Entomopathogenic fungi in soils from Alicante province

We have used Galleria mellonella (Linnaeus, 1758) larvae as a bait for detecting insect pathogens in soils from Alicante (SE Spain). Soil from 61 sites was collected including agricultural fields, forests and a mediterranean shrub ( Nerium oleander L.) growing under natural or garden environments. The most frequently insect pathogens found were fungi (32.8% soils), being Beauveria bassiana (Bals.) Vuill (21% soils) the species most abundant. Metarhizium anisopliae (Metschn.) Sorok (6.4%) and Lecanicillium lecanii (Zimm.) Gams [= Verticillium lecanii Zimm.] (4.8%) were less frequent. B. bassiana also scored the highest infection percentage in a single soil sample (ca. 90% of insects infected), and was also the most frequent (77.8%) entomopathogenic fungus detected in soils under N. oleander .

It is also interesting to compare the presence of microbial antagonists in soil environments with (agricultural soils) or without («natural» soils) human impact. A recent study of microbial entomopathogens of insect pests of apple and pear crops showed a wide variety of entomopathogenic viruses, bacteria, fungi and nematodes from soils of northern and central Europe (Cross et al., 1999). Bidochka et al. (1998) evaluated the presence in soils from Canada of cosmopolitan species of entomopathogenic fungi, such as Metarhizium anisopliae (Metschn.) Sorok. and Beauveria bassiana (Bals.) Vuill., more abundant in agricultural, and natural soils, respectively.
In the present study we have analysed the presence of entomopathogens, mainly fungi, in soils from different ecosystems of Alicante province such as forests, crop fields, natural and garden areas.

Soil sampling
Agricultural, forest, dry river beds and garden soils were sampled in the province of Alicante (SE Spain) to survey microbial antagonists (mainly fungi) of insects (Table 1). We collected 61 samples selected to represent soil and climate variability: 12 from agricultural soils (Prunus amygdalus Batsch., Citrus sp., and other woody crops), 8 from forest soils (Pinus halepensis Mill. and Quercus rotundifolia Lam.), the rest under plants of Nerium oleander L., 20 from natural populations (mostly in dry river beds) and 21 from gardens.
For each sampling site, soil (1.5-2 kg) was collected from 3 points randomly selected and mixed to make an homogeneous sample. Then they were collected from 0-20 cm depth, removing for forest soils the leaflitter layer. Soil samples were homogeneized in the laboratory, 2 mm sieved and stored at 4ºC in the dark before use (less than 12 months).

Physico-chemical analysis of soils
Before analysis, soils were spread on a tray. Soil aggregates were broken by hand or using a hammer. Trays with soil were kept open until soil moisture was equilibrated with that of the laboratory.
Soil texture, pH, conductivity/salinity and carbonate content were determined for all soils collected. Protocols used were those of Anonymous (1979). Three measurements were taken per each physicochemical parameter and soil sample.

Antagonist detection. Isolation of entomopathogenic fungi
Insect antagonists in soil were detected using G. mellonella larvae (from Carolina Biological Supply Co., NC, USA, 27215-3398). Forty grams of soil (freshly collected or 4ºC stored for less than 12 months) were 1 mm sieved and placed in 90 mm diameter sterile Petri dishes (3 replicates per soil). Ten living L 3 -L 4 G. mellonella larvae were buried per plate. Plates were sealed with Paraf ilm (American National Can.) and incubated at 25ºC for 15 days in the dark. After that time the insects were recoved from soil using a dissecting microscope at 40x magnification. Recovered insects were surface sterilised using 1% sodium hypochlorite for 1 min. The sterilizing agent was eliminated and insects were washed three times in sterile distilled water (5 min each time). Insects were finally blotted dry onto sterile filter paper and placed in moist chambers for 1 week at 25ºC in the dark. These conditions usually allowed development (and often sporulation) of insect antagonists (mostly entomopathogenic fungi).
Larvae with cottony f ilaments (probably fungus mycelium) were then observed with a dissecting microscope. Larvae with few hyphae on the surface were assumed to be colonized by fungal saprotrophs. On the contrary, larvae displaying abundant mycelium (i.e. in intersegmental regions) were considered infected. These larvae were plated on corn meal agar (CMA) containing 50 µg ml -1 penicillin, 50 µg ml -1 streptomycin, 50 µg ml -1 rose bengal and 1 mg ml -1 Triton X-100. Slide cultures (Gams et al., 1998) of fungi infecting larvae were carried out and after microscopic observation, genus was assigned according to Domsch et al. (1993). Species identification was determined using specialised bibliography including Lacey (1997). Entomopathogenic fungi were inoculated on CMA plates and then transferred to 10 ml universal bottles with CMA slopes and after 7-15 days under normal incubation, they were stored at 4ºC in the dark. Conidia and conidiophores of each fungal strain were measured (10 estimations per strain and structure). The size of the fun- gal structures was estimated as the average of the measurements taken.

Data analysis
Statistics were applied to analyse differences with respect to the isolation of entomopathogenic fungi in the environments surveyed. For soils, when checking the homogeneity of variances Levene's statistic, we found that they were non homogeneous within the four populations (agricultural, forest and natural or gardens N. oleander soils) tested. We could not then apply an ANOVA test, since the four populations did not show internal variations. The alternative was to apply a Kruskall-Wallis test, used when differences are non-significant, and we carried out an analysis of possible interpopulation differences. Table 1 indicates physico-chemical characteristics of soil samples. Most soils collected displayed in water a basic pH (more than 8). Conductivity was moderate for most soils (over 100 meq l -1 ). However some soil samples taken in Nerium oleander sites had a high conductivity (more than 600 meq l -1 ). The highest value of conductivity (over 1000 meq l -1 ) was recorded from a lemon tree field. Carbonate content was also moderate (40-60%) for most soils and high (100%)

Presence of entomopathogenic fungi in soils
As already described, G. mellonella larvae were used as bait for microbial antagonists of insects in the soils collected. At 32.8% of soils (20 out of 61), entomopathogenic fungi were the most frequent insect pathogens. Results of isolations of fungal strains and the morphological data for conidia and phialides (values are averages for 10 measurements carried out at random) are recorded in Table 2.
A higher number of forest soils analysed (62.5%) displayed entomopathogenic fungi respect to agricultural soils (50%). In the case of soils under N. oleander, this number was higher in soils under natural vegetation (30%) than in those from gardens (14%).
In accordance to this, B. bassiana was mostly isolated from forest soils, and least from garden soils. On the contrary, M. anisopliae was mainly found in agricultural soils, and not found under N. oleander. L. lecanii was equally found in N. oleander soils (both natural and from gardens) and agricultural soils, but not in forest soils. We found L. psalliote in only one forest soil, but with a high (>80%) infection percentage.
Apart from fungal entomopathogens, we also detected colonisation of G. mellonella larvae by common soil fungi (Zygomycetes and Deuteromycetes or Mi- tosporic fungi). The most common were Mortierella sp., Rhizopus sp., Penicillium sp. and Aspergillus sp., although other genera such as Fusarium sp. were also found.

Discussion
In our study we have isolated entomopathogenic fungi from several soil environments, including garden soils with a large antropic influence, although less fungi were recovered from these. This indicates that entomopathogenic fungi can be naturally found from environments close to host plants (either cultivated or growing naturally) that harbour phytophagous insects.
We have not found significative differences in the physico-chemical conditions among soils sampled in our survey. This seems logical since most of soils sampled are basic with a high carbonate content. We have found several species of entomopathogenic fungi, although the most common was B. bassiana. Tarasco et al. (1997) found B. bassiana and M. anisopliae to be the most abundant entomopathogenic fungi in Southern Italy. However, they never recorded simultaneously two species in the same soil sample. In Canadian soils, the most frequent species were also M. anisopliae and B. bassiana (Bidochka et al., 1998). The latter was majority in soils from colder areas. These soils also had larvae infected with Paecilomyces spp.
Diverse factors may affect survival of entomopathogenic fungi in soil. Lingg and Donaldson (1981) found that survival of B. bassiana conidia is dependent on temperature and soil water content. Raid and Cherry (1992) reported that M. anisopliae conidia were pathogenic on the sugar cane pest Lygirus subtropicus under a wide range of soil temperatures and moistures. Moreover, in general terms, M. anisopliae is able to resist in soil longer than B. bassiana, because the latter seems more sensitive to soil microbiota (Bidochka et al., 1998).
We have found entomopathogenic fungi from nearly 15% of the N. oleander soils tested. Natural N. oleander populations develop under mediterranean conditions in streams with very irregular water supply, therefore with poor soils, little prof iled and with low organic matter. The presence of entomopathogenic fungi in those soils shows that these antagonists are able to cope with high environmental stress.
Our study confirms that soils are an important reservoir of strains of entomopathogenic fungi, poten-tial antagonists for controlling insect pests. The strains isolated in this study are being further tested for development as biocontrol agents.