Entomopathogenic Fungi in Soils from Alicante Province L
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Spanish Journal of Agricultural Research (2003) 1 (3), 37-45 Entomopathogenic fungi in soils from Alicante province L. Asensio*1, T. Carbonell1, J. A. López-Jiménez2 and L. V. Lopez-Llorca1 1 Laboratorio de Fitopatología. Departamento de Ciencias Ambientales y Recursos Naturales. Facultad de Ciencias. Universidad de Alicante. Apdo. 99. 03080 Alicante. Spain 2 Departamento de Investigación y Desarrollo. Jardinería Huerto del Cura, S. A. Prolongación de Curtidores, s/n. 03203 Elx (Alicante). Spain Abstract We have used Galleria mellonella (Linnaeus, 1758) larvae as a bait for detecting insect pathogens in soils from Ali- cante (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 we- re 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. Key words: Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii, Galleria mellonella, biocontrol. Resumen Hongos entomopatógenos de suelos de la provincia de Alicante Hemos utilizado larvas de Galleria mellonella (Linnaeus, 1758) como cebo para detectar patógenos de insectos en suelos de Alicante (SE España). Se recogieron muestras de suelo de 61 localidades incluyendo campos agrícolas, bos- ques y matorral mediterráneo (Nerium oleander L.). Estas últimas muestras se tomaron tanto de jardines como de for- maciones de vegetación natural. Los patógenos de insectos más frecuentes (46,8% de los suelos) fueron los hongos, siendo Beauveria bassiana (Bals.) Vuill (21% de los suelos) la especie más abundante. También se encontraron Me- tarhizium anisopliae (Metschn.) Sorok (6,4%) y Lecanicillium lecanii (Zimm.) Gams [= Verticillium lecanii Zimm.] (4,8%), aunque con menor frecuencia. B. bassiana fue también el hongo con mayor porcentaje de infección en una muestra de suelo (90% de insectos infectados), y el más frecuentemente detectado (77,8%) en suelos bajo N. oleander. Palabras clave: Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii, Galleria mellonella, control biológico. Introduction soils containing entomopathogens. Piralid lepidopte- ran Galleria mellonella L. larvae are a suitable bait For most soils, fungi (either saprotrophs, mycor- for detection of insect antagonists (mostly microbial) rhizal or pathogens) are the main component of its mi- present in soil samples (Zimmermann, 1986). Expe- crobiota (Gams, 1992). Soil is also the main source of riments carried out using these larvae showed that fungal entomopathogens, and isolation of these orga- their mobility depended on soil structure and particle nisms involves soil sampling since that is their natu- size (Zimmermann, 1986), factors that have been ral habitat. shown to influence infection of insects by microbial Soil factors (temperature, pH or organic content, entomopathogens (Vänninen, 1995). relative moisture or mineral, organic or biotic com- In the so-called «suppressive soils», without ta- ponents) can affect fungal persistence and activity king any control measures, plant diseases are not eco- (Riba et al., 1991; Charnley, 1997). It is therefore nomically important. For some of these soils, natu- important to know physico-chemical properties of ral enemies have been found to control pathogen populations by affecting several stages of its life * Corresponding author: [email protected] cycle (Kerry, 1990). Chances of finding good can- Received: 22-05-02; Accepted: 11-04-03. didates to be used as biocontrol agents in these soils 38 L. Asensio et al. / Spanish Journal of Agricultural Research (2003) 1 (3), 37-45 are very high (Olivares-Bernabeu and Lopez-Llor- Soil texture, pH, conductivity/salinity and carbo- ca, 2002). nate content were determined for all soils collected. It is also interesting to compare the presence of mi- Protocols used were those of Anonymous (1979). Three crobial antagonists in soil environments with (agri- measurements were taken per each physicochemical cultural soils) or without («natural» soils) human im- parameter and soil sample. pact. A recent study of microbial entomopathogens of insect pests of apple and pear crops showed a wide va- riety of entomopathogenic viruses, bacteria, fungi and Antagonist detection. Isolation nematodes from soils of northern and central Europe of entomopathogenic fungi (Cross et al., 1999). Bidochka et al. (1998) evaluated the presence in soils from Canada of cosmopolitan spe- Insect antagonists in soil were detected using G. cies of entomopathogenic fungi, such as Metarhizium mellonella larvae (from Carolina Biological Supply Co., anisopliae (Metschn.) Sorok. and Beauveria bassiana NC, USA, 27215-3398). Forty grams of soil (freshly (Bals.) Vuill., more abundant in agricultural, and na- collected or 4ºC stored for less than 12 months) were tural soils, respectively. 1 mm sieved and placed in 90 mm diameter sterile In the present study we have analysed the presence Petri dishes (3 replicates per soil). Ten living L3-L4 G. of entomopathogens, mainly fungi, in soils from dif- mellonella larvae were buried per plate. Plates were ferent ecosystems of Alicante province such as forests, sealed with Parafilm (American National Can.) crop fields, natural and garden areas. 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. Recove- Material and methods red insects were surface sterilised using 1% sodium hypochlorite for 1 min. The sterilizing agent was eli- Soil sampling minated and insects were washed three times in steri- le distilled water (5 min each time). Insects were fi- Agricultural, forest, dry river beds and garden soils nally blotted dry onto sterile filter paper and placed were sampled in the province of Alicante (SE Spain) to in moist chambers for 1 week at 25ºC in the dark. The- survey microbial antagonists (mainly fungi) of insects se conditions usually allowed development (and often (Table 1). We collected 61 samples selected to repre- sporulation) of insect antagonists (mostly entomopa- sent soil and climate variability: 12 from agricultural thogenic fungi). soils (Prunus amygdalus Batsch., Citrus sp., and other Larvae with cottony filaments (probably fungus woody crops), 8 from forest soils (Pinus halepensis mycelium) were then observed with a dissecting mi- Mill. and Quercus rotundifolia Lam.), the rest under croscope. Larvae with few hyphae on the surface were plants of Nerium oleander L., 20 from natural popula- assumed to be colonized by fungal saprotrophs. On the tions (mostly in dry river beds) and 21 from gardens. contrary, larvae displaying abundant mycelium (i.e. in For each sampling site, soil (1.5-2 kg) was collec- intersegmental regions) were considered infected. The- ted from 3 points randomly selected and mixed to ma- se larvae were plated on corn meal agar (CMA) con- ke an homogeneous sample. Then they were collected taining 50 µg ml-1 penicillin, 50 µg ml-1 streptomycin, from 0-20 cm depth, removing for forest soils the le- 50 µg ml-1 rose bengal and 1 mg ml-1 Triton X-100. Sli- aflitter layer. Soil samples were homogeneized in the de cultures (Gams et al., 1998) of fungi infecting lar- laboratory, 2 mm sieved and stored at 4ºC in the dark vae were carried out and after microscopic observa- before use (less than 12 months). tion, genus was assigned according to Domsch et al. (1993). Species identification was determined using specialised bibliography including Lacey (1997). Ento- Physico-chemical analysis of soils mopathogenic fungi were inoculated on CMA plates and then transferred to 10 ml universal bottles with CMA Before analysis, soils were spread on a tray. Soil ag- slopes and after 7-15 days under normal incubation, gregates were broken by hand or using a hammer. Trays they were stored at 4ºC in the dark. Conidia and conidio- with soil were kept open until soil moisture was equi- phores of each fungal strain were measured (10 esti- librated with that of the laboratory. mations per strain and structure). The size of the fun- Entomopathogenic fungi from Spanish soils 39 Table 1. Location of soil sampling sites, crop-vegetation types and physico-chemical parameters of soil samples Sampling site pH Conductivity Carbonates Sample Crop-vegetation –1 Texture (UTM) (in H2O) CS (meq l ) (% CaCO3) 1 Maigmó Almond trees 8.93 ± 0.04 142.93 ± 5.94 52.28 ± 5.04 Loamy sand (30SYH141544) 2 Maigmó Almond trees 8.77 ± 0.03 99.45 ± 22.13 55.03 ± 2.40 Loamy sand (30SYH078668) 3 Tárbena Almond trees 8.30 ± 0.145 96.40 ± 1.35 36.78 ± 17.07 Loamy sand (30SYH521876) 4 Callosa Lemon trees 8.39 ± 0.017 166.18 ± 36.87 54.82 ± 8.90 ND (30SXH902425) 5 Orihuela Lemon trees 8.40 ± 0.051 1012.4 ± 185.7 41.26 ± 6.03 Loamy-Clay-silt (30SXH789237) 6 Benjúzar Lemon trees 8.61 ± 0.055 158.50 ± 21.53 41.31 ± 1.61 Loam (30SXH902171) 7 Benferri Orange trees 8.67 ± 0.025 104.96 ± 7.83 67.44 ± 19.83 ND (30SXH786262) 8 Almoradí Orange trees 8.85 ± 0.023 117.67 ± 9.08 46.41 ± 3.11 ND (30SXH935189) 9 Gata Orange trees 8.19 ± 0.040 78.33 ± 13.03 61.04 ± 13.39 Loamy-Clay-silt (31SBC488968) 10 Denia Orange