas Gerais State, Brazil, from July 2017 to January 2018, in low tunnels (short hoop structures covered with white plastic), with black plastic mulching and drip irrigation (tunnel location 4 in Fig. 1). This ield experiment was established in 18 low tunnels representing four blocks, each with three strawberry NC O 228 U 227 TE D Author Proof PR O O F Fig. 1 Experimental ield setup in open-ield locations 1, 2 and 3 in Atibaia (1: 23°04 14.32 S 46°40 58.2 W, 2: 23°04 33.5 S 46°40 30.1 W, 3: 23°08 00.7 S 46°37 04.5 W) and in low tunnel location 4 in Senador Amaral (22°33 12.1 S 46°13 41.8 W). Rows and area used for recording of data are indicated as a rectangle inside each bed beds of each treatment, i.e., 12 strawberry beds per treatment. Each bed was 20 m long, 1.1 m wide and contained 250 plants, totaling 3000 plants per treatment. At location 4, bare root strawberry plants, cultivar Albion (University of California, 2006) were planted at the 4-leaf stage individually in three rows with a distance of 0.27 cm between rows. Preparation of fungal inoculum Article No : 1147 Pages : 14 The two fungal isolates (M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375) were retrieved from the − 80 °C culture collection and plated onto Petri dishes (90 × 15 mm) containing 20 ml Potato Dextrose Agar (PDA; Merck, Darmstadt, Germany). The cultures were then kept in darkness at 25 °C for 10 days until harvesting of conidia. This was done by adding 10 ml sterile 0.05% Tween 80 (Oxiteno, São Paulo, Brazil) to the culture and scraping of the conidia with a sterile spatula. Conidial concentrations were estimated using a Neubauer hemocytometer (Merck, Darmstadt, Germany) and adjusted to 1 × 108 conidia ml−1. Later, 10 ml of each suspension was inoculated with a pipette into individual polypropylene bags (35 cm length × 22 cm width) containing 300 g autoclaved (121 °C, 20 min) parboiled rice, inside an aseptic laminar low chamber. The fungus-inoculated rice kernels were mixed in the plastic bags and incubated in darkness at 25 °C for 10 days. The bags were gently shaken every 2 days to ensure evenly MS Code : 1147 255 256 257 258 259 260 13 Journal : Large 10340 254 Dispatch : 10-8-2019 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 Journal of Pest Science 287 288 Author Proof 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 Fungal inoculation of strawberry roots Rice kernels colonized with the two isolates (M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375) were added into water plus 0.05% Tween 80 as described below. For the open-ield experiments at locations 1, 2, 3, the original conidia concentration per gram of rice kernels for each isolate was estimated to 2.5 × 108 g−1 rice for M. robertsii and 1.3 × 109 g−1 rice for B. bassiana. The concentration was then adjusted to 1.5 × 1012 conidia of M. robertsii on 3.0 kg rice and B. bassiana on 0.56 kg rice. The rice was mixed with 100 l of well water plus 50 ml 0.05% Tween 80, resulting in 1.5 × 106 conidia ml−1. The control consisted of 100 l of well water plus 50 ml 0.05% Tween 80. The inal suspensions for the experiments contained 1.5 × 106 conidia ml−1. For the low tunnel experiment at location 4, the original conidia concentration per gram of rice kernels for each isolate was estimated to 1.8 × 108 g−1 rice for M. robertsii and 7.5 × 108 g−1 rice for B. bassiana. The concentration was then adjusted to 1.5 × 1012 conidia of M. robertsii on 8.3 kg rice and B. bassiana on 2.0 kg rice. The rice was mixed with 750 l well water plus 375 ml 0.05% Tween 80, resulting in 2.0 × 106 conidia ml−1. The control consisted of 750 l of well water plus 375 ml 0.05% Tween 80. Strawberry roots were inoculated by immersing the root system of each plant completely into the respective treatment suspensions for 2 min. The inoculated plants were transported to the correct position in the rows inside plastic trays to avoid dripping suspension, and then, the plants were immediately planted into the row. The suspensions were continuously mixed with a wooden stick during the strawberry root inoculation to ensure homogenized concentrations. PR O O F 286 D 285 TE 284 In the open-ield experiments at locations 1, 2 and 3, we observed 15 lealets (= one leaf from a triplet) and 15 lowers representing 15 plants in each of the central rows of the strawberry beds as indicated in Fig. 1. In the low tunnel experiment at location 4, we observed 15 lealets (= one leaf from a triplet) and 15 lowers from six plants (i.e., 2 or 3 lealets per plant) in each of the central rows per strawberry bed as indicated in Fig. 1. Each lealet was destructively sampled by hand and visually observed, and the arthropod pests were identiied to species level and counted in the ield. The predatory mites were transferred to plastic vials (500 ml, 8.5 cm high, 10 cm diameter) containing 70% ethanol and taken to the laboratory for identiication by observing each specimen under microscope. Each predatory mite was collected with a ine brush from the vial with 70% ethanol and mounted in Hoyer’s medium for identiication to species by comparing their morphology with information from original descriptions and redescriptions provided in Rowell et al. (1978), Chant and Yoshida-Shaul (1991), Moraes et al. (2004) and Tixier et al. (2008). Lealets with characteristic symptoms of the plant pathogenic fungi Mycosphaerella fragariae Tul. (Lindau), Dendrophoma obscurans (Ell & Ev.) and Pestalotia longisetula (Guba) were recorded, and the percentage of lealets with the diseases was calculated. EC 283 R 282 R 281 distributed fungal growth on rice kernels. Prior to use in the experiment, the conidial viability was checked by preparing a conidial suspension by adding 1 g of rice with sporulating fungi from the plastic bag to 10 ml sterile 0.05% Tween 80. From the third dilution, 150 µl of the conidial suspension was transferred with a pipette onto PDA. The percentage of conidia germination was then evaluated according to Oliveira et al. (2015). Suspensions were only used if germination rates were higher than 95%. NC O 280 U 279 Evaluations: arthropod pests, natural enemies and plant pathogens All four ield experiments were evaluated each 30 days for 6 months. However, the results obtained at location 4 (low tunnel experiment) are only reported up to 120 days after inoculation, because the producer applied a synthetic chemical pesticide at this time, which may have inluenced the following observations at 150 and 180 days after inoculation. Evaluation of colonization of strawberry leaves and soil Sampling of strawberry leaves and soil adjacent to plant roots was done 180 days after inoculation to evaluate the presence of entomopathogenic fungi. One strawberry leaf (= three lealets) was randomly and destructively collected from one plant per plot in the center row of each replicate plot treatment at each of the four locations. Collected leaves were placed in separate plastic bags and transferred to the laboratory for evaluation of endophytic colonization. The leaves were cut in sections of 4 cm × 1 cm, and they were then surface sterilized by following the method described by Greenield et al. (2016). Three sections of leaves were plated on one Petri dish (90 × 15 mm) with the following selective media: 20 ml of PDA, 0.5 g l−1 of cycloheximide, 0.2 g l−1 of chloramphenicol, 0.5 g l−1 of dodine (65%) and 0.01 g l−1 of crystal violet (Behie et al. 2015). The sterilization eiciency was conirmed by plating 100 μl of the last rinsing water of the sterilization onto PDA (Parsa et al. 2013). Further, imprints of sterilized leaves were used as an additional method to conirm whether the sterilization was successful. This was done by gently pressing the leaf section with the cut edge onto the PDA medium (Greenield et al. 2016) before placing sections in selective media plates. The Petri dishes were incubated at 25 °C for 15 days before visually observed for fungal outgrowth of Metarhizium or 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 Journal of Pest Science 386 387 388 389 Author Proof 390 391 392 393 394 395 396 397 398 399 400 401 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 EC 385 We itted Poisson generalized linear mixed models to the T. urticae counts obtained from locations 1, 2 and 3 (open ield), including in the linear predictor the efects of block and diferent quadratic polynomials per each treatment and location combination over time (natural log-transformed) as ixed efects, and two random efects, namely the efect of bed (since observations taken over time on the same bed are correlated) and an observation-level random efect to model overdispersion. Hence, the maximal model included 32 ixed efects and 2 variance components, totaling 34 parameters. We then performed backwards selection, using likelihood ratio (LR) tests to assess the signiicance of the ixed efects. Treatments were compared by itting nested models using grouped treatment levels and comparing them using LR tests; a signiicant test statistic means that the treatments cannot be grouped, as they are statistically diferent (see, e.g., Fatoretto et al. 2018). After model selection, the efects of proportion of occurrence of each plant pathogen species present (M. fragariae; P. longisetula; and D. obscurans), damage by Coleoptera (holes in the lealets most likely caused by Colaspis spp.) and number of thrips (F. occidentalis) were added, separately, as covariates in the model and their signiicance was assessed using LR tests. For the other variables observed in locations 1, 2 and 3 (open ield), we worked with the aggregated values across R 384 R 383 NC O 382 U 381 PR O O F Statistical analysis 380 all time points. The proportion of lealets infected by plant pathogens present (M. fragariae, P. longisetula or D. obscurans) and the proportion of lealets damaged by Coleoptera were analyzed by itting quasi-binomial models with a logit link, including the efects of block, treatment, location and the interaction between treatment and location in the linear predictor. The number of thrips was analyzed by itting quasi-Poisson models, also including the efects of block, treatment, location and the interaction between treatment and location in the linear predictor. Signiicance of efects was assessed using F tests, since the dispersion parameter was estimated (Demétrio et al. 2014). Multiple comparisons were performed by obtaining the 95% conidence intervals for the linear predictors. For location 4 (low tunnel), Poisson generalized linear mixed models were itted to the T. urticae counts, including in the linear predictor the efects of block and diferent intercepts and slopes per each treatment over time as ixed efects, and two random efects, namely the efect of bed (since observations taken over time on the same bed are correlated) and an observation-level random efect to model overdispersion. Here, the maximal model included 9 ixed efects and 2 variance components, totaling 11 parameters. As for the models itted for locations 1, 2 and 3 (open ield), we then performed backward selection, using likelihood ratio (LR) tests to assess the signiicance of the ixed efects. Treatments were compared the same way, by itting nested models using grouped treatment levels and comparing them using LR tests. Again, after model selection, the efects of the proportion of occurrence of the number of pests present and plant pathogens were added, individually, as covariates in the model and their signiicance was assessed using LR tests. For the other variables observed at location 4 (low tunnel), we worked with the aggregated values across all time points. The proportion of lealets infected by plant pathogens was analyzed by itting quasi-binomial models with a logit link, including the efects of block and treatment in the linear predictor. The number of cucurbit beetles, white lies, thrips and predatory mites was analyzed by itting quasi-Poisson models, also including the efects of block and treatment in the linear predictor. Signiicance of efects was assessed using F tests, and multiple comparisons were performed by obtaining the 95% conidence intervals for the linear predictors. All analyses were carried out in R (R Core Team 2018). Goodness of it was assessed using half-normal plots with a simulated envelope, using package hnp (Moral et al. 2017). Generalized linear mixed models were itted using package lme4 (Bates et al. 2015). All plots were generated using package ggplot2 (Wickham 2009). D 403 379 TE 402 Beauveria on each plant fragment. The frequency of occurrence was estimated as the number of plant fragments with entomopathogenic fungi present in relation to the total number of plant fragments. Soil samples adjacent to plant roots were collected with a garden spade, from the same plants where leaves were sampled, without removing the plants. Then, soil with roots was placed into individual plastic bags and brought back to the laboratory. Here, the soil was mixed, and subsequently, 1 g was sampled and added to 10 ml of sterile 0.05% Tween 80 and vigorously vortexed for 30 s and serially diluted into distilled water + 0.05% Tween 80 to obtain the following concentrations: 1 × 10, 1 × 10−1, 1 × 10−2 and 1 × 10−3. Petri dishes (90 × 15 mm) containing selective agar medium as described above were divided into four equal quarter sections by marking the bottom part of the Petri dishes with a permanent marker. Then, 100 µl from each soil dilution suspension was pipetted onto the selective media in each of the four sections. After the 100 µl was dried up inside a laminar low chamber, the Petri dishes were incubated in darkness at 25 °C for 15 days, and the presence of Metarhizium or Beauveria was detected according to fungal growth morphology in each plate. The frequency of occurrence was estimated as the number of soil samples with entomopathogenic fungi in relation to the total number of samples. 378 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 Journal of Pest Science 480 Results 481 Efects of M. robertsii and B. bassiana on T. urticae Author Proof 490 491 PR O O F 489 D 488 TE 487 EC 486 R 485 R 484 NC O 483 Root inoculation of strawberry plants with the two fungal treatments (M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375) signiicantly inluenced the number of T. urticae adults over the 6-month period (180 days) in openield locations 1, 2 and 3 (LR = 30.31, df = 2, p < 0.0001) (Fig. 2) and the low tunnel location 4 (LR = 10.39, df = 2, p = 0.0055) (Fig. 3). No diference between plants inoculated with the two entomopathogenic fungi was seen in locations 1, 2 and 3 (LR = 0.07, df = 1, p = 0.3092) nor in location 4 (LR = 0.02, df = 1, p = 0.8793). U 482 There was no signiicant three-way interaction among open-field locations (1, 2 and 3), treatment and time (LR = 4.06, df = 8, p = 0.8516), nor significant two-way interactions between open-ield locations (1, 2 and 3) and treatment (LR = 0.69, df = 4, p = 0.9524) and between treatment and time (LR = 3.00, df = 4, p = 0.5574). However, there was a signiicant interaction between location and time (LR = 49.91, df = 4, p < 0.0001), which means that the population dynamics of spider mites changed diferently between the inoculated and control plants over time at each location, with a signiicantly higher number of adults on the control plants in the three locations (LR = 30.31, df = 2, p < 0.0001) (Fig. 2). For the low tunnel location 4, there was no signiicant interaction between treatment and time (LR = 2.49, df = 2, p = 0.2879); however, there were signiicant efects of time (LR = 43.02, df = 1, p < 0.0001) and Fig. 2 Efect of inoculation of strawberry root with Beauveria bassiana (Bb) isolate ESALQ 3375 or Metarhizium robertsii (Mr) ESALQ 1622 on numbers of adult Tetranychus urticae per lealet 30, 60, 90, 120, 150 and 180 days after inoculation, at the open-ield locations 1, 2 and 3 in Atibaia, São Paulo State, Brazil (Loc 1: 23°04 14.32 S 46°40 58.2 W, Loc 2: 23°04 33.5 S 46°40 30.1 W, Loc 3: 23°08 00.7 S 46°37 04.5 W). The dots represent the observations; the solid lines are the itted curves for the mean number of T. urticae per lealet; and the gray areas represent 95% conidence intervals of the curves 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 513 514 515 516 517 518 519 520 521 522 523 PR O O F D TE 512 EC 511 Besides, in locations 1, 2, 3 there was no signiicant interaction between numbers of T. urticae and thrips in lowers (LR = 1.03, df = 1, p = 0.3092). In low tunnel location 4, there was no signiicant interaction between numbers of T. urticae and thrips in lowers (LR = 0.73, df = 1, p = 0.3929) or whitelies (LR = 3.74 df = 1, p = 0.0532). R 510 treatment (LR = 10.39, df = 2, p = 0.0055), and hence, there was a signiicantly higher number of T. urticae adults on the control plants at diferent times of evaluation, when compared to the two fungal treatments (Fig. 3). There was no signiicant efect of the proportion of leaflets infected by the plant pathogens M. fragariae (LR = 0.20, df = 1, p = 0.6569), P. longisetula (LR = 1.89, df = 1, p = 0.1693) and D. obscurans (LR = 1.90, df = 1, p = 0.1686) on the number of T. urticae in open-ield locations 1, 2 and 3. However, there was a signiicant efect of the proportion of leaves damaged by Coleoptera (holes in the lealets most likely caused by Colaspis spp.) on the number of T. urticae (LR = 5.13, df = 1, p = 0.0235), suggesting that numbers of T. urticae were lower on lealets damaged by Coleoptera (estimate of − 1.60 in the logit scale, with an associated standard error of 0.72, indicating a negative relationship). R 509 tion 4 in Senador Amaral, Minas Gerais State, Brazil (22°33 12.1 S 46°13 41.8 W). The dots are the observations; the solid lines are the itted curves for the mean number of T. urticae per lealet; and the gray areas represent 95% conidence intervals NC O 508 Fig. 3 Efect of inoculation of strawberry root with Beauveria bassiana (Bb) isolate ESALQ 3375 or Metarhizium robertsii (Mr) ESALQ 1622 on numbers of adult Tetranychus urticae per lealet from 30, 60, 90 and 120 days after inoculation at the low tunnel loca- Table 1 Means ± SE of proportion of lealets damaged by Coleoptera (%), cumulative number of thrips in lowers and proportion of lealets with symptoms of the pathogens D. obscurans, P. longisetula and M. Assessmenta Treatmentsb U Author Proof Journal of Pest Science B. bassiana M. robertsii H2O + Tween 80 Test statistic p value Efects of M. robertsii and B. bassiana on other pests and diseases Damage caused by Coleoptera (holes in the lealets) was signiicantly reduced on strawberry plants inoculated with B. bassiana ESALQ 3375 compared to control plants in open-ield locations 1, 2 and 3 (Table 1). There was no significant interaction between location and treatment (F4,34 = 1.68, p = 0.1767), but there was a signiicant efect fragariae (%) representing the diferences in the open-ield locations 1, 2 and 3, with summaries of generalized linear models Locations 1, 2, 3 Coleoptera damage No. of thrips D. obscurans P. longisetula M. fragariae 4.4 ± 0.88b 6.6 ± 1.15ab 8.7 ± 2.02a F2,38 = 4.17 p = 0.0240 24.5 ± 4.67a 21.6 ± 3.34a 30.9 ± 6.27a F2,38 = 1.97 p = 0.1549 2.7 ± 1.23a 2.5 ± 1.10a 4.5 ± 1.58a F2,38 = 1.02 p = 0.3710 1.3 ± 0.37b 1.3 ± 0.48b 3.7 ± 1.24a F2,38 = 4.92 p = 0.0158 6.1 ± 1.66b 4.6 ± 1.35b 9.8 ± 2.69a F2,38 = 5.84 p = 0.0066 Separate analyses were performed for each response variable a Data (mean ± SE) followed by diferent letters within a column are signiicantly diferent (GLM, followed by post hoc Tukey test, p < 0.05) b Treatments included root inoculations of the entomopathogenic fungal isolates Beauveria bassiana ESALQ 3375 (B. bassiana), Metarhizium robertsii ESALQ 1622 (M. robertsii) and control treatment with H2O + 0.05% Tween 80 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 524 525 526 527 528 529 530 531 532 533 534 535 536 537 Journal of Pest Science 546 547 548 549 Author Proof 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 B. bassiana M. robertsii H2O + Tween 80 Test statistic p value Whitelies No. of thrips Diseases 6.6 ± 1.70a 6.0 ± 1.54a 5.9 ± 1.38a F2,30 = 0.07 p = 0.9359 1.9 ± 5.33a 1.6 ± 3.70a 1.8 ± 2.91a F2,30 = 0.18 p = 0.8358 0.5 ± 0.31a 0.5 ± 0.31a 1.2 ± 0.42a F2;30 = 0.95 p = 0.3988 PR O O F 545 Assessmenta Summaries of separate statistical analyses for each response variable using generalized linear models are presented below a Data (mean ± SE) followed by diferent letters within a column are signiicantly diferent (GLM, followed by post hoc Tukey test, p < 0.05) b Treatments included root inoculations of the entomopathogenic fungal isolates Beauveria bassiana ESALQ 3375 (B. bassiana), Metarhizium robertsii ESALQ 1622 (M. robertsii), and control treatment with H2O + 0.05% Tween 80 D 544 Treatmentsb at location 4 was: M. robertsii = 14.3 ± 3.83; B. bassiana = 14.8 ± 3.06; and control = 13.6 ± 2.57 predatory mites per lealet accumulated for all sampling dates. TE 543 Table 2 Means ± SE of cumulative number of whitelies per lealet and thrips per lower, and the mean ± SE proportion of lealets with symptoms of foliar pathogens (combined % incidence of D. obscurans + P. longisetula + M. fragariae) in the low tunnel location 4 EC 542 R 541 R 540 of location (F2,40 = 12.61, p < 0.0001). The mean damage caused by Coleoptera (± SE%) in each location was: location 1 = 10.68 ± 1.57a; location 2 = 3.89 ± 0.84b; and location 3 = 4.54 ± 1.15b. There was no diference in the number of thrips in lowers between fungus-inoculated strawberry plants and the control plants in open-ield locations 1, 2 and 3 (Table 1). There was no signiicant interaction between location and treatment (F4,34 = 0.47, p = 0.7651), but there was a signiicant efect of location (F2,40 = 11.98, p = 0.0001). The mean ± SE (%) in each location was: location 1 = 27.59 ± 4.28b; location 2 = 14.26 ± 2.23c; and location 3 = 40.09 ± 6.78a. Although there was no diference in the proportion of lealets (n = 15 lealets per replicate) with symptoms of the plant pathogenic fungus D. obscurans in open-ield locations 1, 2 and 3 (F2,38 = 1.02, p = 0.3710), the proportion of lealets (n = 15 lealets per replicate) with symptoms of M. fragariae and P. longisetula were signiicantly smaller on plants inoculated with M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375 in all ields (Table 1). Besides, for D. obscurans, there was no signiicant interaction between location and treatment (F4,34 = 0.79, p = 0.5386) and among the three open-ield locations (F2,40 = 1.54, p = 0.2300). For P. longisetula, there was also no signiicant interaction between location and treatment (F4,34 = 0.58, p = 0.5676) and among the three open-ield locations (F2,40 = 0.04, p = 0.8433). Regarding the disease caused by M. fragariae, there was no signiicant interaction between location and treatment (F4,34 = 0.46, p = 0.7640), but there was a signiicant efect of location (F2,40 = 39.84, p < 0.0001). The mean ± SE (%) in each location was: location 1 = 3.83 ± 1.06; location 2 = 14.20 ± 1.90; and location 3 = 0.56 ± 0.29. In low tunnel location 4, in addition to T. urticae, the other major pests were whitelies and thrips in lowers, but there was no diference in the number of any of these among the three treatments (Table 2). In this location, the density of pest was always very low and very few leaves with symptoms of plant pathogens were observed. The cumulative proportion of lealets with symptoms of all the diseases (D. obscurans + P. longisetula + M. fragariae) is viewed in Table 2. NC O 539 U 538 Efects of M. robertsii and B. bassiana on predatory mites At open-ield locations 1, 2 and 3, few arthropod natural enemies were observed, but at low tunnel location 4 there were many predatory mites, mainly of the species Neoseiulus californicus McGregor (Acari: Phytoseiidae). The numbers of these predatory mites at location 4 were not signiicantly diferent on plants inoculated with M. robertsii and B. bassiana, compared to the control (F2,30 = 0.04, p = 0.9642). The mean ± SE (%) for the three treatments Colonization of M. robertsii and B. bassiana in strawberry leaves and soil Article No : 1147 Pages : 14 591 593 Low colonization levels of the plants by both Metarhizium spp. and Beauveria spp. were observed 180 days after inoculation of strawberry roots. At open-ield location 1, neither Metarhizium spp. nor Beauveria spp. were recovered on selective media from leaf samples, but Metarhizium spp. was found in all soil samples while Beauveria spp. was not recovered from soil. From samples collected at open-ield location 2, 33.3% (2 out of 6) of leaf sections and 16.7% (1 out of 6) of soil samples were found to harbor Beauveria spp., while Metarhizium spp. was recovered from 16.7% (1 out of 6) of the soil samples but not from the leaves. At open-ield location 3, Beauveria spp. was recovered from 25% (1 out of 4) of leaves and soil samples while Metarhizium spp. was found in 75% (3 out of 4) of the soil samples and not in leaves. At low tunnel location 4, Beauveria spp. was recovered from 41.7% (5 out of 12) of leaf samples and from 8.3% (1 out of 12) of soil samples. At this location, Metarhizium spp. was not recovered from the leaves, but the recovery from soil samples was 75% (9 out of 12). None of the leaves or samples from the control plots were found to contain any of the target fungi at any of the four locations. MS Code : 1147 590 592 13 Journal : Large 10340 589 Dispatch : 10-8-2019 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 Journal of Pest Science 624 625 626 Author Proof 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 PR O O F 623 D 622 colonized with B. bassiana (NATURALIS) and M. brunneum (BIPESCO5) showed signiicantly reduced incidence and severity of three Fusarium species (F. oxysporum, F. culmorum and F. moniliforme) used in planta bioassays in controlled greenhouse settings with sterile soil. So far, B. bassiana is the most studied entomopathogenic fungal species against plant pathogens and it has been reported to protect tomato and cotton seedlings against the plant pathogens Rhizoctonia solani and Pythium myriotylum (Ownley et al. 2008). Furthermore, Sasan and Bidochka (2013) reported a 59.4% inhibition of Fusarium solani f. sp. phaseoli in bean, when co-cultured in pretreated sterile potting mixture with M. robertsii. In another study, the co-inoculation of wheat seeds with Metarhizium brunneum Petch and the mycoparasitic fungus Clonostachys rosea (Link) Schroers et al. (Hypocreales: Bionectriaceae) resulted in infections by M. brunneum in root-feeding Coleopteran larvae and provided protection against the plant pathogen F. culmorum (Keyser et al. 2016), but M. brunneum did not afect the plant pathogen individually. The present strawberry ield study suggests that the tested isolates of B. bassiana and M. robertsii can provide long-term protection of strawberries against both arthropod pests and foliar pathogens using a single root application at the time of planting. Our data also suggest that natural populations of predatory mites, most of them identiied as N. californicus, remained unafected on strawberry plant inoculated with M. robertsii ESALQ 1622 or B. bassiana ESALQ 3375. The ield experiments therefore indicate a limited nontarget efect on arthropod natural enemies when the fungi are applied as root inoculants. Few studies have investigated the efects of plant-associated entomopathogenic fungi on arthropod natural enemies and mostly focus have been on efects on parasitoids (Bixby-Brosi and Potter 2012; Akutse et al. 2014; Jaber and Araj 2018). One of the few studies reporting on efects of plant–fungi interactions on predatory mites was by Schausberger et al. (2012), who showed that bean (P. vulgaris) colonized by the mycorrhizal fungus Glomus mosseae and infested with T. urticae changed the composition of herbivore-induced plant volatiles. This caused the fungus-inoculated plants to become more attractive to the predatory mites, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae), than non-mycorrhizal plants. It was suggested that the predatory mites associated the plant response with the presence of prey (Patiño-Ruiz and Schausberger 2014) and hence showed a higher oviposition rate on these plants resulting in more eicient T. urticae suppression (Hofmann et al. 2011). Canassa et al. (2019) reported in short-term leaf disk experiments that P. persimilis showed no diference in the predation rate on spider mites from inoculated plants with B. bassiana (ESALQ 3375) and M. robertsii (ESALQ 1622) compared to control plants. The use of B. bassiana (NATURALIS) and M. brunneum (BIPESCO5) as inoculants in sweet pepper combined with the aphid endoparasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae) also indicated TE 621 EC 620 R 619 R 618 Our ield experiment, replicated at four locations, shows that root inoculations of strawberry plants with M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375 resulted in lower numbers of T. urticae adults compared to non-inoculated control plants. Few studies have investigated the potential of plant inoculated with entomopathogenic fungi as microbial control agents under natural ield conditions (reviewed by Jaber and Ownley 2018; Vega 2018), and the present study is the irst report of the efect on T. urticae numbers on strawberry plants inoculated with M. robertsii and B. bassiana evaluated in the ield under commercial cultivation regimes. The two fungal isolates were previously found to reduce T. urticae populations on bean P. vulgaris (Canassa et al. 2019), and since our strawberry ield study shows a similar efect, this may suggest that these isolates may be used as root inoculants of other crops to control T. urticae. Further, predatory mite populations were not negatively afected by strawberry plants inoculated with M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375, indicating that adverse nontarget efects on arthropod natural enemies may be limited or non-existing. The potential of B. bassiana as an endophyte for pest management has been reported in ield studies with other crops. For example, Gathage et al. (2016) reported lower infestation levels of Liriomyza leafminers in bean leaves (P. vulgaris) in a bean ield experiment in Kenya where bean seeds had been inoculated with B. bassiana G1LU3 and Hypocrea lixii Patouillard (syn. Trichoderma lixii) F3ST1. Further, Castillo-Lopez et al. (2014) reported lower numbers of A. gossypii on cotton plants grown in the ield in Texas, USA, from seeds inoculated with the commercial product Botanigard® (BioWorks Inc, Victor, NY) based on the GHA strain of B. bassiana. Our ield experiments also suggest that strawberry plants inoculated with M. robertsii ESALQ 1622 and B. bassiana ESALQ 3375 reduced the proportion of leaf damage caused by Coleopteran pests, while no efects on other pest damage, such as whitelies or thrips in lowers, were observed. Mantzoukas et al. (2015) reported from ield studies of Sorghum bicolor that B. bassiana and M. robertsii suppressed tunneling Sesamia nonagrioides Lefébvre (Lepidoptera: Noctuidae) larvae by 60% and 87% and increased larval mortality by 80% and 100%, respectively, compared to control plants after spray inoculations of plants. We also recorded a reduction in the prevalence of the foliar plant pathogenic fungi M. fragariae and P. longisetula in strawberry plants inoculated with B. bassiana ESALQ 3375 or M. robertsii ESALQ 1622. According to Jaber and Alananbeh (2018), only few studies have been conducted on the efects of plant inoculated with entomopathogenic fungi afecting plant pathogens, and so far, no ield studies have been carried out. Jaber and Alananbeh (2018) reported, however, that sweet pepper Capsicum annum L. (Solanaceae) endophytically NC O 617 Discussion U 616 13 Journal : Large 10340 Article No : 1147 Pages : 14 MS Code : 1147 Dispatch : 10-8-2019 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 Journal of Pest Science 728 729 730 731 Author Proof 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 PR O O F 727 D 726 performed in natural soils (Parsa et al. 2018), as was the case in the present study. It should therefore be expected that end-point measurements of endophytic colonization will be limited in ield studies, particularly over the 6-month time period. Given that negative efects were broadly observed against both T. urticae and selected plant pathogens in the foliage after the single inoculation events of strawberry roots with isolates of either B. bassiana or M. robertsii, and considering the inconsistent re-isolation of fungi from leaf samples, it seems most likely that plant-induced defenses were responsible for the reductions, but this will require further studies to elucidate and conclude. It has been widely suggested that the mechanisms used by entomopathogenic fungi as plant associates and endophytes to antagonize plant pests or pathogens may result through the production of secondary metabolites by the associated fungus (Vidal and Jaber 2015; Yan et al. 2015; McKinnon et al. 2017; Jaber and Alananbeh 2018). Alternatively, another mechanism could be through induced systemic defense mechanisms of the inoculated plants, because the endophyte can be irst recognized as a potential invader, which leads the plants to trigger its immune responses and consequently synthesize speciic regulatory elements that may afect the arthropod pests and plant pathogen (Brotman et al. 2013; McKinnon et al. 2017). In conclusion, the present study demonstrates that entomopathogenic fungi can be applied as root inoculants in commercial strawberry ields to simultaneously control important arthropod pests, particularly T. urticae, and plant pathogenic fungi. There were no indications that the inoculations of strawberry plant with the entomopathogenic fungal isolates tested had negative nontarget efects on naturally occurring predatory mites, particularly N. californicus. Hence, inoculation of strawberry plants with entomopathogenic fungi through root dipping may be used in combination with predatory mites for the control of T. urticae. This may represent a new tool and an innovative biological control strategy that could be implemented in IPM and organic strawberry production. TE 725 EC 724 R 723 R 722 compatibility in control of Myzus persicae Sulzer (Homoptera: Aphididae) (in a greenhouse study (Jaber and Araj 2018). In another recent study, González-Mas et al. (2019) reported that the numbers of A. gossypii parasitized by A. colemani were not inluenced by whether the aphids had been feeding on seed-inoculated melon plants with B. bassiana (isolate EABb 01/33-Su) or not. Further, application of B. bassiana on melon leaves did not inluence the number of aphids consumed by larvae of the lacewing, Chrysoperla carnea Stephens (Neuroptera: Chrysopidae), and C. carnea showed preference to feed on aphids reared on inoculated rather than control plants in a choice bioassay (González-Mas et al. 2019). All these indings indicate that plant inoculated with entomopathogenic fungi may be used in combination with parasitoids and predators to enhance the biocontrol eicacy of several plant pests in diferent crops. In our study, we were able to recover Metarhizium and Beauveria from strawberry leaves and soil adjacent to the roots at the end of the experiment and cropping cycle, meaning 180 days (for locations 1, 2, 3) and 120 days (for location 4). The main aim of the present study was not to evaluate in depth the dynamics of endophytism of the inoculated fungal isolates using a close-to-practice inoculation method in strawberry production systems, and the use of commercial farm settings did not allow for repeated and complete destructive sampling of plant material. However, Castro et al. (2016) have previously reported the persistence in strawberry soil and rhizospheres in Brazil of the isolates M. anisopliae (ESALQ1037) and M. robertsii (ESALQ1426) for up to 12 months after soil drench application. Further, Klingen et al. (2015) report that two Norwegian isolates, one B. pseudobassiana and one M. brunneum, and an Austrian isolate of M. brunneum had long-term persistence (> 1 year) in bulk soil and rhizosphere soil of strawberries in a semiield experiment in Norway. It has previously been reported that B. bassiana is a more extensive colonizer of foliar tissues than Metarhizium spp., when seed inoculations were used, while Metarhizium spp. have been reported as almost exclusively colonizing the rhizosphere of various plant species (Ownley et al. 2008; Quesada-Moraga et al. 2009; Akello and Sikora 2012; Akutse et al. 2013; Behie et al. 2015), and similar results have been observed in our study. Although the observed efects of the inoculation on herbivore densities were consistent, endophytic colonization was not consistently detected in strawberry plants in our study. It has been previously reported that endophytic establishment may be inluenced by several variables, such as host plant, fungal strain, environmental conditions, substrate and soil (Sánchez-Rodríguez et al. 2018). Moreover, previous research has showed that the establishment of entomopathogenic fungi within plant tissues may be transient (GarridoJurado et al. 2017) and the establishment success of fungal isolates is signiicantly reduced when inoculations are NC O 721 U 720 Author contributions Article No : 1147 Pages : 14 FC, IDJ, IK and NVM conceived and designed research. FC and FCNE conducted experiments. RAM analyzed data, prepared igures and wrote the statistical analysis section. FC, IK, IDJ and NV wrote the manuscript. All authors reviewed and approved the manuscript. Acknowledgements Daniela Milanez Silva and Vitor Isaias are thanked for technical assistance. We thank the strawberry producers Claudio Donizete dos Santos, Rafael Maziero, Mario Inui and Maurício dos Santos for letting us perform the experiments in their ields. We also thank Dr. Fagoni Fayer Calegario for helping to ind the farmers MS Code : 1147 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 13 Journal : Large 10340 773 Dispatch : 10-8-2019 813 814 815 816 817 818 819 820 821 822 Funding This work was supported by the National Council for Scientific and Technological Development (CNPq) [Process No. 141373/2015-6] and by The Research Council of Norway through the SMARTCROP Project [Project Number 244526]. 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Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the field

Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the field

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