Microbial control of cotton pests. Part I: Use of the

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Microbial control of cotton pests. Part I: Use of the naturally occurring entomopathogenic fungus Aspergillus sp. (BC 639) in the management of Creontiades dilutus (Stal) (Hemiptera: Miridae) and beneficial insects on transgenic cotton crops a

Robert K. Mensah & Leah Austin

a

a

NSW Department Primary Industries and Australian Cotton CRC, Australian Cotton Research Institute, Narrabri, NSW, Australia Available online: 05 Mar 2012

To cite this article: Robert K. Mensah & Leah Austin (2012): Microbial control of cotton pests. Part I: Use of the naturally occurring entomopathogenic fungus Aspergillus sp. (BC 639) in the management of Creontiades dilutus (Stal) (Hemiptera: Miridae) and beneficial insects on transgenic cotton crops, Biocontrol Science and Technology, 22:5, 567-582 To link to this article: http://dx.doi.org/10.1080/09583157.2012.670199

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Biocontrol Science and Technology, Vol. 22, No. 5, May 2012, 567582


RESEARCH ARTICLE Microbial control of cotton pests. Part I: Use of the naturally occurring entomopathogenic fungus Aspergillus sp. (BC 639) in the management of Creontiades dilutus (Stal) (Hemiptera: Miridae) and beneficial insects on transgenic cotton crops Robert K. Mensah* and Leah Austin NSW Department Primary Industries and Australian Cotton CRC, Australian Cotton Research Institute, Narrabri, NSW, Australia (Received 6 November 2011; final version received 23 February 2012) The development and adoption of transgenic (Bt) crops that express the Bacillus thuringiensis (Bt) toxin has reduced the use of synthetic insecticide on transgenic crops to target Helicoverpa spp., the major insect pest of cotton in Australia. However, it has also increased the threat posed by sucking pests, particularly Creontiades dilutus (green mirid), which are unaffected by the Bt toxins in transgenic cotton crops. Here we report the efficacy of the entomopathogenic fungus Aspergillus sp. (BC 639) in controlling the infestation of transgenic cotton crops by C. dilutus and promoting interactions of transgenic cotton with beneficial insects. The results showed that the number of C. dilutus adults and nymphs recorded on plots treated with 1000, 750, 500, 250 ml/ha BC 639 fungus formulation were the same as on plots treated with the recommended concentration of the commercial insecticide Fipronil. The fungus was found to have minimal effect on predatory insects compared with Fipronil and was most effective against C. dilutus when applied at the rate of 500 ml/ha (equivalent to 50 g spores/ha). At this rate, the fungus was as effective as Fipronil for controlling C. dilutus populations and ensured the survival of predatory beetles, lacewings and spiders compared with Fipronil treatment. The yield from fungus-treated plots was 5.24 bales per acre compared with 5.40 and 3.88 bales per acre for Fiproniltreated and unsprayed plots, respectively. The ability of the BC 639 strain to control C. dilutus infestations of transgenic cotton crops while conserving beneficial insect populations suggests its potential for supplementing integrated pest management programs to reduce the use of synthetic insecticides for transgenic cropping systems. Keywords: transgenic cotton; entomopathogenic fungus; integrated pest management; Creontiades dilutus; Helicoverpa spp; Bacillus thuringiensis (Bt)

Introduction The introduction and adoption of transgenic (Bollgard II† ) cotton crops has reduced the importance of Helicoverpa spp. as major cotton pests in Australia (Wilson, Mensah, and Fitt 2004) but has increased the threat posed by sucking pests such as green mirids (Creontiades dilutus) to cotton crops. The first commercial transgenic cotton crops, which express a Cry1Ac toxin from Bacillus thuringiensis *Corresponding author. Email: [email protected] ISSN 0958-3157 print/ISSN 1360-0478 online # 2012 Taylor & Francis http://dx.doi.org/10.1080/09583157.2012.670199 http://www.tandfonline.com


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(Bt) and are marketed as Ingard† , were introduced in Australia in 1996 for the control of Helicoverpa spp. (Davidson 2003; Greenplate, Mullins, Penn, Dahm, and Reich 2003; Olsen, Daly, Holt, and Finnegan 2005). The level of toxin in Ingard† cotton plants declines as the plants age (Greenplate et al. 2003; Olsen et al. 2005; Downes, Parker, and Mahon 2010). Therefore, in 20042005, the Ingard† cotton was replaced with Bollgard II† cotton, which expresses Cry1Ac and Cry 2Ab toxins for season-long control of Helicoverpa spp. (Fitt, Daly, Mares, and Olsen 1998; Greenplate et al. 2003; Jackson, Bradley, Van Duyn, and Gould 2004; Mahon, Olsen, Garsia, and Young 2007). The sustained control of Helicoverpa spp. by Bt cotton crops has reduced synthetic insecticide use against these pests by approximately 90% (Wilson et al. 2004; Pyke 2007). The synthetic insecticides used in controlling Helicoverpa spp. prior to the introduction of transgenic cotton crops inadvertently suppressed green mirid populations in conventional (non-transgenic) cotton crops, causing C. dilutus to be dismissed as a secondary pest in cotton crops. Nonetheless, given that C. dilutus is unaffected by Bt toxins, the introduction and adoption of transgenic cotton, has increased the sizes of populations of green mirids and other sucking pests (Wilson et al. 2004). Creontiades dilutus can cause severe damage to cotton plants from seedling emergence to late boll formation (Adams and Pyke 1982; Khan and Bauer 2001; Khan et al. 2004). The damage to cotton terminal shoots, branch primordia and young leaves can increase branching or delay the growth of cotton seedlings (Khan et al. 2004). From the squaring and early boll setting stages of the cotton plant, feeding by C. dilutus usually causes abortion of the square and the shedding of young bolls, resulting in delayed maturation and yield loss (Mensah and Khan 1997; Khan et al. 2004). The extensive damage incurred by C. dilutus on Bt cotton crops has resulted in increased use of synthetic insecticides (usually 45 sprays per season) to manage this major pest (Fitt and Wilson 2000). Over-use of synthetic insecticides against C. dilutus could result in pest resistance, disruption of the activities of natural enemies of the pest and human health problems. Additionally, the increased use of synthetic insecticides and associated disruption of beneficial insect activities is counter-productive to integrated pest management (IPM) efforts (Mensah 2002; Wilson et al. 2004) made possible by the adoption of transgenic cotton technology. Thus, the continued use of synthetic insecticides may offset the benefits of IPM enabled by transgenic cotton lines. Entomopathogenic fungi are among the natural enemies of a wide range of insect pests that are potential candidates for microbial control in many cropping systems (Butt, Jackson, and Magan 2001; Meyling and Eilenberg 2007; Eken and Hayat 2009; Hajek and Delalibera 2010). All stages of insect development, from egg to adult are susceptible to fungal infection (Ferron 1981). Several crop pests succumb to attack by entomopathogenic fungi (Gillespie 1986), and unlike bacteria and viruses, fungal conidia can infect insects without the need for ingestion (Hajek and St. Leger 1994). The fungal conidia can infect insects not only through the gut, but also through the spiracles and the surface of the insect’s integument. As a result, both chewing and sucking insect pests can be targeted either through direct contact with the fungus or by secondary infection from infected insects or sprayed vegetation (Moore and Prior 1993). Therefore, to sustain future cotton production in Australia, it is crucial that the industry have access to microbial control agents, such as entomopathogenic fungi, for conservation biological control of cotton pests (Lacey,


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Frutos, Kaya, and Vail 2001; Meyling and Eilenberg 2007) to support IPM of transgenic cotton crops. This paper reports on a series of studies aimed at determining the efficacy of an entomopathogenic fungus Aspergillus sp. (BC 639) oil-based product, in controlling populations of both C. dilutus and beneficial insects in a cotton cropping system. Specifically, the objectives of the study were to (1) assess the efficacy of different rates of application of the BC 639 product, (2) identify the optimum rate of the BC 639 formulated product needed to effectively control C. dilutus populations, and (3) assess the impact of BC 639 product on beneficial insects and cotton yields.

Materials and methods Isolation and formulation of the entomopathogenic fungus Aspergillus sp. (BC639) We isolated Aspergillus sp. (BC 639) fungus from infected C. dilutus adult in a commercial cotton farm at Yarral in Narrabri, New South Wales. The BC 639 spores were cultured on potato dextrose agar (PDA) medium. Spores were scraped from the PDA medium, dried and formulated into a product (Becker Underwood, Australia). The spore concentration was estimated using a haemocytometer to be 1.0 107 spores/ml. This spore concentration was used throughout the studies, unless stated otherwise. Conidial viability was determined by optical microscope 24 hours after placing in potato dextrose broth culture. The per cent germination was determined from 100 spore counts, and 100% germination was observed. The concentration of the Fipronil insecticide (Nufarm Pty Ltd) in the stock solution used to prepare the solution employed in the field studies was 200 g/L SC.

Experiment 1: Efficacies of different rates of BC639 on the abundances of C. dilutus and predatory insects on commercial transgenic (Bollgard II† ) cotton crops The trial was conducted on commercial Bollgard II† cotton on a farm at Norwood (29828?S, 149850?E) near Moree, New South Wales, Australia, from 5 December 2006 to 19 February 2007. The cotton used for the trial was at the early squaring stage, when it is attractive to green mirids (Khan et al. 2004). We evaluated the efficacy of the following treatments against green mirids and predatory insects: (1) 1000 ml/ha of BC 639 (Aspergillus sp.), (2) 750 ml/ha of BC 639 (Aspergillus spp.), (3) 500 ml/ha of BC 639, (4) 62.5 ml/ha of Fipronil and (5) unsprayed (negative) control. The treatment plots were arranged in a randomized complete block design with six replicates per treatment. Each replicated plot measured 40 m wide (i.e., 40 rows) and 90 m long. Foliar application of each treatment was made on 18 January 2007 using a ground rig spray equipment fitted with flat fan nozzles to achieve a droplet size of 200 mm. The treatment was applied in the mornings when temperature was between 208C and 288C. The timing of treatment was based on the IPM Guidelines and recommendations by CottonLogic to use an economic threshold of 0.5 green mirids per metre (Deutscher and Wilson 1999; Khan et al. 2004). Pre-treatment counts of green mirid adults and nymphs, predatory beetles, predatory bugs, predatory lacewings and spiders were made by visual inspection of whole cotton plants. Both adults and nymphs of beneficial insects were

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counted. Plants were assessed in randomly selected 1-m lengths of row of each treatment replicate (i.e., 6 m per treatment). Post-treatment counts were made weekly from 5 December 2006 until 19 February 2007. Data are expressed as numbers of individuals of that species counted per metre for each treatment. Cotton in each treated plot was harvested separately using a four-row picker (John Deere, Model 9965, USA) at the end of the season and the average lint yields (bales/acre) were compared between treatments. Experiment 2: Efficacy of lower rates of BC 639 on the abundance of C. dilutus (green mirids) and beneficial insects on commercial cotton crops Following the results of experiment 1, the efficacy of a lower (250 ml/ha) application rate of BC639 was compared with (1) 750 ml/ha of BC 639 and (2) 500 ml/ha of BC639 to assess differences in the efficacy of control of C. dilutus, predatory beetles, predatory bugs, predatory lacewings and spiders. This was compared with data from unsprayed transgenic cotton crops (negative control) and transgenic cotton crops treated with 62.5 ml/ha of Fipronil. The trial was conducted in an irrigated field of commercial Bollgard II† cotton crops at the Australian Cotton Research Institute farm in Narrabri during the 20082009 season. Trials were conducted from 8 January to 12 February 2009. The treatment plots were arranged in a randomized complete block design with eight replicates per treatment. Each replicated plot measured 16 m (i.e., 16 rows) wide and 250 m long. Foliar applications of each treatment were made on 8 and 22 January 2009. The decision to apply the treatment was based on the IPM Guidelines and the CottonLogic recommended economic threshold of 0.5 green mirids per metre. Pre-treatment counts were made by visual counting of the numbers of green mirid adults and nymphs, as well as beneficial insects such as predatory beetles, predatory bugs, predatory lacewings and spiders on cotton plants in each treatment. Posttreatment counts were made on 3, 7 and 14 days after the first spray application, and 3, 7, 14 and 21 days after the second spray application. For all counts, we assessed a randomly selected 1-m length of row of each treatment replicate (i.e., a total of 8 m). Data are expressed as numbers of individuals per metre for each treatment. Experiment 3: Comparisons of the efficacies of BC639 and commercial insecticides in managing the abundances of C. dilutus on commercial transgenic (Bollgard II† ) cotton crops The experiment was conducted using dryland transgenic (Bollgard II† ) cotton crops at Getta Getta near Goondiwindi (28855?S, 150831?E) from 19 January to 2 March 2010. The efficacy of the following treatments against C. dilutus and predatory insects was assessed: (1) 500 ml/ha (2) 62.5 ml/ha Fipronil and (3) unsprayed (negative control). The plots were arranged in a randomized complete block design with three replicates per treatment. Each replicated plot measured 8 m (i.e., 8 rows) wide and 250 m long. Foliar applications of each treatment were made on 19 January and 9 February 2010 using ground rig spray equipment fitted with flat fan nozzles to achieve a droplet size of 200 mm. All insecticide formulations were water miscible. All treatments were applied using a spray volume of 100 L/ha. The untreated (negative control) plot was left unsprayed and the plot treated with synthetic insecticide



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received two applications similar to and at the same date as the plots treated with the fungus. For each treatment replicate, visual counts of C. dilutus (adults and nymphs) and predatory insects on whole cotton plants were made in a randomly selected 1 m length of row of cotton plants (i.e., 6 m per treatment). Pre-treatment counts were made 24 hours before treatment, and post-treatment counts made 4, 7, 14 and 21 days after treatment (DAT). The counts at 21 DAT were used as pre-treatment counts for the second set of treatments. One-metre lengths of row were randomly selected in each treatment replicate, (i.e., a total of 6 m was examined per treatment). The predatory insects were classified as being either predatory beetles, bugs, lacewings or spiders. Data were expressed separately for C. dilutus adults and nymphs, predatory beetles, predatory bugs, predatory lacewings and spiders. Each insect group was expressed both as total numbers of individuals per metre and numbers of individuals per metre per sampling date. Cotton in each treated plot was harvested separately using a four-row picker (John Deere, Model 9965, USA) at the end of the season and the average lint yields (bales/acre) were compared between treatments.

Data analysis All experimental data were analyzed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, CA, USA). Treatment and sample dates were the independent variables. TukeyKramer multiple comparisons tests were used to separate means.

Results Experiment 1: Efficacy of different rates of application of BC639 on green mirids and beneficial insects on commercial Bollgard II† cotton crops The number of green mirid adults and nymphs recorded in plots treated with different rates of BC 639 were not significantly different (P 0.05) from plots treated with Fipronil (Figure 1). The number of green mirid adults and nymphs per metre recorded in the unsprayed plot was significantly higher (P B0.008) than for the plots treated with BC639 fungus and Fipronil insecticide (Figure 1). In contrast, the number of green mirids per metre in plots treated with Fipronil insecticide was not significantly different (P 0.05) from plots treated with the different rates of BC639 fungus (Figure 1). Overall, approximately 2.5 times as many green mirid adults and 2.2 times as many green mirid nymphs were recorded in the untreated plots than in plots treated with fungal insecticides (Figure 1). Predatory insects identified from the study plots are listed in Table 1. No significant difference (P 0.05) in the number of predatory beetles, bugs, lacewings and spiders per metre per sample date was found between BC 639-treated plots and the unsprayed (control) plots (Table 2). In contrast, the number of predatory beetles, lacewings and spiders per metre recorded on the Fipronil-treated plots was significantly lower (P B0.001, P B 0.01 and P B0.01, respectively) than on the unsprayed and BC 639-treated plots, except for the numbers of predatory bugs per metre per sample date, which were identical (P 0.05) on the Fipronil and the

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Figure 1. The efficacies of different rates of application of BC 639 in reducing numbers of Creontiades dilutus (green mirid adults and nymphs) on Bollgard II† cotton crops at Norwood near Moree, 20062007. The arrow indicates the date of treatment with BC 639 and error bars indicate standard error of the mean.

BC639-treated plots (Table 2). Significantly higher (P B0.09) yields were harvested from the synthetic (Fipronil) and biological (BC 639) insecticide-treated plots than the unsprayed plots (Table 3).

Experiment 2: Efficacy of lower rates of BC 639 on the abundance of C. dilutus (green mirids) and beneficial insects on commercial cotton crops Significantly fewer (P B0.001) green mirids per metre were found on plots treated with different rates of the BC 639 and Fipronil insecticide than on unsprayed (control) plots (Figure 2). At the first spray application, the number of green mirids per metre recorded on plots treated with BC 639 fungus and Fipronil insecticide was


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Table 1. Predators of cotton pests sampled and identified from study sites from 20042010. Order


Coleoptera

Family

Species

Coccinellidae

Group

Coccinella transversalis (Fabricius) Diomus notescens (Blackburn) Melyridae Dicranolauis bellulus (Guerin-Meneville) Hemiptera Nabidae Nabis capsiformis (Germar) Lygaeidae Geocoris lubra (Kirkaldy) Pentatomidae Cermatulus nasalis (Westwood) Ochelia schellenbergii (Guerin-Meneville) Coranus triabeatus (Horvath) Neuroptera Chrysopidae Chrysopa spp. Hemerobiidae Micromus tasmaniae (Walker) Araneida Lycosidae Lycosa spp. Oxyopidae Oxyopes spp. Salticidae Salticidae spp. Araneidae Araneus spp.

Predatory beetles

Predatory bugs

Predatory lacewings Spiders

not significantly different (P 0.05) between 8 and 22 January 2009 (i.e., 3, 7 and 14 DAT) (Figure 2). For the second spray application (22 January 2009), plots treated with BC 639 and Fipronil using different rates of application had significantly fewer (P B 0.0001) green mirids per metre than the unsprayed plot when counts were taken at 3, 7, 14 and 21 DAT (25 January to 12 February 2009) (Figure 2). Predatory insects identified from the study plots are given in Table 1. The results showed that application of 750, 500 and 250 ml/ha of BC 639 to cotton plants did not affect the numbers of predatory beetles (Figure 3). No significant difference (P 0.05) in the numbers of predatory beetles per metre was found among BC 639-treated and unsprayed (control) plots after the first and second spray Table 2. Numbers of predators per metre per sample date for untreated commercial cotton crops and comparable crops either treated with a synthetic chemical insecticide or different levels of a fungal insecticide at the Australian Cotton Research Institute in Narrabri, 20062007. Treatment 1000 ml/ha BC639 750 ml/ha BC639 500 ml/ha BC639 62.5 ml/ha Fipronil Control (unsprayed)

Predatory beetles/ Predatory bugs/m/ Predatory lacewings/ m/sample date sample date m/sample date

Spiders/m/ sample date

0.3990.06 a

0.1790.03 ab

0.02590.006 a

1.2690.08 a

0.4590.07 a

0.1990.04 ab

0.01590.005 a

1.2890.08 a

0.4190.02 a

0.1890.03 ab

0.02590.005 a

1.2090.08 a

0.2490.02 b

0.1590.02 b

0.01090.005 b

1.0090.08 b

0.4290.02 a

0.2490.04 a

0.02290.005 a

1.1990.09 a

Note: Means within columns followed by the same letters are not significantly different (P0.05; TukeyKramer multiple comparison test).

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Table 3. Cotton yield (bales/acre) harvested from commercial cotton crops managed without any treatment, using a commercial synthetic insecticide, or three levels of application of a fungal insecticide at Norwood near Moree, 2007. Yield (bales/acre)

1000 ml/ha BC 639 750 ml/ha BC 639 500 ml/ha BC 639 62.5 ml/ha Fipronil Unsprayed (Control)

3.2890.11 3.3590.09 3.3790.19 3.4290.13 2.8490.05

a a a a b

Note: Means within columns followed by the same letter are not significantly different (P 0.05; TukeyKrammer multiple comparison test).

applications (Figure 3). Although after the first spray (8 January 2009), plots treated with Fipronil insecticide had fewer predatory beetles per metre (from 11 to 22 January 2009) than either plots treated with BC639 or unsprayed plots, the differences were not significant (P0.05) (Figure 3). However, after the second spray application (22 January 2009), the number of predatory beetles per metre recorded on the Fipronil-treated plots was significantly lower (P B0.001) at 3, 7 and 14 DAT than for either the BC 639-treated plots or the unsprayed plots (Figure 3). The numbers of predatory bugs and spiders were not significantly different (P0.05) on BC639-treated, Fipronil-treated and control plots after two treatment applications (Figure 3). Similarly, the number of predatory lacewings recorded on plots treated with BC639 was not significantly different (P0.05) from the unsprayed plots (Figure 3). In contrast, the number of predatory lacewings recorded on plots treated with Fipronil was not significantly different (P0.05) 750 ml/ha BC639 500 ml/ha BC639

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Figure 2. Efficacies of different rates of application of BC 639 in reducing the numbers of Creontiades dilutus (green mirids) per metre on cotton crops at ACRI in Narrabri, 20082009. Arrows indicate dates of treatment application with BC 639 and error bars indicate standard error of the mean.

Biocontrol Science and Technology 22-1-09

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Figure 3. Numbers of predatory beetles, bugs, lacewings and spiders per metre on transgenic (Bollgard II† ) cotton treated with BC 639 and synthetic insecticides at ACRI in Narrabri in NSW, 20082009. Arrows indicate dates of treatment application with BC 639 and error bars indicate standard error of the mean.

from BC639-treated and unsprayed plots on 15 and 22 January 2009 only (Figure 3). Thereafter, plots treated with Fipronil insecticide had fewer lacewings per metre than both BC639-treated and unsprayed plots. This indicates that BC639 was more selective against predatory lacewings than was Fipronil (Figure 3).

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Significantly fewer (P B0.0001) green mirid adults and nymphs per metre were found on plots treated with BC639 and Fipronil than on unsprayed plots (Figure 4A and 4B). No significant differences (P 0.05) were found between BC639- and Fiproniltreated plots, indicating that the ability of BC639 to control green mirid populations was similar to that of Fipronil, which is the most common commercial insecticide used in the cotton industry to manage green mirids. Application of 500 ml/ha of BC639 fungus to cotton plants did not have a significant negative effect against predatory beetles (Figure 5). No significant difference (P 0.05) was detected between the number of predatory beetles per metre in plots treated with BC639 and on unsprayed (control) plots (Figure 5). In contrast, plots treated with Fipronil had the lowest number of predatory beetles per metre (Figure 5). The number of predatory bugs per metre was recorded in plots 500 ml/ha BC 639 62.5 ml/ha Fipronil

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Experiment 3: Comparison of the efficacy of BC 639 and commercial insecticides on the abundance of green mirids on commercial cotton crops

Date of assessments Figure 4. Comparisons of efficacies of BC 639 and conventional synthetic insecticides in reducing the number of Creontiades dilutus (green mirid) adults (A) and nymphs (B) on commercial transgenic (Bollgard II† ) cotton crops at Getta Getta near Goondiwindi, 2009 2010. Arrows indicate dates of treatment and error bars indicate standard error of the mean.

Biocontrol Science and Technology 500 ml/ha BC 639

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Figure 5. Comparison of numbers of predatory beetles, bugs, lacewings and spiders per metre on commercial transgenic (Bollgard II† ) cotton crops treated with BC 639 fungus and conventional insecticides at Getta Getta near Goondiwindi, 20092010. Arrows indicate dates of treatment and error bars indicate standard error of the mean.

treated with BC639 fungus was not significantly different (P 0.05) from the unsprayed plots (Figure 5). In contrast, the Fipronil-treated plots had the lowest number of predatory bugs from 23 January to 2 February, after the first spray application. Thereafter, no significant difference (P0.05) was detected among treatments (Figure 5). The number of spiders per metre recorded in the fungustreated and control (unsprayed) plots was not significantly different (P 0.05)


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throughout the study (Figure 5). However, spider numbers recorded in the Fiproniltreated plots were significantly lower on 26 January (P B0.0001) and 16 February (PB0.001) (7 days after the first and second spray applications, respectively) than the BC 639 treated and unsprayed plots (Figure 5). However, the number of spiders per metre on the Fipronil-treated plots recovered within 14 days after application of the synthetic insecticide (Figure 5). Cotton yields harvested from the fungus- and insecticide-treated plots were not significantly different (P 0.05), but were significantly different (P B0.01) from the unsprayed plots. The fungus-treated plots yielded 5.2490.01 bales per acre compared with 5.4090.08 bales and 3.8890.12 bales per acre in the Fiproniltreated and unsprayed plots, respectively. Discussion Application of an oil-based formulated product of the entomopathogenic fungus BC 639 (Aspergillus sp.) effectively controlled populations of C. dilutus adults and nymphs on commercial transgenic cotton crops. The fungus killed green mirids within 37 days after application although infected green mirids were found in the treated plots 2128 days after treatment. This indicates that the BC639 fungus can persist and cause secondary infection of green mirids. The death of secondarily infected insects produces more spores able to infect and kill more insects. The effect of the BC 639 fungus on predatory insects was not significantly different (P 0.05) from the unsprayed (control) treatment. In contrast, the conventional synthetic insecticide Fipronil had a significant negative effect (PB0.01) on most predatory insects, except predatory bugs where no significant difference (P0.05) was observed when plots treated with the BC 639 fungus were compared with those treated with the conventional synthetic insecticide Fipronil. There was no difference among treatments in the impact of Fipronil on predatory bugs because these predators, particularly Nabis kingbergii, are usually present on cotton crops that are infested with Helicoverpa spp. larvae (Deutscher, Vogel and Wilson 2011). Soft-bodied insects, particularly caterpillars, are major prey, and on transgenic cotton crops, Helicoverpa spp. larvae (prey availability) is very low due to increased mortality of these stages by the Bt toxin. In this study, most of the predatory bugs found on the cotton crops were N. kingbergii adults, which are extremely mobile and move in and out of the treated cotton crops, making it difficult to determine mortality caused by the individual treatments. In addition, N. kingbergii adults tend to hide in the lower canopies of cotton plants, searching for prey, and such a behaviour might have helped these predators to avoid pesticide sprays. When BC639 was applied at rates of 250 and 500 ml/ha (equivalent to 25 and 50 gram (g) spores/ha, respectively; Gary Bullard, BioCare Pty Ltd (now known as Becker Underwood Pty Ltd), the treatment involving the 500 ml/ha rate decreased the number of green mirids compared with the 250 ml/ha treatment (not significant; P 0.05). However, the 500 ml/ha rather than the 250 ml/ha application rate had similar efficacy against green mirids as the 1000 ml/ha fungal treatment and treatment with 62.5 ml/ha of the commercial conventional synthetic insecticide Fipronil. This result suggests that the optimum effective rate for the application of BC639 to control infestations of green mirids on cotton crops was 500 ml/ha. We found no negative effect of the BC639 fungus against predatory beetles, bugs,



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lacewings and spiders, in comparison with the unsprayed (control) plots. In contrast, the synthetic insecticide killed the beneficial insects. Butt and Goettel (2000) reported that entomopathogenic fungi can negatively affect the survival of soft-bodied beneficial arthropods; however, in our studies, no soft-bodied predatory insects infected by the fungus were found on either treated or control plots, preventing us from speculating on whether the fungus affects only soft-bodied beneficial arthropods, causing direct mortality of these insects. One advantage of using BC639 for the biological control of green mirids on transgenic cotton crops is that infected green mirids were found in the treated plots at 2128 days after treatment, suggesting that the BC 639 fungus could be selfperpetuating and can potentially provide ongoing protection as the cotton plant grows. We observed that the oil-based formulation of the BC639 spores used in our study adhered to the cotton plant’s foliage after application, and this may explain the long residual effect of the fungal isolate on the targeted pest. Nonetheless, other explanations could be localized transmission to other parts of the same field-grown plant or even neighbouring plants due to rain splash (Bruck and Lewis 2002), the migration of infected insect hosts from the site of infection to another plant or plant part where they die (Hajek 1997; Feng, Chen, and Chen 2004), or the infection of predatory insects after coming into contact with innoculum from sporulating cadavers on treated plants (Meyling and Eilenberg 2006; Meyling, Pell, and Eilenberg 2006), causing secondary infections of green mirids that were not initially treated. In Australia, the adoption of transgenic cotton crops in 1996 has caused a drop in synthetic insecticide use against major pests (Helicoverpa spp.), from 10 kg active ingredient/ha in the 1998/1999 season to 0.5 kg in the 2002/2003 season (Wilson et al. 2004). However, there has been a rise in synthetic insecticide usage (mostly Fipronil) against sucking pests from zero to one spray per season in 2003/2004 to approximately five sprays in 2005/2006 (Whitehouse 2011). Approximately 40, 55 and 65% of all the insecticide sprays used on transgenic cotton in 2003/2004, 2005/ 2006 and 2006/2007, respectively, were Fipronil and targeted green mirids (Whitehouse 2011). The over-reliance on insecticides and the potential of green mirids and other sucking pests to develop resistance to insecticides (particularly Fipronil) will be a major obstacle in the reduction of synthetic insecticide use and the adoption of IPM on cotton. Hence, the use of entomopathogenic fungi BC 639 to manage green mirids on transgenic cotton crops would be advantageous not only to cotton production in Australia but worldwide. Presently, with Australia’s monoculture practices in agriculture and the use of synthetic insecticides against green mirids, we are inadvertently acting against beneficial insects. Many areas where crops are grown, especially cotton growing areas in Australia, are remote from wide areas of vegetation and therefore lack ecological diversity, resulting in instability in the cotton agro-ecosystem (Mensah 1999, 2002). Such areas are often without trees, bushes or weeds, and often lay fallow for most of the year. With no natural refuges and food sources for adult natural enemies of pests, especially on transgenic cotton crops, beneficial insect populations decline rapidly, becoming ineffective (Mensah 1999). Therefore, the use of BC639 fungus in IPM programmes in cotton crops may help to conserve and utilize beneficial insects effectively to manage pests, particularly green mirids on transgenic crops. Gardezi (2006) evaluated five entomopathogenic Aspergillus species for efficacy against the maize stem borer and found that the fungi killed larvae, pupae and adults of the pest


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in maize fields. In another study, Bin, Li, and Feng (2008) reported that Aspergillus spp. caused higher mortalities than other fungi (Beauveria bassiana and Entomophthora planchoniana) on migrating alate aphids that were trapped on cabbages and wheat in China. Although no apparent detrimental effect was found in terms of efficacy and conservation of beneficial insects in the use of BC 639 fungus against green mirids on transgenic crops, the major hurdle for the commercialization of BC 639 (Aspergillus spp.) for use against agricultural pests is the risk assessment of the product by regulatory authorities. The spores of Aspergillus spp. are prevalent in the air and can be inhaled by humans (Pasqualotto 2009), but the spores are known only to cause serious infection in immuno-depressed individuals (Hedayati, Pasqualotto, Warn, Bowyer, and Denning 2007). In addition, there are important geographical variations in the distribution of Aspergillus spp worldwide (Pasqualotto 2009). For example, the distribution of airborne Aspergillus spp. will vary between tropical, semi-arid and arid weather conditions (Pasqualotto 2009), indicating that the risks of using these species as biocontrol agents will vary among countries. Regardless of where BC639 is used, adherence to appropriate precautions would be essential. In this study, protective clothing (overalls), gloves, goggles and nose filter masks were used during BC639 application and during assessment of green mirids on treated cotton plants. No workers were found sick after using the product during the study period. In general, the risk associated with microbial biocontrol agents, particularly entomopathogenic fungi, is lower than that associated with chemical pesticides, as several species of fungi have been developed as environmentally benign alternatives to replace synthetic insecticides to which pests have developed resistance and that are known to pose risks to humans and the environment (Zimmermann 2007a, b; Scheepmaker and Butt 2010). Consequently, an important challenge in efforts to ensure the sustainable production of transgenic cotton is to document the benefits of microbial pest-control measures (e.g., the use of entomopathogenic fungi) relative to more traditional approaches, such as the use of synthetic insecticides to control populations of commercially important sucking pests such as green mirids. In conclusion, the ability of the entomopathogenic fungus BC639 to control C. dilutus infestations of transgenic cotton crops while conserving beneficial insect populations, especially predatory insects, suggests its potential for use in supplementing IPM programs to reduce the use of synthetic insecticides on transgenic cropping systems. Acknowledgements We wish to thank Mr and Mrs Peter Glennie, Sarah Ball, Kylie May and all past and present agronomists of Glennie & Sons Property at Norwood near Moree for co-operating with the small plot field trials. Many thanks to Mr Iain Macpherson (Macpherson Agricultural Services Pty Ltd), Geoff Phelps (Merah North) and the late Christopher Lehmann for their cooperation. Special thanks also to research and technical staff of Becker Underwood Pty Ltd for assistance in product formulation and manufacturing, which enabled the small plot trials to be conducted. Our sincere thanks are given to the IPM team at the Australian Cotton Research Institute in Narrabri: Angela Singleton, Ms Ruth Coates, Ray Morphew, Stacey Cunningham, Carolyn Palmer, Katinka Atkins and Lori Nemec for their technical support. Funding for this project was provided by the Cotton Research and Development Corporation (CRDC), and Becker Underwood Pty Ltd provided technical support for the mass production of fungal isolate. This work forms part of the CRDC project 03DAN001.


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