Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and...

For Peer Review O

nly

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and Metarhizium anisopliae against adult Guava

Weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Journal: Biocontrol Science & Technology

Manuscript ID: CBST-2008-0041.R1

Manuscript Type: Research Article

Date Submitted by the Author:

n/a

Complete List of Authors: Brito, Eliane; UENF, Entomology and Plant Pathology de Paula, Adriano; UENF, Entomology and Plant Pathology Vieira, Laerciana; UENF, Entomology and Plant Pathology Dolinski, Claudia; UENF, Entomology and Plant Pathology Samuels, Richard; State University of North Fluminense, Entomology and Plant Pathology

Keywords: efficiency, fungus, insecticide, oil, adjuvants, insect

URL: http://mc.manuscriptcentral.com/cbst

Biocontrol Science & Technology

For Peer Review O

nly

1

Biocontrol Science and Technology

Increasing fungal efficiency against guava weevil

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with

Beauveria bassiana and Metarhizium anisopliae against adult Guava Weevil

Conotrachelus psidii (Coleoptera: Curculionidae)

Eliane Souza Brito, Adriano Rodrigues de Paula, Laerciana Pereira Vieira, Claudia

Dolinski and Richard Ian Samuels*

Department of Entomology and Plant Pathology – CCTA – Universidade Estadual do

Norte Fluminense - Darcy Ribeiro

Av. Alberto Lamego 2000, Campos dos Goytacazes, RJ. 28013-602, Brasil.

*corresponding author: [email protected]

Page 1 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

2

Abstract

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii

Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil.

Control methods for this pest are based on the application of insecticides. In search of

alternatives, this study was performed to select fungal isolates of Beauveria bassiana

and Metarhizium anisopliae as potential candidates for the control of adult C. psidii.

Tests were carried out using three products applied with the entomopathogenic fungi

:Tween 80, Sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI

was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the

most effective isolates when used in combination with all of the products. The least

virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality,

however, this isolate showed significantly improved efficiency when applied together

with either Sunflower oil or IMI, causing 57.3% and 88.6% mortality respectively. The

efficiency of all the isolates tested here improved when applied together with IMI, with

LT50 values of 5.3 - 10.3 days when compared to LT50 values in Tween alone of 9.5 - 17

days. The isolate that produced the highest number of conidia on the cadavers of adult

C. psidii was LPP138, independent of the product used, however conidial production

was slightly reduced when fungi were applied together with IMI. These results are

promising for developing new formulations of the isolates to be tested in the field.

Key words: efficiency; fungus, insecticide; oil; adjuvants; insect

1. Introduction

Page 2 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

3

Guava (Myrtaceae: Psidium guajava L.), native to tropical regions of the

Americas, is of economic importance in over 50 tropical and subtropical countries

(Menzel, 1985). However, insect pests and plant diseases are responsible for huge

annual losses in production. Of the insect pests that attack guava plants, the guava

weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is considered

to be one of the most important pest species, damaging up to 90% of the fruits in Rio de

Janeiro State (Boscán de Martinez and Cásares, 1982). Control methods for this pest are

based on applications of chemical insecticides during the peak infestation period

(October-March), with unwanted residual effects, low efficiency against the adults and

inefficient for the control of larvae within the fruit or soil (Sampaio, 1975; Barelli &

Galli, 1998). A more rational approach is therefore needed.

However, the importance of conventional chemical insecticides cannot be

underestimated and the possibility of combining insecticides with biological control

agents is an interesting approach for controlling certain pest species, allowing the

reduction of chemical applications and reducing the development of resistance. Some

insecticides have the capacity to increase stress and affect insect behaviour that may

lead to improved performance of entomopathogens. This has been demonstrated in

recent studies that showed the advantages of applications of sub-lethal doses of

Imidacloprid (IMI) together with fungi for the control of leaf-cutting ants (Santos et al.,

2007). IMI is a systemic insecticide of the neonicotinoid group, with contact and oral

toxicity. The site of action of this insecticide is the nicotinic receptor of acetylcholine,

therefore acting as a neurotoxin (Elbert et al., 1991). IMI was found to act

synergistically with Beauveria bassiana and Metarhizium anisopliae for the control of

Diaprepes abbreviatus (Quintela and McCoy 1997).

Page 3 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

4

Adult C. psidii can be targeted with Integrated Pest Management strategies whilst

4th instar larvae could be controlled by applications of entomopathogenic nematodes to

the soil (Dolinski et al., 2006, Del Valle et al., 2008). One of the main drawbacks of

biological control is the slow kill time and dependence on climatic conditions. In the

case of entomopathogenic fungi, high humidity is normally needed for infection to

occur. However, the success of the Lubilosa Program, where oil formulations of fungi

were used to control the desert locust, has shown that at least humidity limitations can

be overcome (Lomer et al., 2001). Another possible advantage of using

entomopathogenic fungi is the potential for secondary cycling within insect pest

populations, thus reducing the need for continuous applications. This was found to be

an important factor in the control of locusts and grasshoppers (Thomas et al., 1995;

Thomas, 1996).

Given that the adult stage of this insect only showed moderate infection rates

when exposed to fungal isolates suspended in tween under laboratory conditions

(current study), experiments were carried out to improve efficiency by combining fungi

with vegetable oil or sub-lethal concentrations of an insecticide Imidacloprid.

2. MATERIAL AND METHODS

2.1 Insects

Adult C. psidii were collected from commercial guava orchards in the

municipality of São Francisco de Itabapoana in North Rio de Janeiro State and insects

were maintained in the laboratory in plastic boxes (11 x 11 x 3.2 cm) lined with filter

paper at 25°C, 70 ± 10 % RH and 12L: 12D photoperiod. Insects were fed with slices of

fresh guava fruit.

Page 4 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

5

2.2 Fungal isolates and preparation of suspensions

Isolates of M. anisopliae and B. bassiana detailed in Table 1 were obtained from

the collection at CENARGEN (CG 24) in Brasilia, ESALQ (ESALQ 818) in Piracicaba

(São Paulo), and the Laboratory of Entomology and Plant Pathology State University of

North Fluminense (LPP 19; 91; 110; 138; 144). Fungi were cultured on Sabouraud

Dextrose Agar (Dextrose 10g; Peptone 2.5g; Yeast Extract 2.5g; Agar 20g in 1L H20) at

270C for 15 days before being used in experiments. Fungal suspensions were prepared

in Tween 80 (0.05% in sterile distilled water) and conidial concentration determined

using a Neubauer hemocytometer.

2.3 Effect of Imidacloprid on C. psidii

The commercial product Confidor 700 GrDA (Bayer) composed of IMI (70%)

and inert ingredients (30%) was used in all experiments. Different concentrations of

Confidor® (1000; 500; 100 and 50 ppm in 1 mL of water) were applied to adult C. psidii

using a Potter spray tower (Burkard, UK). Fifteen insects were placed in a Petri dish and

sprayed simultaneous for each concentration of IMI. Following applications, insects

were maintained in plastic boxes lined with filter paper and offered fresh slices of guava

over the 5 day experimental period. Observations of insect behavior (locomotion and

feeding) and mortality were carried out every 24h. A sub-lethal concentration was

defined as that which did not cause insect mortality but provoked an increase in host

susceptibility to fungal infections (determined from preliminary experiments; data not

shown).

2.4 Isolate selection and application of fungi in Sunflower oil or Imidacloprid

Page 5 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

6

Isolates of B. bassiana and M. anisopliae were tested against C. psidii by spray

application of fungal suspensions mixed with Tween 80 (0.05%), 8% sunflower oil or

IMI (100ppm). The fungal concentration used in all cases was 1 x 108 conidia mL-1 and

suspensions (1 mL) applied to the insects using a Potter tower. For each treatment 15

insects were used and each experiment was repeated three times. Control insects were

treated as above but without addition of the fungus. Following treatment, groups of 5

insects were maintained in plastic boxes (11 x 11 x 3.2 cm) with a regular supply of

fresh guava during a 20 day observation period. Mortality was evaluated on a daily

basis. Dead insects were removed and surface sterilized in 70% alcohol for 30 seconds

and immediately rinsed in sterile distilled water for a further 30 seconds before being

placed in Petri dishes lined with damp filter paper and maintained at 25°C to evaluate

whether the fungi had caused host death.

Mortality data was calculated as percentages and the normal distribution of the

data verified using Lilliefors test. As the data was shown to have a normal distribution,

it was deemed unnecessary to use Arc Sin corrections. The data was analyzed using

two-way Analysis of Variance. Mortality was compared between isolates and between

treatments using one-way Analysis of Variance and Duncan’s post-hoc test. Probit

analysis was used to determine LT50, corrected according to Throne et al. (1995).

2.5 Conidial production on C. psidii cadavers

The production of conidia on insect cadavers was evaluated 5 days after death.

Five cadavers were chosen from each treatment and each repetition and conidial

concentration evaluated for each cadaver. The cadaver was placed in an Eppendorf tube

Page 6 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

7

containing 1 mL of Tween 80 (0.05%) and the tube was the agitated rigorously using a

vortex mixer. The conidial concentration was estimated using a Neubauer chamber.

3. RESULTS

3.1 Effect of different concentrations of Imidacloprid on C. psidii.

Observations of mortality showed that a concentration of 1000 ppm caused the

highest mortality, whilst 100 ppm and 50 ppm did not result in insect mortality during

the experimental period (Fig. 1). However, all concentrations used caused alterations in

behavior (locomotion and feeding), when compared to control insects (results not

shown). The most obvious changes were seen in locomotion, with insects appearing

initially moribund shortly after IMI application. Over time (four days), the insects

treated with the 2 lower concentrations returned to normal activity, whereas the two

higher concentrations resulted in insect death. No control mortality was seen during the

period of the experiment.

3.2 Isolate virulence

The two-way ANOVA showed that there was an interaction effect when

comparing isolates x formulation (F 12, 42 = 3.22; p< 0.01), an interaction between

isolates (F 6, 42 = 11.44; p< 0.01), and interaction between formulations (F 12, 42 = 34.8;

p< 0.01). To further analyze these interactions, one-way ANOVAs followed by

Duncan’s multiple tests were carried out.

The results shown in Table 2 demonstrate the virulence of the isolates tested,

with mortalities ranging from 26.6% (M anisopliae ESALQ 818) to 73.3% (M.

Page 7 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

8

anisopliae LPP 91 and B. bassiana LPP 19) when formulated in Tween 80. The use of

oil significantly increased mortality of 5 of the isolates tested here (ESALQ 818, LPP

110; LPP 138; CG 24, LPP 19) when compared to the application of these isolates in

Tween only.

Inoculations of isolates together with applications of IMI at a concentration of

100 ppm, resulted in higher host mortality than when formulated in oil, with 100%

mortality for five of the isolates tested here and 86.6% mortality when using the least

virulent isolate (M. anisopliae ESALQ 818). No statistical differences were seen when

comparing isolates applied with IMI.

Isolates B. bassiana LPP110 and M. anisopliae ESALQ 818 showed marked

differences in LT50 values when using different products (Table 3). For example, LT50

values for LPP110 in Tween, Sunflower oil and IMI were 17.0 days, 13.1 days and 8.9

days respectively. For ESALQ 818 LT50 values in Sunflower oil and IMI were 14.2

days and 10.3 days respectively. The lowest value for LT50 was recorded for B.

bassiana isolate LPP114 (5.31 days) when applied with IMI. No control mortality was

seen during the period of the experiment. Control insects survived under laboratory

conditions for on average 45-50 days.

3.3 Evaluation of conidial production following infections

These results demonstrate the differences between isolates when considering the

factor of conidial production following host colonization. The results show the

variability between the isolates tested, with B. bassiana LPP 138 producing the highest

conidial concentrations in Tween and oil, however, the use of insecticide reduced

Page 8 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

9

conidial production for this isolate (Table 4). Interestingly, the most virulent isolate, B.

bassiana LPP 114, produced the lowest number of conidia (0.9 x 108 ml-1) in both

Tween and oil, with a slight increase when formulated with insecticide (1.8 x 108 ml-1).

4. DISCUSSION

The results show that all of the isolates tested were pathogenic to adult C. psidii,

although with variations in virulence among these isolates. The most virulent isolates

when applied in Tween did not cause 100% host mortality over the experimental period.

However, an increase in efficiency was observed following application in Sunflower oil

and moreover when the fungi were applied together with a sub-lethal concentration of

IMI. As it is common that isolates show higher virulence under laboratory conditions

than under field conditions, maximization of the efficiency of the isolates is important

before starting field trails. A model for predicting the performance of entomopathogenic

fungi has been recently proposed for locust control (Klass et al. 2007), which might be

applicable to other insect-fungus interactions. The use of oil formulations has been

shown to improve efficiency under field conditions, stimulating fungal germination

independently of the ambient humidity (Batta, 2003). The use of insecticides in

combination with entomopathogenic fungi is also an interesting approach that can be

used against certain species of insects that are difficult to control with fungi alone. The

guava weevil shows a high tolerance to fungal infections, with the lowest LT50 values

seen here being approximately 10 days (current study). Previous studies have shown

that IMI has no adverse effects on germination of five fungal isolates and acted

synergistically with entomopathogenic fungi to rapidly kill the leaf-cutting ant Atta

sexdens (Santos et al., 2007). Here C. psidii mortality was only seen with concentrations

Page 9 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

https://www.researchgate.net/publication/248416442_Production_and_testing_of_novel_formulation_of_the_entomopathogenic_fungus_Metarhizium_anisopliae_Metch_Sorokin_Deuteromycetes_Hyphomycetes?el=1_x_8&enrichId=rgreq-86159b0316eaacc98683d78854d4cf4e-XXX&enrichSource=Y292ZXJQYWdlOzI0ODk2NDAwOTtBUzoxNjI5OTYyNDAyNjUyMTZAMTQxNTg3Mjc0MDE3Mg==

For Peer Review O

nly

10

of 500 ppm and 1000 ppm, demonstrating the tolerance of this insect to insecticides,

especially when compared to results for toxicity to leaf-cutting ants, where LC50 was

estimated to be 145.3 ppm (Santos et al. 2007).

Vegetable oil when used here in combination with all isolates tested caused an

increase in mortality, which was more obvious when comparing the isolate of lowest

virulence (ESALQ 818) in Tween (26% mortality) and in oil (57% mortality). Similar

results were obtained by Maranga et al. (2005), for the control of the tick Amblyomma

variegatum, with mortality reaching 95% when B. bassiana or M. anisopliae were

formulated in oil as opposed to 49% mortality when formulated in water.

IMI, when applied at a concentration that had no significant effects on insect

survival, promoted increased efficiency of all the isolates tested here, with five of the

isolates causing 100% host death over the experimental period. The insecticide

treatment also provoked a reduction in LT50 values for all of the isolates tested, reducing

to 5 days the time taken to kill 50% of the insects for the most virulent isolate. This is a

significant advance over the use of either oil or Tween and should be tested under field

conditions. Quintela & McCoy (1998), suggested that the synergistic effect of IMI and

entomopathogenic fungi in the case of joint applications against Diaprepes abreviatus,

was due to a reduction in insect motility, leading to an increased adhesion of the conidia

to the host integument, that would normally be removed by friction as the larvae move

through their tunnels in the soil. In the case of the guava weevil, C. psidii, it is likely

that the insecticide acted as a general stressor, lowering the insect's tolerance to fungal

infections.

Hiromor and Nishigki (2001) showed that joint applications of M. anisopliae and

the insecticide Fenitrothion, increased mortality of Anomala cuprea larvae, when

Page 10 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

11

compared to using these agents individually. These authors attributed this to an

inhibitory effect of the insecticide to the humoral defence system of the host insect.

Jaramillo et al. (2005) also found additive effects of joint applications of IMI and M.

anisopliae for the control of bug Cyrtomenus bergi, increasing host mortality from 40%

with fungal inoculations only, to 88% when combining fungi with this insecticide.

The reduction in total conidial concentration per cadaver associated with

infections following application of fungi together with insecticide for some of the

isolates tested here indicates that although this combination increased mortality, it may

have a negative effect when considering possible secondary infections in the field. In

this case it is necessary to determine the importance of secondary infections in relation

to the damage caused by the pests. Production of secondary inoculum on infected

insects is considered to be important in the spread of infection throughout a population

(Carruthers and Soper, 1987). The possible use of an isolate not considered to the most

virulent of those tested here but found to be the highest conidial producer (LPP 138),

with mean concentrations of 4.6 x 108 conidia ml-1 insect-1 when applied in Tween only

and 4.8 x 108 conidia ml-1 insect-1 when applied in Tween + oil, may therefore have

certain advantages, although it is difficult to estimate the importance of secondary

cycling for the control of this pest. It is interesting to note that Beauveria bassiana

isolate LPP 138 is a natural pathogen of the guava weevil, however, the other Beauveria

bassiana isolate found naturally infecting C. psidii, LPP 114, produced 5 times less

conidia than LPP 138 following application in Tween or Tween + oil.

This study shows that applications of fungi alone are unlikely to be efficient for

the control of the guava weevil C. psidii, given its high tolerance to fungal inoculations

under ideal laboratory conditions, however, significant increases in mortality and speed

Page 11 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

12

of host death were obtained using vegetable oil and IMI. We are currently assessing

these two combinations under field conditions.

Acknowledgements:

We thank Ederaldo Azevedo da Silva and Paulo César de Oliveria Pedra Jr. for

technical assistance and Dr. James Throne for help with Probit Analysis. We would also

like to thank the anonymous referees for their useful comments. This work was funded

by a grant from CNPq (473803/03-8). Richard Samuels is a CNPq research fellow.

REFERENCES

Barelli NL, Galli JC, 1998. Avaliação de danos causados por Anastrepha spp. (Dip.

Tephritidae) e por Conothrachelus psidii (Marshall, 1922) (Coleoptera:

Curculionidae) em frutas de goiaba da cultivar `paluma'. XVII Congresso Brasileiro

de Entomologia p. 12. Rio de Janeiro.

Batta YA. 2003. Production and testing of novel formulations of the entomopathogenic

fungus Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina:

Hyphmycetes).Crop Protection 22: 415-422.

Bateman RP. 1997. Methods of application of microbial pesticide formulations for the

control of grasshoppers and locusts. Memoirs of the Entomological Society of

Canada 171: 67-79.

Page 12 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960




https://www.researchgate.net/publication/272456113_Methods_of_application_of_microbial_pesticide_formulations_for_the_control_of_grasshoppers_and_locusts?el=1_x_8&enrichId=rgreq-86159b0316eaacc98683d78854d4cf4e-XXX&enrichSource=Y292ZXJQYWdlOzI0ODk2NDAwOTtBUzoxNjI5OTYyNDAyNjUyMTZAMTQxNTg3Mjc0MDE3Mg==



For Peer Review O

nly

13

Boscán de Martinez N Cásares R. 1982. Distribución en el tiempo de las fases del

gorgojo de la guayaba Conotrachelus psidii Marshall (Coleoptera: Curculionidae)

en el campo. Agro Tropical. 31: 123–130.

Carruthers RI, Soper RS. 1987. Fungal Diseases, In: J.R. Fuxa and Y. Tanada, (Eds.),

Epizootiology of Insect Diseases, New York, pp. 357-417.

Dell Valle EE, Dolinski C, Barreto ELS, Souza RM, Samuels RI. 2008. Efficacy of

Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied in Galleria

mellonella (Lepidoptera: Pyralidae) insect cadavers to Conotrachelus psidii,

(Coleoptera: Curculionidae) larvae. Biocontrol Science and Technology 18: 33 – 41.

Dolinski C, Del Valle EE, Stuart RJ. 2006. Virulence of entomopathogenic nematodes

to larvae of guava weevil, Conotrachelus psidii (Coleoptera:Curculionidae), in

laboratory and greenhouse experiments. Biological Control 38: 422–427.

Elbert A, Becker B, Hartwig J, Erdelen C. 1991. Imidacloprid - a new systemic

insecticide. Pflanzenschutz-Nachrichten Bayer 44: 113-136.

Hiromori H, Nishigaki J, 2001. Factor analisys of synergistc effect between the

entomopathogenic fungus Metarhizium anisopliae and synthetic insecticides.

Applied Entomology and Zoology 36: 321-236

Jaramillo J, Borgemeister C, Ebssa L, Gaigl A, Tobón R, Zimmermann G. 2005. Effects

of combined applications of Metarhizium anisopliae (Metsch.) Sorokin

(Deuteromycotina: Hyphomycetes) strain CIAT 224 and different dosages of

imidacloprid on the subterranean burrower bug Cyrtomenus bergi Foreschner (

Hemiptera: Cydnidae). Biological Control 34: 12-20.

Page 13 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

14

Klass JI, Blanford S, Thomas, MB. 2007. Use of a geographic information system to

explore spatial variation in pathogen virulence and the implications for biological

control of locusts and grasshoppers. Agricultural and Forest Entomology 9: 201–

208.

Lomer CJ, Bateman RP, Dent D, De Groote H, Douro-Kpindou O-K, Kooyman C,

Langewald J, Ouambama Z, Peveling R, Thomas M. 1999. Development of

strategies for the incorporation of biological pesticides into the integrated

management of locusts and grasshoppers. Agricultural Forest Entomology 1: 71-88.

Lomer CJ, Bateman RP, Johnson DL, Langwald J, Thomas, M. 2001. Biological

Control of Locusts and Grasshoppers. Annual Review of Entomology 46: 667 -

702.

Maranga RO, Kaaya GP, Mueke MJ, Hassanali, A. 2005. Effects of combining the

fungi Beauveria bassiana and Metarhizium anisopliae on the mortality of the tick

Amblyomma variegatum (ixodidae) in relation to seasonal changes. Mycopathologia

159: 527-532

Menzel CM. 1985. Guava: an exotic fruit with potential in Queensland. Queensland

Agricultural Journal 111: 93-98.

Prior C, Jollands P, Le Patourel G. 1998. Infectivity of oil and water formulations of

Beauveria bassiana (Deuteromycotina: Hyphomycetes) to the cocoa weevil pest

Pantorhytes plutus (Coleoptera: Curculionidae). Journal of Invertebrate Pathology

52: 66-72.

Page 14 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

15

Quintela ED, McCoy CW. 1997. Pathogenicity enhancement of Metarhizium anisopliae

and Beauveria bassiana to first instars of Diaprepes abbreviatus (Coleoptera:

Curculionidae) with sublethal doses of imidacloprid. Environmental Entomology

26: 1173-1182.

Quintela ED, McCoy CW. 1998. Conidial attachment of Metarhizium anisopliae and

Beauveria bassiana to the larval cuticle of Diapreps abbreviatus (Coleoptera:

Curculionidae) treated with imidacloprid. Journal of Invertebrate Pathology 72:220-

230.

Sampaio A. 1975. Gorgulho-da-goiaba tem agora um moderno controle. Correio

Agrícola. 2: 20-21.

Santos AV, Oliveira BL, Samuels RI. 2007. Selection of entomopathogenic fungi for

use in combination with sub-lethal doses of imidacloprid: perspectives for the

control of the leaf-cutting ant Atta sexdens rubropilosa Forel (Hymenoptera:

Formicidae). Mycopathologia 163: 233-240.

Thomas MB, Wood SN, Lomer, CJ. 1995. Biological control of locusts and

grasshoppers using a fungal pathogen: the importance of secondary cycling.

Proceedings of the Royal Society of London B 259: 265-270.

Thomas MB, Gbongboui C, Lomer, CJ. 1996. Between-season survival of the

grasshopper pathogen Metarhizium flavoviride in the Sahel. Biocontrol Science and

Technology 6: 569-573.

Thomas MB, Langewald J, Wood SN. 1996. Evaluating the effects of a biopesticide on

populations of the variegated grasshopper, Zonocerus variegates. Journal of Applied

Ecology 33: 1509-1600.

Page 15 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

16

Throne JE, Weaver DK, Chew V, Baker JE 1995. Probit analysis of correlated data:

Multiple observations over time at one concentration. Journal of Economic

Entomology 88:1510-1512.

Figure legend

Figure 1. Effect of Imidacloprid on adult Conotrachelus psidii mortality following spray

application of four concentrations of insecticide.

SDW: sterile distilled water control.

Page 16 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nlyFigura 1

1 2 3 4 5

0

1

2

3

4

+

Num

ber

of D

ead

Inse

cts

(mea

n

SE

)

Experimental Period (Days)

1000 ppm 500 ppm 100ppm 50 ppm SDW

Page 17 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

1

Table 1 Details of the fungal isolates used in tests for pathogenicity and virulence

against adult C. psidii

Accession

Number

Species Original Host Geographical Origin

ESALQ 818 M. anisopliae Isolated from soil Piracicaba, São Paulo,

Brazil

LPP 110 B. bassiana Acromyrmex sp. (Hymenoptera:

Formicidae)

Belém, Pará, Brazil

LPP 138 B. bassiana Conotrachelus psidii (Coleoptera:

Curculionidae)

Campos, Rio de Janeiro,

Brazil

LPP 114 B. bassiana Conotrachelus psidii (Coleoptera:

Curculionidae)

Campos, Rio de Janeiro,

Brazil

CG 24 B. bassiana Euschistus heros Fabr.(Heteroptera: Pentatomidae)

Londrina, Paraná, Brazil

LPP 91 M. anisopliae Isolated from soil Rondônia, Brazil

LPP 19 B. bassiana Blissus antillus ( Hemiptera:

Lygaeidae)

Ibitióca, Rio de Janeiro,

Brazil

Page 18 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

2

Table 2. Mortality of adult C. psidii infected with B. bassiana and M. anisopliae when

applied in Tween 80, Sunflower oil +Tween 80 and joint applications of fungi

and Imidaclorprid (IMI).

Percentage Mortality (Mean ± SD)

Isolate/Product Tween 80 Oil+ Tween 80 IMIESALQ 818 26.6 ± 17.3 A a 57.3 ± 10.2 A b 86.6 ± 13.3 A b

LPP 110 48.7 ± 16.6 AB a 77.3 ± 10.2 ABC ab 100 A b

LPP 138 62 ± 10 BC a 89.0 ± 16.7 BC b 100 A b

LPP 114 62 ± 10 D1a2 95.3 ± 3.8 C a 100 A a

CG 24 66.6 ± 13.3 BC a 77.6 ± 13.7 ABC ab 93.3 ± 3.8 A b

LPP 91 73.3 ± 6.6 CD a 84.3 ± 17.0 AB c 100 A a

LPP 19 73.3 ± 6.7 CD a 90.6 ± 3.9 C ab 100 A b

Note: Fungal concentration 1 x 108 conidia mL-1; Tween concentration 0.05%; Sunflower oil concentration 8%; IMI concentration 100 ppm.Control mortality for all treatments was zero.

1Values followed by the same capital letter indicated that the results were not

statistically different between isolates when using the same formulation (Tukey test

at the 5% probability level).

2Values followed by the same lower case letter indicate that the results were not

statistically different when comparing formulations with the same isolate using the

Tukey test at the 5% probability level

Page 19 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

3

Table 3. Lethal times (LT50) for B. bassiana and M. anisopliae (1 x 108 conidia ml-1)

infecting adult C. psidii, applied in Tween 80, Sunflower oil +Tween 80 and

joint applications of fungi and Imidaclorprid (IMI)

IsolateTWEEN 80

LT50 days [CL]OIL

LT50 days [CL]IMI

LT50 days [CL]

ESALQ 818 - 14.2 [12.9 – 15.8] 10.3 [9.0 –11.5]

LPP 110 17.0 [15.4 – 19.5] 13.1 [12.1 –14.2] 8.9 [8.1 – 9.7]

LPP 138 12.7 [11.7 – 14.0] 9.6 [9.1 –10.6] 6.7 [6.1 – 7.2]

LPP 114 9.5 [8.3 – 10.5] 8.5 [7.2 – 9.6] 5.3 [4.7 – 5.6]

CG 24 12.3 [10.2 – 14.5] 12.4 [10.1 – 14.5] 7.3 [5.5 – 8.7]

LPP 91 12.4 [12.1 – 14.2] 16.0 [15.4 – 19.5] 9.3 [8.1 – 9.7]

LPP 19 9.7 [7.4 – 11.6] 8.3 [5.9 – 10.3] 5.7 [5.3 – 6.1]

CL – Upper and lower confidence Limits

Note: Fungal concentration 1 x 108 conidia mL-1; Tween concentration 0.05%; Sunflower oil concentration 8%; IMI concentration 100 ppm.Control mortality for all treatments was zero.

Page 20 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Table 4. Conidial production (mean conidia ml-1 x 108 /cadaver Ꮚ� SD) on C. psidii cadavers following B. bassiana and M. anisopliae infections.

Isolate/Product Tween 80 Oil +Tween 80 IMI

ESALQ 818 0.6 ± 0.3 0.9 ± 0.5 1.1 ± 0.5

LPP 110 1.8 ± 0.3 1.1 ± 0.5 1.2 ± 0.6

LPP 138 4.6 ± 1.5 4.8 ± 1.2 3.1 ± 1.5

LPP 114 0.9 ± 0.4 0.9 ± 0.3 1.8 ± 0.4

CG 24 3.6 ± 0.9 2.4 ± 0.6 2.9 ± 0.6

LPP 91 1.7 ± 0.5 1.9 ± 1.8 1.8 ± 1.3

LPP 19 2.1 ± 1.6 2.3 ± 1.8 2.4 ± 1.3

Page 21 of 21



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and...

Documents

Transcript of Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and...