Persistence and efficacy of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) and diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) on wheat and maize

ARTICLE IN PRESS Crop Protection 27 (2008) 1303– 1311

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Persistence and efficacy of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) and diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) on wheat and maize Christos G. Athanassiou a, Nickolas G. Kavallieratos b,, Basileios J. Vayias a, Johanna B. Tsakiri c, Nickoleta H. Mikeli d, Constantin M. Meletsis e, Zˇeljko Tomanovic´ f a

Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75 Iera Odos Street, 11855 Athens, Attica, Greece Laboratory of Agricultural Entomology, Department of Entomology and Agricultural Zoology, Benaki Phytopathological Institute, 8 Stefanou Delta Street, 14561 Kifissia, Attica, Greece c Department of Farm Management, School of Agricultural Technology, Technological Educational Institute of Thessalonica, Sindos 54101, Thessalonica, Greece d Department of Greenhouse Crops and Floriculture, Technological Educational Institute of Kalamata, 24100 Antikalamos, Kalamata, Greece e Department of Crop Production, Technological Educational Institute of Kalamata, 24100 Antikalamos, Kalamata, Greece f Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia b

a r t i c l e in fo

abstract

Article history: Received 14 September 2007 Received in revised form 5 March 2008 Accepted 6 March 2008

Wheat and maize were treated with 8  106 and 8  108 conidia/kg of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin, alone and in combination with 250 ppm Diatomaceous Earth (DE). Bioassays were conducted after application and monthly for 5 months using adult Sitophilus oryzae (L) and Rhyzopertha dominica (F.). Both treated grains and the bioassay samples were held at 2771 1C and 6575% r.h. Mortality assessments were made at 7 and 14 d, and after the 14 d counts all adults were removed and the samples incubated at the same environmental conditions for 60 d to record progeny production. Mortality of S. oryzae exposed for 7 d on both grains decreased during the 5-month period, however, maximum mortality usually occurred in the combination of the highest fungal rate with the DE. Mortality of S. oryzae was generally lower in maize than in wheat. Mortality of R. dominica in both grains was generally greater than mortality observed for S. oryzae. Progeny production of S. oryzae increased gradually during the 5 months, but this increase varied depending on the treatment. Similar results were noted for R. dominica with generally lower progeny production compared with S. oryzae. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Metarhizium anisopliae Diatomaceous earth Sitophilus oryzae Rhyzopertha dominica Wheat Maize

1. Introduction The potential of using entomopathogenic fungi as an alternative to traditional grain protectants has received increased attention during recent years (Moore et al., 2000; Lord, 2001). Entomopathogenic fungi have low mammalian toxicity, and many studies have shown that they are very effective against several stored-grain insect pests (Moore et al., 2000). Furthermore, these substances can persist on the product and recycle in the cadavers, reintroducing more inoculum in the grain. Hence, while persistence is generally considered as a drawback in the case of traditional grain protectants, due to residues on the product, it is a desired characteristic in the case of fungi (Stathers, 2002).

 Corresponding author. Tel.: +30 2108180215; fax: +30 2108077506.

E-mail address: [email protected] (N.G. Kavallieratos). 0261-2194/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2008.03.004

Previous studies suggest that Metarhizium anisopliae (Metschnikoff) Sorokin can be used with success against several pest species (Batta, 2004, 2005; Michalaki et al., 2006; Kavallieratos et al., 2006). Another potential alternative in the use of grain protectants is the use of desiccant dusts, particularly diatomaceous earths (DEs), which can be effective against a wide range of stored-grain insects (Korunic, 1998; Subramanyam and Roesli, 2000; Arthur, 2000, 2002; Fields and Korunic, 2000). Diatomaceous earths have low mammalian toxicity, and can easily be removed from the product before milling (Korunic et al., 1996, 1998). Also, as inert materials, DE particles remain unaffected and can persist on the product, providing long-term protection (Athanassiou et al., 2005; Vayias et al., 2006). However, the main drawback for using DEs is that they can affect the physical properties of grain, particularly bulk density (Korunic et al., 1996; Korunic, 1998). Since both fungi and DEs act on the insect cuticle, their combined use has been

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proposed as a means of reducing application rate (Lord, 2001; Akbar et al., 2004; Vassilakos et al., 2006). Akbar et al. (2004) reported that the combination of Beauveria bassiana (Balsamo) Vuillemin with the DE Protect-It, had a synergistic effect against larvae of the red flour beetle, Tribolium castaneum (Herbst). Moreover, since both substances persist on the grain, it seems that such a combination may have a certain advantage as a long-term protectant. The rice weevil, Sitophilus oryzae (L.) and the lesser grain borer, Rhyzopertha dominica (F.) are two of the most destructive insect pests of stored grains worldwide (Aitken, 1975). They are classified as primary pests, which means they are capable of infesting sound grain kernels. Larvae of S. oryzae and R. dominica develop inside the kernel and therefore are protected from grain protectant residues on the exterior of the kernel. Both species have developed resistance to traditional grain protectants and fumigants (Champ and Dyte, 1976; Arthur, 1996). Recent studies indicate that S. oryzae and R. dominica are susceptible to M. anisopliae and DE (Batta, 2004, 2005; Athanassiou et al., 2004; Kavallieratos et al. 2005; Michalaki et al., 2006). However, very little data are available on the long-term insecticidal activity of these two substances against these two species. In the present study, we evaluated the potential of a long-term protection of wheat and maize against S. oryzae and R. dominica through the combined use of a M. anisopliae strain and a DE formulation. In addition to parental mortality, we also evaluated progeny production on the treated grains.

2. Materials and methods 2.1. Test insects Adults of R. dominica and S. oryzae were used in the tests. These adults were taken from cultures that was reared in the laboratory on whole wheat at 2771 1C and 6575% r.h. The cultures had been kept at the Benaki Phytopatological Institute since 1994 and 2002, respectively. All adults used in the tests were o2 weeks old.

The fungus was subcultured on plates with oatmeal agar (O3506, Sigma, Munich, Germany) for mass production of the fungal conidia. During conidial production, plates were incubated at 2071 1C and 16 h illumination per day. After 14 days of incubation, the fungal conidia were collected by scraping the conidial layers formed on the plate surface using a sterilized scalpel. The conidia were added to 100 ml sterile distilled water, stirred and filtered through muslin. A formulation containing fungal conidia and a dust carrier at 1:4 ratio (W/W) was prepared. The dust carriers were a mixture of flour, oven ash (completely burned paper sheets), chalk powder (finely ground board chalks) and charcoal (finely ground coal pieces) (Batta, 2004; Kavallieratos et al., 2006). Harvested conidia were thoroughly mixed with the carrier in screw-capped bottles. The concentration of fungal conidia in the conidial suspension was determined using a haemocytometer (Precicolor, HBG, Giessen-Luetzellinden, Germany). Two formulations, corresponding to two concentrations of fungal conidia were prepared, containing 8  106 and 8  108 conidia/g of the formulation, respectively (Michalaki et al., 2006; Kavallieratos et al., 2006).

2.5. Grain treatment There were five fungus/DE combinations: the lower dose (8  106) of the fungus alone, the higher dose (8  106) of the fungus alone, the lower dose with DE, the higher dose with DE and the DE alone. For each grain treatment, lots of 1 kg were prepared. In each lot, the respective quantity of fungus (1 g for each dose corresponding to 8  106 and 8  108 conidia/kg of wheat or maize) and DE (0.25 g) was added. Each of these lots was introduced in glass jars (15 cm in diameter, 35 cm in height), which were shaken manually for approx. 5 min to achieve an equal distribution of the dust on the entire grain mass. For each grain, there was an additional lot, which was untreated and served as a control. During the entire experimental period (see below) all jars were kept in incubators set at 26 1C and 65% r.h.

2.2. Grains

2.6. Bioassays

Untreated, clean and infestation-free hard wheat (var. Mexa) and maize (var. Dias) were used in the tests. Both grains came from the 2003 harvest in Greece. The moisture content of the grains, as determined by a Dickey-John moisture meter (DickeyJohn Multigrain CAC II, Dickey-John Co., Auburn, IL, USA), was 11.2 and 11.7 for wheat and maize, respectively. Before the beginning of the experiments, the grain quantities were held at ambient conditions (see below) for 7 d, to equilibrate to the desired r.h.

The residual efficacy of the above treatments was monitored for a period of 5 months (from June to December 2003) after the grain treatment, by conducting bioassays at 30-d intervals. The first bioassay was carried out at the day the grains were treated with fungus and/or DE in the containers. In this bioassay, six samples, of 50 g each, were taken from each jar (treatments and controls). Each sample was placed in a small cylindrical glass vial (7.5 cm in diameter, 12.5 cm in height, with a hole 1.5 cm in diameter at the top covered with a tulle for aeration). Then, 50 adults of S. oryzae were introduced into each vial. The vials were then placed in incubators, set at 26 1C and 65% r.h (Kavallieratos et al., 2006). The desired r.h. level in the incubators was maintained by using saturated salt solution of sodium chloride, as recommended by Greenspan (1977). The number of dead adults on each vial was counted after 7 and 14 d of exposure in the treated or untreated grain. The same procedure was also followed in the case of R. dominica. The exposed adults were classified as dead (unable to move) or alive (indicating even minimal mobility signs); however, cadavers were not transferred to humid chambers to assess conidial presence. Hence, the mortality measured during the entire test was due to the application of fungus/DE during the first bioassay; there were no re-isolation from the dead individuals. The same experimental protocol was repeated at 30-d intervals; thus, 6 bioassays were totally carried out, during the above-mentioned experimental period. The entire procedure was

2.3. DE formulation The DE formulation used in the experiments was Protect-It (Hedley Technologies Inc., Mississauga, Ontario, Canada) a DE that contains 83.7% SiO2 with 10% silica aerogel. This DE was used at a dose rate of 250 ppm (equivalent to 0.25 g/kg of wheat or maize). 2.4. Fungal formulations The M. anisopliae isolate used in the tests was strain Meta 1, obtained by Dr. Y.A. Batta [Laboratory of Plant Protection, Department of Plant Production and Protection, Faculty of Agriculture, An-Najah National University, P.O. Box 425 (Tulkarm), West Bank, Palestine, Via Israel]. This strain was first isolated from an infected individual of Harpalus caliginosus (F.) (Batta, 2003).

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replicated six times by preparing new grain lots each time (6  6 vials on each case).

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and 154.6722.3 and 29.777.7 on untreated maize, respectively. All statistical analysis (calculations and figures) were performed using the statistical package Statistica v.7.0 (StatSoft, Inc.).

2.7. Progeny production counts In each bioassay, after the last (14 d) mortality count, all adults (dead and alive) were removed from the vials, and the vials were left in the incubators for an additional period of 60 d. After the termination of this interval, the emerged adults in each vial were counted. 2.8. Data analysis The mortality counts were corrected as recommended by Abbott (1925). Before the analysis, mortality and progeny production data were arcsine and logarithmically [ln(x+1)] transformed, respectively. GLM-repeated measures Anova with two repeated factors (experimental period with 6 levels and insect exposure with 2 levels) was applied with mortality (or progeny with one repeated factor) as the response variable. The mortality counts of the 14-d exposure interval and the bioassays after treatment were subjected to non-linear regression analysis (Lane and Payne, 1997), and exponential or logistic equations were calculated where appropriate. The same procedure was followed in the case of the progeny production counts. Progeny production in the control vials was not included in this analysis, since a preliminary ANOVA revealed that, in all cases, the number of progeny in the control vials was significantly higher than the respective figures in the treated vials (Po0.01). The number of progeny/vial (adult7SE) in the control vials for S. oryzae and R. dominica were 96.5729.2 and 58.779.5 on untreated wheat Table 1 MANOVA parameters for mortality levels of S. oryzae adults (repeated factors: bioassay and exposure interval) Source of variation

df

F

P

Commodity Treatment Commodity  Treatment Bioassay Bioassay  Commodity Bioassay  Treatment Exposure interval Exposure interval  Commodity Exposure interval  Treatment Bioassay  Exposure interval

1 4 4 5 5 20 1 1 4 5

55.2 20.5 1.0 319.3 8.1 2.1 1516.5 34.5 2.3 7.5

o0.01 o0.01 0.40 o0.01 o0.01 o0.01 o0.01 o0.01 0.07 o0.01

3. Results 3.1. Mortality of S. oryzae All main effects were significant at the Po0.01 level (Table 1). Mortality in both grains tested and in all treatments decreased with the experimental period, and this decrease was best described by a logistic equation (Table 2). Mortalities on wheat, after 7 d of exposure, were higher with the combination of the higher fungal rate with DE (Fig. 1A). In this treatment, during the first two bioassays, mortality was 480%, and remained 470% until the 4th bioassay. However, mortality was o60% in the 5th bioassay and did not exceed 33.3% in the last bioassay. At the combination of the lower fungal rate with DE, mortalities ranged between 24.6% and 84.8%, in the first and last bioassay, respectively. The lowest mortality levels were recorded in the case of the wheat treated with the lower fungal rate alone, where mortality was o60% in the 1st bioassay, while o14% of the exposed adults were dead in the last bioassay. Finally, when DE was applied alone, mortality was 58.8% at the 1st bioassay, but remained 438% until the 5th bioassay. After 14 d of exposure, mortality increased further and, in the first two bioassays, reached almost 100% on wheat treated with both rates of fungus, in combination with DE (Fig. 1B). In these two treatments, from the 3rd bioassay and on, mortality was decreased gradually, but remained 480% until the 4th bioassay. Mortalities were 482% early in the experimental period for the higher fungal rate alone or DE alone, while o50% of the exposed adults were dead in the last bioassay. Mortality in treated maize, after 7 d of exposure, was lower than on wheat (Fig. 2A). The highest mortality levels were noted on maize treated with the fungal/DE combination, where both fungal rates gave similar levels of mortality. However, mortality did not exceed 73.5% even in the 1st bioassay. Moreover, in the last bioassay, mortality did not exceed 24% in any of the treatments tested. At the 14-d exposure interval, mortality was 479% on maize treated with the fungus/DE combination, at both fungal rates and remained at those levels until the 3rd bioassay (Fig. 2B). The lower mortality levels were noted on maize treated with the lower fungal rate alone, and DE alone. In these two treatments, o18% of the exposed adults were dead in the last bioassay.

Table 2 Logistic equations, Y ¼ 100/(1+Exp(b1  X+b2)), fitted to the data, for mortality of S. oryzae and R. dominica, for each commodity Days

Treatment

Wheat

Maize

b17s.e.

p (b1)

b27s.e.

p (b2)

R2

b17s.e.

p (b1)

b27s.e

p (b2)

R2

Sitophilus oryzae 14 DE alone 8  106 conidia/kg 8  108 conidia/kg 8  106 conidia/kg +DE 8  108 conidia/kg +DE

0.01470.002 0.01470.001 0.01970.001 0.02670.002 0.02570.001

0.001 0.000 0.000 0.000 0.000

2.40770.207 2.04470.143 3.41370.156 4.39170.367 4.80670.201

0.000 0.000 o0.001 0.000 o0.001

0.967 0.982 0.993 0.985 0.996

0.01370.002 0.01370.002 0.01270.002 0.01570.003 0.01170.002

0.004 0.006 0.007 0.007 0.013

1.01470.224 1.04870.256 1.60070.303 2.60170.426 2.07670.333

0.011 0.015 0.006 0.004 0.003

0.924 0.900 0.886 0.892 0.840

Rhyzopertha dominica 14 DE alone 8  106 conidia/kg 8  108 conidia/kg 8  106 conidia/kg +DE 8  108 conidia/kg +DE

0.00470.002 0.01470.003 0.01870.002 0.01970.003 0.01470.007

0.093 0.008 0.001 0.002 0.093

0.61770.196 2.63370.390 4.00470.336 4.52470.450 4.93971.048

0.035 0.003 0.000 0.001 0.009

0.550 0.899 0.971 0.959 0.702

0.00770.002 0.01170.002 0.01170.002 0.00570.001 0.01870.005

0.012 0.008 0.006 0.001 0.030

1.16470.210 1.62470.288 2.57070.286 2.40770.079 4.75470.867

0.005 0.005 0.001 o0.001 0.005

0.834 0.878 0.897 0.952 0.814

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(7d)

(14d)

S. oryzae mortality (%) on wheat

100

80

60

40 DE MA1 20

MA2 MA1+DE MA2+DE

0 1

2

3 4 Bioassay

5

6

2

1

3 4 Bioassay

5

6

Fig. 1. (A) Mean mortality of S. oryzae adults, exposed for 7 d on DE-treated wheat during a 5-month period and (B) mean mortality of S. oryzae adults, exposed for 14 d on DE-treated wheat during a 5-month period. (Abbreviations: DE ¼ 0.25 g/kg of wheat, MA1 ¼ 0.8  106 conidia/kg of wheat, MA2 ¼ 0.8  108 conidia/kg of wheat, MA1+DE ¼ 0.8  106 conidia/kg of wheat+0.25 g/kg of wheat).

(7d)

(14d)

S. oryzae mortality (%) on maize

100

80

60

40 DE MA1 20

MA2 MA1+DE MA2+DE

0

1

2

3 4 Bioassay

5

6

1

2

3 4 Bioassay

5

6

Fig. 2. (A) Mean mortality (7SE) of S. oryzae adults, exposed for 7 d on DE-treated maize during a 5-month period and (B) mean mortality of S. oryzae adults, exposed for 14 d on DE-treated maize during a 5-month period. (Abbreviations: DE ¼ 0.25 g/kg of maize, MA1 ¼ 0.8  106 conidia/kg of maize, MA2 ¼ 0.8  108 conidia/kg of maize, MA1+DE ¼ 0.8  106 conidia/kg of maize+0.25 g/kg of maize).

3.2. Mortality of R. dominica As in the case of S. oryzae, all main effects were significant (Table 3), while mortality was best described by a logistic model (Table 2). Generally, for both grains, mortality of the exposed R. dominica adults was higher than that of S. oryzae (Figs. 3 and 4). After 7 d of exposure on wheat, the greatest mortality occurred in the combination of the high fungal rate with the DE (Fig. 3A). In this case, 496% of the total number of the exposed adults were

dead during the first two bioassays, and remained close to 82% until the last bioassay. Although the initial mortality was 480% on wheat treated with the combination of the lower fungal rate with DE or the higher fungal rate alone, the decrease was more rapid and reached 59.1% and 46% in the last bioassay, respectively. Finally, the application of DE alone gave the lowest mortality levels ranging between 57.2% (for the 1st bioassay) and 20.9% (for the last bioassay) of the total number of the exposed individuals.

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3.3. Progeny production of S. oryzae

After 14 d of exposure, all adults were dead on wheat treated with the combination of the higher fungal rate with DE during the first bioassay (Fig. 4A). Moreover, in all fungal rates, with or without DE, mortality during the first two bioassays was 490%. In fact, mortality remained at those levels until the 4th bioassay in the case of the lower fungal rate with DE. In the case of DE alone, mortality increased on the 3rd and 4th bioassay, but then dropped dramatically at o45% in the last bioassay. Mortality of the exposed R. dominica adults was lower on treated maize than on wheat, but this difference was not as high as in the case of S. oryzae (Figs. 2A and 3B). After 7 d of exposure, the highest levels of mortality were noted in the case of the combination of the higher fungal rate with DE. After 14 d of exposure, mortality increased further compared with levels after 7 d (Fig. 4B). On maize treated with the higher fungal rate in combination with DE, mortality was 491% in the first five bioassays. For the combination of the higher fungal rate with DE, mortality was o92% in the first 3 bioassays, and 495% in the 4th bioassay. Finally, the lowest mortality was noted on maize treated with the lower M. anisopliae rate alone, which was o35.6% in the last bioassay.

All main effects were significant (Table 4), while the relation between progeny production counts and the testing period was best described by an exponential curve (Table 5). Generally, for both grains, progeny production increased gradually during the experimental period, but this increase varied according to the treatment. On wheat, progeny production in all treatments did not exceed 1 adult/vial during the first three bioassays, while for the combination of the higher fungal rate with DE it remained at that level during the entire experimental period (Fig. 5A). In the last bioassay, the highest offspring number was recorded on wheat treated with both rates of M. anisopliae alone. On maize, progeny production was higher than on wheat in most of the treatments. During the first three bioassays, p1 adult/ vial were found in al treatments, while for the combination of the higher fungal rate with DE progeny production remained at those levels during the entire experimental period (Fig. 5B). In the last bioassay, the highest offspring numbers were recorded in the case of maize treated with DE or with the lower fungal rate alone.

3.4. Progeny production of R. dominica As above, all main effects were significant (Table 6), and an exponential model described better the variation of progeny counts during the testing period (Table 5). As in the case of S. oryzae, progeny production of R. dominica increased with the experimental period (Fig. 6A). However, almost in all cases progeny production of R. dominica was lower than that of S. oryzae. On wheat, p1 adult/vial were found during the first five bioassays in all treatments, with the exception of the lower fungal rate alone in the 5th bioassay. On wheat treated with the higher fungal rate in combination with DE progeny production remained low (o1 adult/vial) during the entire experimental period, while no progeny was found in the first three bioassays. In the last bioassay, the highest progeny numbers were noted at the lower fungal rate alone.

Table 3 MANOVA parameters for mortality levels of R. dominica adults (repeated factors: bioassay and exposure interval) Source of variation

df

F

P

Commodity Treatment Commodity  Treatment Bioassay Bioassay  Commodity Bioassay  Treatment Exposure interval Exposure interval  Commodity Exposure interval  Treatment Bioassay  Exposure interval

1 4 4 5 5 20 1 1 4 5

10.8 34.1 0.8 155.2 6.9 2.4 1252.6 1.0 2.7 0.7

o0.01 o0.01 0.53 o0.01 o0.01 o0.01 o0.01 0.33 0.04 0.64

(14d)

(7d)

R. dominica mortality (%) on wheat

100

80

60

40 DE MA1 20

MA2 MA1+DE MA2+DE

0 1

2

4 3 Bioassay

5

6

1

2

4 3 Bioassay

5

6

Fig. 3. (A) Mean mortality (7SE) of R. dominica adults, exposed for 7 d on DE-treated wheat during a 5-month period and (B) mean mortality of S. oryzae adults, exposed for 14 d on DE-treated wheat during a 5-month period (abbreviations as in Fig. 1).

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(14d)

(7d)

R. dominica mortality (%) on maize

100

80

60

40 DE MA1 20

MA2 MA1+DE MA2+DE

0

1

2

3 4 Bioasay

5

6

1

2

3 4 Bioasay

5

6

Fig. 4. (A) Mean mortality (7SE) of R. dominica adults, exposed for 7 d on DE-treated maize during a 5-month period and (B) mean mortality of S. oryzae adults, exposed for 14 d on DE-treated maize during a 5-month period (abbreviations as in Fig. 2).

Table 4 MANOVA parameters for progeny production of S. oryzae adults (repeated factor: bioassay). Source of variation

df

F

P

Commodity Treatment Commodity  Treatment Bioassay Bioassay  Commodity Bioassay  Treatment

1 4 4 5 5 20

5.4 11.9 1.8 87.9 3.2 3.3

0.03 o0.01 0.15 o0.01 o0.01 o0.01

On maize, progeny production was higher than on wheat. During the first five bioassays, p1 adults/vial were found in all treatments, with the exception of DE alone in the 5th bioassay (Fig. 6B). As in the case of wheat, a complete progeny suppression was recorded during the first three bioassays on maize treated with the higher fungal rate in combination with DE, and remained low (p1 adults/vial) in all bioassays. In the last bioassay, the highest progeny number was recorded on maize treated with the lower fungal rate alone.

4. Discussion Previous studies indicated that the specific M. anisopliae strain could be used with success against several stored-grain pests. For instance, Kavallieratos et al. (2006) found that a dry conidial preparation of Meta 1 provided high mortality of S. oryzae adults while an aqueous preparation of the same strain was significantly less effective. Furthermore, the authors found that both aqueous and dry preparations were equally effective in the case of R. dominica adults. Based on the results of the present study, M. anisopliae was effective against these two species, but its effectiveness varied notably according to the exposure interval, dose rate and grain. Moreover, the simultaneous use of Protect-It increased the fungal efficacy at both rates examined. Lord (2005)

reported that the use of desiccant dusts increased the mortality of R. dominica larvae when applied with B. bassiana. Lord (2001) and Akbar et al. (2004) also found similar results for neonate R. dominica larvae and T. castaneum larvae, respectively. Vassilakos et al. (2006) observed, under certain circumstances, an additive effect when B. bassiana was used with the DE formulation SilicoSec in stored wheat against S. oryzae and R. dominica adults. However, in that study no synergism was recorded, indicating that this effect is manifested more easily in the case of beetle larvae. Furthermore, Athanassiou et al. (2006) found that this additive effect was not affected by the type of the DE formulation, suggesting that all DEs can be used for this purpose. However, the data available in the case of M. anisopliae were few and less clear than those obtained for B. bassiana. Batta (2004) reported that the addition of several inert materials, in a M. anisopliae dry conidial formulation increased the mortality of S. oryzae adults. However, Michalaki et al. (2006) found no synergism when testing M. anisopliae with SilicoSec against larvae of the confused flour beetle, Tribolium confusum Jacquelin du Val on wheat and flour. Further experimentation is required on the potential synergistic effect between M. anisopliae and DEs. Since different fungal strains vary remarkably on their virulence against different stored-grain pests (Moore et al., 2000; Stathers, 2002; Wakefield et al., 2002) the addition of an inert material that increases efficacy may be preferable than seeking and mass producing each time the strain of fungal pathogen that is most effective against a given pest. Apart from the increase in beetle mortality, in many cases the presence of DE in the fungus-treated grain increased the persistence of M. anisopliae during the experimental period. For instance, although the higher dose rate of M. anisopliae with or without DE gave approx. the same 14-d mortality levels (497%) on wheat for R. dominica in the first bioassay, in the last bioassay the combination gave 93.1% mortality while the corresponding level for the fungus alone was only 69.6%. This trend is evident at both fungal rates tested here, indicating that this effect of DE is not related to conidial concentration. As silicaceous materials, DEs react very little with the environment, and can persist for a long

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Table 5 Exponential equations, Y ¼ b1(Exp(b2  X), fitted to the data, for progeny production of S. oryzae and R. dominica for each commodity Days

Treatment

Wheat

Maize

b17s.e.

p (b1)

b27s.e.

p (b2)

R2

b17s.e.

p (b1)

b27s.e.

p (b2)

R2

Sitophilus oryzae 14 DE alone 8  106 conidia/kg 8  108 conidia/kg 8  106 conidia/kg +DE 8  108 conidia/kg +DE

0.004370.0026 0.049870.0234 0.060470.0250 0.012070.0054 0.018270.0124

0.1707 0.1004 0.0732 0.0904 0.2176

0.034670.0034 0.026870.0027 0.022770.0024 0.030370.0026 0.021170.0040

0.0005 0.0006 0.0007 0.0003 0.0063

0.9889 0.9845 0.9802 0.9906 0.9412

0.006370.0008 0.025870.0138 0.026070.0048 0.133170.0493 0.000270.0005

0.0016 0.1358 0.0054 0.0542 0.6726

0.042270.0007 0.035270.0030 0.029670.0010 0.016970.0022 0.046270.0123

o0.0001 0.0003 o0.0001 0.0016 0.0199

0.9997 0.9917 0.9984 0.9670 0.9428

Rhyzopertha dominica 14 DE alone 8  106 conidia/kg 8  108 conidia/kg 8  106 conidia/kg +DE 8  108 conidia/kg +DE

0.012570.0081 0.021470.0072 0.026570.0229 0.011770.0063 0.008970.0063

0.1993 0.0406 0.3116 0.1382 0.2332

0.024470.0038 0.026870.0019 0.019670.0051 0.026470.0031 0.025470.0041

0.0030 0.0002 0.0188 0.0011 0.0035

0.9670 0.9930 0.9004 0.9817 0.9656

0.010670.0069 0.002670.0022 0.005670.0021 0.090170.0751 0.020570.0149

0.2014 0.3078 0.0545 0.2967 0.2424

0.032470.0037 0.040770.0048 0.033170.0021 0.014070.0052 0.021970.0043

0.0010 0.0011 0.0001 0.0537 0.0069

0.9848 0.9864 0.9956 0.7854 0.9451

16 DE 14

MA1

S. oryzae mean reproduction (adults/vial)

MA2 12

MA1+DE MA2+DE

10 8 6 4 2 0 1

2

3 4 Bioassay

5

6

1

2

3 4 Bioassay

5

6

Fig. 5. Mean progeny production (adults per vial) of S. oryzae adults, 60 d after the removal of the parental S. oryzae adults, during a 5-month period on: (A) wheat and (B) maize (abbreviations as in Figs. 1 and 2).

Table 6 MANOVA parameters for progeny production of R. dominica adults (repeated factor: bioassay) Source of variation

df

F

P

Commodity Treatment Commodity  Treatment Bioassay Bioassay  Commodity Bioassay  Treatment

1 4 4 5 5 20

4.1 3.5 0.9 67.7 1.7 2.1

0.05 0.02 0.46 o0.01 0.15 o0.01

period in the DE-treated grain. Athanassiou et al. (2005) found that the DEs SilicoSec, PyriSec and Insecto could retain their insecticidal effect for 8–9 months after their application on wheat or barley against S. oryzae. Also, Stathers et al. (2008) showed the DE Protect-Its maintained its insecticidal efficacy for 20 months on farm-stored maize in the field in Tanzania. Similar results have also been reported by Vayias et al. (2006) for the same DEs on

wheat and maize against T. confusum. DEs gradually absorb moisture from the air, which is negatively related with their insecticidal effect (Subramanyam and Roesli, 2000). Persistence is also an important characteristic in the case of fungal pathogens (Thomas et al., 1995, 1997; Stathers, 2002), despite the fact that there are no data available for M. anisopliae in the case of stored products. Conidial viability is decreased with time (Moore et al., 2000; Batta, 2004), and this is an apparent explanation for the gradual decrease in efficacy against the two species tested here. Although the assessment of conidial viability during the experimental period might had helped the interpretation of the residual effect, previous studies clearly indicate that conidial germination is not always correlated with insecticidal efficacy. For instance, Batta (2004), by using the same M. anisopliae strain (Meta 1), found that, although conidial viability of M. anisopliae decreased with time the insecticidal effect against S. oryzae was not seriously affected. This trend has also been reported in the case of some field pests, such as the whitefly Bemisia tabaci (Gennadius) (Batta, 2003). Hence, conidial germination may be a poor indicator of

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5 DE R. dominica mean reproduction (adults/vial)

MA1 4

MA2 MA1+DE MA2+DE

3

2

1

0

1

2

3 4 Bioassay

5

6

1

2

3 4 Bioassay

5

6

Fig. 6. Mean progeny production (adults per vial) of R. dominica adults, 60 d after the removal of the parental S. oryzae adults, during a 5-month period on: (A) wheat and (B) maize (abbreviations as in Figs. 1 and 2).

insecticidal value, at least in the case of some fungal isolates. Moreover, fungal pathogens may be recycled in the cadavers, reintroducing more inoculum into the system (Thomas et al., 1995, 1997; Stathers, 2002). Hence, under field conditions in a given storage facility, this persistence is likely to be even more increased in comparison with the persistence levels reported here, given that insects existed only in the experimental vials. One additional factor that seems to play an important role in the conidial viability characteristics is the presence of grain. Lord (2005) found that the presence of grain had a certain detrimental effect on the germination of B. bassiana conidia, which indicates a possible metabolic kernel–conidia interaction. Consequently, germination estimation from conidia applied to grains may differ from initial conidial germination level. Rice and Cogburn (1999) using a B. bassiana strain, found that R. dominica was more susceptible than S. oryzae. In light of our findings, this is also evident in the case of M. anisopliae. This stands in accordance with data previously published by Batta (2004, 2005) and Kavallieratos et al. (2006) for the same fungal strain. Differences in cuticular composition and in behavioral responses in the treated grain may be responsible for this effect. Apart from mortality, the reduction of fungal efficacy with the increase of the storage season was lower for R. dominica than for S. oryzae. Conversely, mortality of R. dominica was lower than that of S. oryzae on wheat treated with DE only. It is well known that S. oryzae adults were more mobile than R. dominica adults, thus the possibility of picking up more DE particles is increased. Nevertheless, this difference is eliminated when DE was used with M. anisopliae. Both fungus and DE were generally more effective on wheat than on maize. This result is most evident with S. oryzae especially during the last bioassays. Previous studies document that the type of the grain is determinative of the efficacy of a given DE (Aldryhim, 1993; Subramanyam and Roesli, 2000; Athanassiou et al., 2003, 2004, 2005; Athanassiou and Kavallieratos, 2005; Kavallieratos et al., 2005). In a series of laboratory bioassays, Athanassiou et al. (2003) found that the efficacy of the DE SilicoSec against S. oryzae was significantly lower on maize than

on barley or rice. McGaughey (1972) also reported that the dose rate of DE required for 100% mortality of S. oryzae on rough rice, did not provide complete suppression on milled and brown rice. The author suggested that this could be attributed to the absorption of kernel lipids by the DE particles, or interaction of these particles with oils that exist on the external kernel part. Athanassiou and Kavallieratos (2005) and Kavallieratos et al. (2005) found that DE retention was considerably lower on maize than wheat. Since conidial powder was used in the present tests, it is likely that the lower fungal efficacy on maize might be attributed to decreased powder retention, as in the case of DEs. Furthermore, interactions of conidia with maize kernel lipids or oils may be responsible for these variations. Also, some species are able to develop, infest and oviposit better in certain grains than in others (i.e. better on maize than on wheat), and thus, increased survival may be chiefly influenced by this factor. For instance, Michalaki et al. (2006) found that survival of T. confusum larvae was higher on flour than on wheat treated with M. anisopliae and DE; the authors suggested that, the presence of flour might have reduced the contact of insects with DE or conidia or remove them from their body. Both fungal pathogens and silicaceous dusts are slow-acting insecticides (Golob, 1997; Moore et al., 2000). Consequently, insects may partially recover, move to a less treated layer of the grain mass, mate oviposit and continue to cause grain damage. Hence, the suppression of progeny production is equally important with parental mortality. The present results indicate that, in most cases, progeny production was not totally avoided, but was notably reduced at least during the first bioassays. The simultaneous presence of fungus and DE, at both rates and species tested, decreased progeny production in comparison with the application of fungus alone. This can be considered as a direct consequence of the increased and faster parental mortality caused by the combination treatments, rather than potential ovicidal effect. The present results suggest that M. anisopliae and DE can be effective against S. oryzae and R. dominica on both wheat and maize. Although no clear synergistic effect was found between

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these two substances, both substances are capable of long-term protection in stored grains. Practically, the simultaneous use of two low-toxicity naturally occurring substances that can persist on the product may have some certain advantages over the use of traditional grain protectants where residual effect is not desirable.

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