The effect of application time and soil factors on the ...

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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 15–23 www.elsevier.com/locate/yjipa

The effect of application time and soil factors on the occurrence of Beauveria brongniartii applied as a biological control agent in soil Philip Kessler,a,* Horst Matzke,b and Siegfried Kellera a

Swiss Federal Research Station for Agroecology and Agriculture, FAL Reckenholz, Reckenholzstrasse 191, CH-8046 Z€urich, Switzerland b Eric Schweizer Seeds Ltd., P.O. Box 150, CH-3602 Thun, Switzerland Received 3 September 2002; accepted 6 August 2003

Abstract The effects of abiotic and biotic soil factors on occurrence of the entomopathogenic fungus Beauveria brongniartii after application at different times of the year were examined in Switzerland. Applications made from May to August generally resulted in an increase of 1–5 103 CFU g1 dry soil compared to untreated control plots. Conversely, soils treated in October and November yielded no increase. Soil temperatures between 20 and 25 °C, and high clay content of the soil had a positive effect on the occurrence and density of B. brongniartii whereas increased catalase activity and temperatures above 27 °C had a negative influence. Laboratory experiments revealed that a higher number of CFUs developed after one month of incubation at 22 °C than at 12 °C. Differences were not detected after three months of incubation, indicating that growth rate was simply slower at sub-optimal temperatures. The increase was different in three native soils, but was not correlated with different clay contents of the soil. In sterilized soil, though, the differences were not detected, suggesting that biotic factors have a greater influence rather than soil texture. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Melolontha melolontha; Biological control; Occurrence; Soil temperature; Soil texture; Soil moisture; Biological soil activity; Time of application

1. Introduction Beauveria brongniartii (Sacc.) Petch (Deuteromycotina: Hyphomycetes) is an entomopathogenic fungus, which has been used for several years in Switzerland as a biological control agent (BCA) against the larvae of the European cockchafer, Melolontha melolontha L. (Coleoptera: Scarabaeidae). Melolontha melolontha has a three year life cycle and most of the damage is caused by the larvae during the first two years of the life cycle. They feed on the roots of numerous plant species and can cause heavy damages in grassland, arboriculture and vineyards. Since 1991, a product based on sterilized barley kernels colonized with B. brongniartii has been commercially available. Application of the BCA is achieved by tilling barley kernels overgrown with fungal mycelium into the soil. Following application the fungus

* Corresponding author. Fax: +41-1-377-72-01. E-mail address: philip.kessler@fal.admin.ch (P. Kessler).

0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.08.003

grows and sporulates on the kernels. As insect mortality is dose-related, large numbers of conidia have to be produced on the kernels to provide sufficient inoculum in order to initiate an epizootic and control the host population successfully. Keller et al. (2002) calculated, that for the control of M. melolontha an initial density of B. brongniartii of 103 –104 CFU g1 soil is necessary. In general treatments are successful, but under some circumstances they fail. Possible reasons for failure include late application during autumn, application in sandy soils or after inappropriate handling of the drill machine (Keller, 2000; Matzke, 2000). This indicates that climatic factors and soil factors might be implicated in the success or failure of the treatments. It is therefore important to understand the impact of abiotic and biotic soil factors on the growth and sporulation of B. brongniartii at sites where the BCA is applied. The patterns of growth are determined by soil characteristics such as changes of soil temperature and moisture, soil texture, pH, availability of nutrients, and interactions with soil microorganisms.

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P. Kessler et al. / Journal of Invertebrate Pathology 84 (2003) 15–23

The influence of some of these factors on fungal development and fate in soil has been studied in detail for several entomopathogenic hyphomycetes. This group of fungi grows at a wide range of temperature, but the optimal temperature for growth of most species lies between 20 and 30 °C (Fargues et al., 1997; Roberts and Campbell, 1977; Studdert and Kaya, 1990; Walstadt et al., 1970). Soil moisture is another important constraint on fungal growth and sporulation. Most entomopathogenic fungi require a relative humidity (RH) of 95–100% for growth (Ferron, 1977; Walstadt et al., 1970), whereas studies on water activity requirements (aw ) revealed values between 0.97 and 0.99 (Hallsworth and Magan, 1999; Humphreys et al., 1989). The soil water potential, which gives a more accurate measurement of the energy of water involved (Griffin, 1963), was evaluated to determine water requirements in the soil (Li and Holdom, 1993; Studdert and Kaya, 1990). Changes of pH values between 4 and 7 only slightly affect the growth of fungi, but a lower pH is generally more favorable for their development (Griffin, 1994; WeymanKaczmarkova and Pedziwilk, 2000). The objective of our study was to examine effects of abiotic factors, such as soil temperature and moisture, soil texture, pH and salt content, as well as biotic soil factors, such as organic matter and catalase activity, on the occurrence of B. brongniartii after the application of the BCA. We conducted field trials to assess the influence of these factors, following experiments in the laboratory, where the most crucial factors were studied under controlled conditions. These results are important in order to improve our knowledge of possible constraints to use of the BCA and to optimize application strategies.

2. Materials and methods 2.1. Fungus For both field trials and laboratory experiments we used the commercial product Beauveria-Schweizer from E. Schweizer Seeds Ltd. (Thun, Switzerland). The

product is based on sterilized barley kernels that are overgrown with B. brongniartii. For all experiments the strain FAL 546 (BIPESCO 4) was used. 2.2. Field trials Field trials were conducted at 8 different locations in Switzerland ranging from 47°320 N, 9°190 E in the northeast to 46°120 N, 7°170 E in the south. All sites were natural meadows with the exception of VS 3 and VS 4, which were orchards (Table 1). Treatment plots were set up at each location, each plot measuring 20 20 m2 , except at VS 1, where individual plot sizes measured 20 15 m2 ; and at VS 3 and VS 4 where plots corresponded to the tracks between the tree rows (measuring 4 40 m2 , 4 20 m2 , respectively). Different treatments were applied during 1999: BCA application in spring (May–June), summer (July–August) and autumn (October–November). Treatments were replicated in 4 plots at each study site. Applications were made by tilling 40– 50 kg ha1 of the BCA into the soil (to a depth of 5– 10 cm) using an adapted tilling machine. Four plots per study site were not treated to serve as controls. Soil samples were obtained using a soil core sampler with an internal diameter of 5.5 cm. Core samples were taken from a depth of 5 to 18–20 cm. On each sampling date we collected 10 samples per plot, each consisting of 2 soil cores. Samples were taken before application, and 1 month and 3 months after application. Each sample was stored in a plastic bag and held at 4 °C for no longer than 6 months before processing. The Galleria Bait Method (GBM) was applied to each soil sample to isolate B. brongniartii and document the presence of infective propagules in the soils (Zimmermann, 1986). Stones and roots were first removed from the soil; the soil was then sieved (5 mm mesh) and 60 g of each sample placed in a cylindrical plastic container (4.5 cm Ø, 6 cm high). Four late instar larvae of Galleria mellonella were then added to each container. Baiting was conducted in darkness at 22 °C. For the first five days the containers were turned daily to keep the larvae moving in the soil. After 18–21 days the larvae

Table 1 Description of soil characteristics at the experimental field sites in Switzerland Site

Region in Switzerland

Land use

Claya (%)

Sanda (%)

Organica (%)

pH

Salt (lSi)

Catalaseb (O2 /min)

TG 1 TG 2 TG 3 TG 4 UR VS 1 VS 3 VS 4

Northeast Northeast Northeast Northeast Central South South South

Meadow Meadow Meadow Meadow Meadow Meadow Orchard Orchard

19.7 11.0 12.7 42.5 29.0 14.8 3.4 32.2

40.4 54.5 56.6 20.5 31.7 22.2 57.5 23.0

3.8 2.8 3.4 8.1 9.0 2.1 1.5 4.9

7.0 7.6 6.7 6.3 6.4 7.7 7.5 7.3

239.4 251.3 242.8 259.9 194.0 625.8 217.1 226.4

2.80 1.58 3.03 6.08 4.30 1.79 0.98 3.20

a b

Dried at 40 °C. Measured after Beck (1971).


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Table 2 Soil water contenta and rainfallb at field sites following application of B. brongniartii in different seasons Site

Spring

Summer

Autumn

Water content (%) Rainfall (mm)



1999 Avrg TG 1 TG 2 TG 3 TG 4 UR VS 1 VS 3 VS 4

24.7 19.6 24.6 39.2

Ba Ca BCa Aab

364 319 428 407

306 306 306 306

16.7 Da 25.6 BCa

201 201

136 136

Overall

25.0 a

26.9 Ca 18.1 Da 19.2 CDa 36.5 Bb 35.8 Aa 21.0 CDa

26.2 a

1999

Avrg

229 190 232 170 362 198

296 296 296 296 400 151

26.8 22.6 23.2 40.8 35.7 20.0 17.5 25.5

BCa CDa DEa Aa Ba DEa Ea CDa

1999

Avrg

270 270 280 282 196 149 147 147

188 188 188 188 251 160 160 160

26.5 a

a

Mean of measured soil water contents (dried at 105 °C for 8 h) from 4 treatment plots over a 3 months sampling period. The values were subjected to a logarithmic transformation prior to an analysis of variance. Means followed by different uppercase letters in a column and by different lowercase letters in a row are significantly different (p < 0:05) according to Tukeys HSD test. b Data from the Swiss Meteorological Institute (SMI).

were examined and assigned to one of the following categories: ‘‘healthy,’’ ‘‘infected with Beauveria’’ or ‘‘death from other reasons,’’ Beauveria species were isolated and identified under the microscope. The abundance (frequency) of B. brongniartii in a trial plot was determined from the percentage of soil samples giving a positive result with the GBM. To assess the density of B. brongniartii in each plot, the number of colony forming units (CFU) per gram of dry soil were determined on a selective medium (SM). Soil samples taken within each plot were pooled, mixed and sieved (5 mm mesh). Twenty grams of the homogenized sample of fresh soil were added to a 200 ml Erlenmeyer flask containing 100 ml tap water plus 1.8 g L1 of tetraSodiumpyrophosphate (Na4 P2 O7 *10H2 0) to promote disaggregation of the soil (Fornallaz, 1992). All flasks were shaken for 3 h at 120 rpm on a longitudinal shaker. After shaking, flasks were allowed to stand for 15 s and a 0.1 ml sample of the suspension was spread over the surface of a SM using a Drigalsky spatula (Fornallaz, 1992). Five replicates per sample were prepared and incubated in the dark at 22 °C. After 8–10 days the number B. brongniartii CFUs, which have a typical appearance and stain the medium a red-brownish color, were counted. To express the number of CFU g1 dry soil, the moisture content of the soil samples was determined by drying 100 g of ÔwetÕ soil at 105 °C for 8 h and then re-weighing. The SM was adapted from that of Strasser et al. (1996): 10 g peptone from meat pancreatically digested, 20 g glucose, and 18 g agar were all dissolved in 1 liter of distilled water and autoclaved for 20 min at 120 °C. The medium was cooled to 60 °C and 0.6 g streptomycin, 0.05 g tetracycline, and 0.05 g cyclohexamide (previously dissolved in 20 ml sterile distilled water) and 0.1 ml Dodine (AS: 490 g L1 ) were added. Soil factors such as moisture content, pH, clay content, sand, organic material, and salt were analyzed by

Eric Schweizer Seeds Ltd. (Table 1). Catalase activity was measured to assess microbiological activity in the soil (Beck, 1971). Soil temperature in the field plots was measured every hour for three months after application either by temperature probes directly placed at the study sites to a depth of 10 cm (HOTDOG DT1, ELPROBUCHS AG, CH-9471 Buchs, Switzerland) or by a neighboring meteorological station of the Swiss Meteorological Institute (SMI). Daily rainfall was measured by the SMI stations. 2.3. Laboratory experiments Soils were selected from three meadows in the northeast region of Switzerland (TG 1, TG 3, and TG 4). The soil originating from TG 1 was characterized as a sandy loam (19% clay and 40% sand), and the soil originating from TG 3 was characterized as a loamy sand (12% clay and 57% sand). Both of these soil samples had a low content of organic matter. The third soil type originating from TG 4 was described as a loamy clay (42% clay, 23% sand) with a high content of organic material (8%) (Table 1). The soils were sampled in May 2001 and sieved (5 mm mesh) prior to use. One experiment was performed using native (non-sterilized) soils and a second experiment was performed with soils that had been autoclaved twice for 20 min at 120 °C. Fifty grams of each soil type were added to a cylindrical plastic container (4.5 cm Ø, 6 cm high). Three B. brongniartii kernels were placed in each container to a depth of 2 cm. Five replicate containers were prepared for each soil type and test temperature. The containers were held in a larger plastic box together with a container filled with water to ensure high ambient humidity. Boxes were incubated in the dark at 12 or 22 °C and at 80% RH for 1 and 3 months, respectively. After incubation, the entire content of each container was transferred into a

)1 a )10 a 1344 b 1429 b

1305 Bb 4376 Bb 145 896

Mean of maximal increase in the number of colony forming units from four treatment plots. The values were subjected to a logarithmic transformation prior to an analysis of variance. Means followed by different uppercase letters in a column and by different lowercase letters in a row are significantly different (p < 0:05) according to Tukeys HSD test.

—

—

—

—

—

—

0 33

1305 4409 Total

a

0 218 0 0 8 0 9 0 0 292 84 0 68 0 3 0 0 420 10 0 29 0 0 0 0 Aa )75 Aab )140 Aa 0 Aa 73 Aa 13 Aa 3 Aa 47 Aa 0 199 0 0 81 13 3 50 —

—

0 275 140 0 8 0 0 3 ABa Bb ABa Aa Cb Cb 48 846 33 0 2245 4892 0 1126 17 0 529 4764 48 282 39 0 2254 4892 0 280 6 0 8 0 74 Aa 2874 Bc )57 Aa 0 Aa 74 2858 10 0 0 3681 152 0 0 808 209 0


Autumn Summer

No application

Spring 3 months Maximal after increasea 1 month after

Application in autumn

Before 3 months Maximal after increasea 1 month after Before

Application in summer

Maximal increasea

We detected native populations of B. brongniartii at TG 1, TG 2, TG 3, UR, and VS 4. The density of B. brongniartii ranged from 3 to 808 CFU g1 dry soil, and the occurrence ranged between 5 and 40% of the soil samples sampled before application of the fungus (Tables 3 and 4). During the three months following the application of the BCA, we detected differences of the density of B. brongniartii between trial sites as well as between seasonal application time points. Overall, the density in the plots treated in spring and summer increased on average by 1.4 103 CFU g1 and 1.3 103 CFU g1 dry soil, respectively; and the frequencies of soil samples with infected Galleria mellonella increased by 19 and 21%, respectively (Tables 3 and 4). The application in autumn did not result in a detectable increase in B. brongniartii density in the soil of the isolation methods. After the spring and summer application the density increased significantly at TG 2, UR, VS 1, VS 3, and VS 4 compared to the control, whereas at TG 1, TG 3, and TG 4 application in the

3 months after

3.1. Field trials

1 month after

3. Results

Before

The abundance (frequency) of B. brongniartii was calculated by the percentage of soil samples per plot from which the fungus was isolated using the GBM. The density of B. brongniartii was calculated using the mean of the number of CFU counted per gram of soil. The dataset on CFU g1 dry soil and frequency of B. brongniartii was transformed by natural log function prior to analysis using a one-way ANOVA. Significant contrasts were analyzed using the Tukey HSD test. A multiple regression analysis was performed to obtain several models to assess the influence of soil factors on the increase in CFU g1 dry soil of B. brongniartii after application. Independent variables with non-significant partial regression coefficients were eliminated by a stepwise backward elimination procedure. Simplified regression models were verified by residual analyses. All data analyses were performed using the software package STATISTICA 5.5 (Statsoft, 1999).

Table 3 Beauveria brongniartii density (CFU g1 dry soil) before application, one month and three months after application at different locations and in different seasons

2.4. Data analysis

Maximal increasea

300 ml Erlenmeyer flask and 100 ml of tap water with 0.1% Tween 80 added (Fornallaz, 1992). The flasks were shaken on a horizontal shaker at 110 rpm for 3 h. A 1 ml sample of each suspension was diluted 1:1000 for the native soil and 1:10000 for the sterile soil, and 0.1 ml of each dilution was plated onto a selective medium (Sabouraud-2-Glucose-agar, Strasser et al., 1996). Five replicate dishes were prepared and incubated at 22 °C. After 10 days the number of CFUs/plate was counted.

0 Aa )128 Aa 74 Aa 0 Aa 39 Aa 0 Aa 9 Aa 0 Aa


Location Application in spring

18

Table 4 Abundance of B. brongniartii (percentage of positive soil samples per plot) before application, one month and three months after application at different locations and in different seasons revealed by the Galleria bait method Location Application in spring

Total

Application in autumn

No application Spring

Before

1 month after

3 months Maximal after increasea

Before

1 month after


Before

1 month after


0.0 15.0 0.0 0.0

10.0 42.5 0.0 0.0

10.0 47.5 2.5 0.0

5.0 32.5 5.0 0.0. 7.5 0.0

15.0 35.0 5.0 27.5 22.5 12.5

15.0 50.0 2.5 12.5 42.5 40.0

5.0 40.0 12.5 0.0 0.0 0.0 n.d. 0.0

—

5.0 30.0 2.5 2.5 10.0 0.0 n.d. 0.0

0.0 0.0

15.0 15.0

20.0 50.0

10.0 32.5 2.5 0.0

Aa ABb Aa Aa

10.0 ABa 17.5 ABa )2.5 Aa 27.5 ABb 35.0 Bb 40.0 Bb

20.0 ABa 50.0 Bb 19.2 b

21.3 b

— — — — — — —

0.0 Aa 5.0 )10.0 Aa 25.0 )10.0 Aa 7.5 2.5 Aa 0.0 10.0 Aa 5.0 0.0 Aa 0.0 n.d. 0.0 0.0 Aa 0.0 )1.1 a

Summer

Autumn

Maximal increasea

5.0 22.5 0.0 0.0 7.5 0.0 0.0 0.0

5.0 12.5 2.5 5.0 7.5 0.0 0.0 0.0

0.0 )2.5 )5.0 5.0 2.5 0.0 0.0 0.0

Aa Aab Aa Aa Aa Aa Aa Aa

0.0 a

a

Mean of changes in the percentage of soil samples from four treatment plots from which B. brongniartii was isolated using the Galleria bait method. Values were subjected to a logarithmic transformation prior to an analysis of variance. Means followed by different uppercase letters in a column and by different lowercase letters in a row are significantly different (p < 0:05) according to Tukeys HSD test.

19

same seasons did not result in any significant density increase. Accordingly, the application in summer and spring at TG 2, UR, VS 1, VS 3, and VS 4 significantly increased the abundance of B. brongniartii (with the exception at TG 2 in the summer) compared to the control. In contrast to the CFU method at TG 4 we detected significantly more B. brongniartii in the soil after using the GBM after the summer application. But overall, the results of both re-isolation methods were consistent. Data on soil temperature over the trial period are presented in Fig. 1. The number of hours with measured temperatures between 20 and 25 °C ranged between 483 and 774 h after the spring application, and between 174

Fig. 1. Average temperature and mean number of hours recorded at different temperature ranges measured at different locations over three months following the application of B. brongniartii at different times of the year.



Application in summer

20


and 843 h after the summer application. After the spring application the soil temperature was >27 °C for 45–94 h at the trial sites in the northeast region of Switzerland (TG 1–4), but only for 0 and 4 h at the trial sites in the Valais. After the summer application the soil temperature was >27 °C for 16–37 h at the trial sites in TG 2, TG 3, and UR. During the same period soil temperatures also fell below 10 °C for more than 500 h at trial sites in TG 1 and TG 4. During the three months following the autumn application, soil temperatures were predominantly below 10 °C and never higher than 20 °C at any of the locations. The spring of 1999 in Switzerland was characterized by heavier-than-average rainfall (Table 2) that produced waterlogged soils. As a result, at site TG 4, large parts of the experimental site were flooded for more than 2 weeks. While there was great variation in soil water content among the 8 locations (16.7–40.8%), no seasonal changes within a particular location were detected except at TG 4 (Table 2). Using a multiple regression analysis, we defined models using a minimal number of soil and climatic factors to explain the variance in the measured growth data. A first model was based on data obtained from the SM (R2 ¼ 0:61; F ¼ 7:77; p < 0:005). The variance was described with the factors ‘‘optimal temperature’’ (number of hours between 20 and 25 °C) (b ¼ 0:48; p < 0:05), ‘‘catalase activity’’ (b ¼ 1:20; p < 0:05) and ‘‘clay content’’ (1.00, p < 0:05). The second model was based on the data from the GBM (R2 ¼ 0:68; F ¼ 7:66; p < 0:005) and included the factors ‘‘optimal temperature’’ (b ¼ 0:87; p < 0:05), ‘‘catalase activity’’ (b ¼ 0:52; p > 0:05!), ‘‘clay content’’ (b ¼ 0:70; p < 0:1) and the ‘‘number of hours with temperatures >27 °C’’ (b ¼ 0:39; p < 0:1). The second model was simplified further and reduced to two factors: ‘‘optimal temperature’’ (b ¼ 0:97; p < 0:05) and ‘‘temperature

>27 °C’’ (b ¼ 0:56; p < 0:05) (R2 ¼ 0:60; F ¼ 11:99; p < 0:0005). Optimal soil temperature was a major factor in all models and the only factor that showed direct correlation with the growth variables obtained with both isolation methods (GBM: r ¼ 0:65; p < 0:05; SM: r ¼ 0:53; p < 0:05, Spearmans R correlation). 3.2. Laboratory experiment 3.2.1. Effect of temperature on occurrence (Table 5) Growth of B. brongniartii was significantly higher at 22 °C than at 12 °C [F ðdf ¼ 1; n ¼ 115Þ ¼ 39:23; p < 0:0005]. In native soils after 1 month of incubation at 12 °C an average of 2.3 105 CFU g1- soil was produced, whereas at 22 °C an average of 8.6 105 CFU g1 soil was produced. Two months later, the average number of CFU increased to 6.2 105 CFU g1 soil at 12 °C and up to 1.4 106 CFU g1 soil at 22 °C. The difference in soil CFUs at the two test temperatures was greater after one month than after three months of incubation [F ð1; 47Þ ¼ 4:97; p < 0:03]. The growth of B. brongniartii was significantly greater in sterile soil than in native (non-sterile) soil at both incubation temperatures [F ð1; 92Þ ¼ 463:07; p < 0:0005]. After 1 month at 12 °C 1.1 106 CFU g1 soil was produced, and 3.4 107 CFU g1 soil at 22 °C. After 3 months the number of CFUs g1 soil of B. brongniartii was 3.3 106 g1 soil at 12 °C and 2.2 107 at 22 °C. As with the native soils, the difference in CFUs g)1 soil at the two test temperatures was significantly greater after 1 month than after 3 months [F ð1; 46Þ ¼ 32:20; p < 0:00005]. 3.2.2. Occurrence in different soils There were significant difference in fungal growth in the three different native soils [F ð2; 47Þ ¼ 5:28; p < 0:01]. After 1 month, the highest density was

Table 5 The effect of soil type, incubation temperature and sterilization on the number of B. brongniartii CFU g1 soila 12 °C

22 °C 1

Numbers of CFU g

Numbers of CFU g1 soil

soil

After 1 month

After 3 months

After 1 month

After 3 months

Native TG 3 (sand) TG 1 (loam) TG 4 (clay)

1.3 105 Aa 1.5 105 Aa 4.0 105 ABa

8.8 105 ABb 4.2 105 Abc 5.4 105 Aa

1.3 106 Bb 3.9 105 Aab 8.7 105 ABa

1.9 106 Ab 1.3 106 Ac 1.0 106 Aa

Average

2.3 105 a

6.2 105 b

8.6 105 b

1.4 106 b

Sterile TG 3 (sand) TG 1 (loam) TG 4 (clay)

6.7 105 BCa 9.5 105 Ca 1.5 106 Ca

2.8 106 BCb 3.8 106 Cb 3.4 106 Ca

3.8 107 Cc 3.5 107 Cc 2.9 107 Cb

2.7 107 Bc 1.7 107 Bc 2.0 107 Bb

1.1 106 a

3.3 106 b

3.4 107 c

2.2 107 c

Average a

1

Mean of CFU g dry soil counted on SM. CFU values were subjected to a logarithmic transformation prior to an analysis of variance. Means followed by different uppercase letters in a column and by different lowercase letters in a row are significantly different (p < 0:05) according to Tukeys HSD test.


obtained in the TG 4 soil (loamy clay) (8.7 105 CFU g1 soil), whereas the maximum was reached after 3 month in TG 3 (loamy sand) (1.9 106 g1 soil). The occurrence of B. brongniartii could not be directly correlated with clay content in the soil. However, there was a significant increase of the numbers of CFUs from month 1 to 3 in soils with less organic matter (TG 1 and TG 3), but this did not occur in the soil with a higher content of organic material (TG 4). These differences were not observed when the experiments were done in sterilized soil, with the exception of TG 3 and TG 1 at 12 °C [F ð2; 46Þ ¼ 0:37; p > 0:69] (Table 5).

4. Discussion In most cases there was a significant increase in the number of CFUs of B. brongniartii in the soil following application of the BCA in the spring and summer. Generally, we can expect an average increase of 1– 5 103 CFU g1 dry soil three months after application of the ‘‘Beauveria-Schweizer’’ product (40 kg ha1 ). In contrast, applications in autumn did not increase the fungus density in the soil. This highly significant seasonal effect suggests that there is a strong influence of climatic factors such as soil temperature and humidity, on growth and establishment of the fungus in the soil. 4.1. Influence of soil temperature Temperature is known to be one of the most important factors influencing the development of an organism. On Sabouraud-dextrose-agar as well as on barley kernels, B. brongniartii is able to vegetatively grow at temperatures between 2 and 33 °C, to germinate between 2 and 27 °C and to sporulate between 5 and 28 °C. The optimal growth temperature for B. brongniartii lies between 20 and 25 °C (Aregger-Zavadil, 1992). Our observations in the field indicated a correlation between the number of hours at which the soil temperature was between 20 and 25 °C, and the number of CFUs produced in the soil. These optimal temperatures were never achieved after the application of the BCA in the autumn, indicating that soil temperature is a major factor affecting the success or failure in the production and establishment of fungal inoculum for control of Melolontha. Our laboratory study further strengthen this hypothesis by showing there was a reduced fungal growth in soils at 12 °C compared to 22 °C. Since we observed this effect in native soil as well as in sterile soil, we conclude that temperature has a direct influence on the growth of B. brongniartii and is not an indirect consequence of temperature effects on potentially antagonistic microbial populations in the soil. The same conclusion was made for the effect of temperature on

21

CFUs of B. bassiana on Spodoptera pupal cadavers (Studdert and Kaya, 1990). The difference in CFUs recovered from soils incubated at 12 vs. 22 °C decreased over time, suggesting that although sub-optimal temperatures will slow down the growth rate of the fungus, they ultimately do not decrease the level of inoculum that can potentially be attained in the soil; this has been demonstrated for B. brongniartii (Aregger-Zavadil, 1992) as well as for other hyphomycetous fungi (Fargues et al., 1992; Ferron, 1978; Studdert and Kaya, 1990). Cool temperatures in the autumn probably inhibit the development of B. brongniartii, going into a period when the temperatures are even more unfavorable to growth and sporulation (27 °C have a lethal effect on the spores (Aregger-Zavadil, 1992). This corresponds to our model where we showed a negative effect of the number of hours at temperatures above 27 °C on the frequency of infection of G. mellonella from treated soil samples. The failure of the spring applications at TG 1 and TG 3 may be attributed to the extended periods that these soil were exposed to temperatures >27 °C in the 3 months after treatment. In contrast, the density of B. brongniartii increased in the two orchards (VS 3 and VS 4), where soil temperatures never reached 27 °C after the spring application. The treated areas are shaded by the surrounding fruit trees and not directly exposed to the sun, unlike an area of open grassland. 4.2. Influence of soil moisture and rainfall Soil moisture is another important factor affecting the development of microorganisms. Most entomopathogene fungi need 95–100% RH for optimal development (Hallsworth and Magan, 1999; Luz and Fargues, 1997, 1998). As RH values in soil range around 99%, even at the permanent wilting point, and can drop under 95% only in the uppermost layers, conditions are generally suitable for fungal development (Griffin, 1963). Another variable that might influence fungal growth is the water content of the soils. As no seasonal changes in soil water content within a particular trial site were detected (with the exception in TG 4), we conclude that soil water content is not a major factor influencing growth and establishment of the fungus. Neither water content nor rainfall were considered as crucial factors in our proposed regression

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models. In fact, water content has been reported to have no direct effect on fungal growth (Griffin, 1969) and no major influence on growth and sporulation of B. brongniartii in native soil (Aregger-Zavadil, 1992), or insect infection (Ferron, 1967). Generally, the humidity conditions should be favorable for fungal growth, as long as the application slits in the soil are properly closed and the fungus colonized barley kernels are completely embedded in the soil after treatment. The weather conditions in the spring of 1999 were characterized by heavy rainfall, which lead to waterlogged soils. Since it has been suggested that the development of B. brongniartii is inhibited in very wet and heavy soil as a result of an oxygen deficiency (M€ ullerK€ ogler, 1965), we assume that the failure of the spring application at TG 4 was a result of poor soil aeration due to the flooding that occurred 2 weeks after treatment. 4.3. Influence of pH Soils with extreme pH values are known to be highly suppressive to several plant diseases, whereas soil microbiota are only slightly affected at intermediate pH values (Alabouvette et al., 1992; Griffin, 1994). As the pH values at the field trial sites ranged between 6.3 and 7.6, the growth of B. brongniartii was not expected to be adversely affected. Indeed, no correlation was found between soil pH and growth of B. brongniartii. The effect of low soil pH on fungal growth, as exists, for example, in forest soils (pH