- Email: [email protected]
Survival and feeding activity of Protaphorura armata in different composts
ARTICLE IN PRESS Pedobiologia 50 (2006) 185—190
www.elsevier.de/pedobi
PROCEEDINGS OF THE XITH INTERNATIONAL COLLOQUIUM ON APTERYGOTA, ROUEN, FRANCE, 2004
Survival and feeding activity of Protaphorura armata in different composts Maria Agnese Sabatinia,, Gloria Innocentib, Matteo Montanarib, Sonia Ganassia a
Dipartimento di Biologia Animale, Universita ` degli Studi di Modena e Reggio Emilia, via Campi, 213/D; I-41100 Modena, Italy b Dipartimento di Protezione Valorizzazione Agroalimentare, Alma Mater Studiorum Universita ` degli Studi di Bologna, viale Fanin, 46, 1-40127 Bologna, Italy Received 10 May 2005; accepted 21 December 2005
KEYWORDS Springtail; Compost; Trichoderma atroviride; Conidia viability; Gut content analysis; Ca-Lignosulphonate
Summary Effects of compost products, enriched or not-enriched with a strain of the mycoparasitic fungus Trichoderma atroviride, on the survival of the collembolan Protaphorura armata and the viability of fungal conidia after the transit through the springtail gut were investigated. The effect of compost enriched with CaLignosulphonate (Ca-Ls), a low cost by-product of the acid sulphite pulping process, with lignin-like structure, on P. armata was also evaluated. All compost products enriched or not with the mycoparasitic fungus or Ca-Ls did not affect P. armata survival. No statistical differences were found in animal survival for different types of product or in enriched and not-enriched products. In addition to adults, live juveniles were also observed in all compost products. The gut content of animals, collected at the end of the survival test from compost enriched with T. atroviride, was examined under the light microscope, and in a few cases observations revealed the presence of some T. atroviride conidia. Subsequent tests carried out to study the viability of conidia after the transit through the springtail gut showed that colonies of the fungus developed from all faecal pellets produced by adult and juveniles specimens of P. armata previously fed on conidia of T. atroviride. These results suggest compatibility between Collembola and Trichoderma or Ca-Ls in the composts. & 2006 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: 0592055550; fax: 0592055548.
E-mail address: [email protected] (M.A. Sabatini).
Composting in part solves the problem of disposal and reuse of wastes, and the use of compost is important in modern sustainable agriculture,
0031-4056/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2005.12.005
ARTICLE IN PRESS 186 particularly in southern Europe where soils have a poor organic content as well as in areas continuously used for arable production where organic matter levels are decreasing. Compost is a hygienic humus-rich product used as an amendment to improve soil structure and promote plant growth (Popkin 1995). Compost can also provide biological control against plant diseases (Hoitink and Fay 1986); the suppressive effect seems to be related to indigenous microbial consortia (Hoitink 1990; Postma et al. 2003) and depends on the origin and quality of the compost. In particular, the nature of organic matter, maturity level and salinity strongly influence the activity of microorganisms (Hoitink and Bohem 1999). To enhance suppressiveness to plant diseases, compost can be enriched with selected strains of microbial antagonists or with different products such as Ca-Lignosulphonate (Ca-Ls), a by-product of the pulping process, available in large amounts at low cost. Ca-Ls is used to improve chemical–physical parameters and microbial biocontrol activity of soil to enhance plant growth and health (Meier et al. 1993; Lazarovitis 2001; Soltani et al. 2002). Enrichment of compost with Ca-Ls and the mycoparasitic fungus Trichoderma atroviride improved its suppressiveness against melon Fusarium wilt disease (Montanari et al. 2004a). In soils enriched or not-enriched composts come in contact with components of the soil community; however, the effects of composts on soil animals have been scarcely investigated (Mueller et al. 1993; Pfotzer and Schuler 1997; Crouau et al. 2002; Petersen et al. 2003). It has been shown in microcosm studies that the collembolan Protaphorura armata may significantly control disease caused by Gaeumannomyces graminis var. tritici and Fusarium culmorum, two of the most important soil borne pathogenic fungi of cereals (Sabatini and Innocenti 2001). Previous studies also demonstrated the absence of negative interactions between springtails and T. harzianum, a fungus controlling fungal plant diseases, in tests carried out in Petri dishes or under controlled conditions in the glasshouse (Curl 1979; Wiggins and Curl 1979; Lartey et al. 1994; Innocenti et al. 2001; Sabatini et al. 2002). The aim of this study was to investigate (i) the interactions between the collembolan P. armata, a species used also in ecotoxicological tests (Hopkin 1997), and compost products, enriched or not enriched with a strain of the mycoparasitic fungus T. atroviride or Ca-Ls and (ii) the effect of the feeding activity of this fungivorous collembolan species on the viability of conidia of T. atroviride.
M.A. Sabatini et al.
Materials and methods Test organisms Collembola: The Collembola used belonged to the species P. armata (Tullberg, 1869) sensu Gisin, 1952. Springtails, derived from specimens collected in a cultivated cereal field of the University of Bologna located in the Po Valley near Carpi (Modena, Italy), were reared for several generations in the laboratory. They were maintained in glass jars containing clay saturated with distilled water and kept in a thermostatic chamber at 20 1C. Animals were fed on brewer’s yeast. Fungi: The benomyl-tolerant strain of T. atroviride Karsten 312 B2 (Ta 312 B2) was used. The fungus is kept in the collection of the ‘‘Dipartimento di Protezione Valorizzazione Agroalimentare,’’ University of Bologna. Cultures were stored in tubes on PDA amended with 5 mg l1 of benomyl at 5 1C. This fungus is characterised by a high antagonistic activity (Montanari et al. 2004a), and it is suitable for establishment in composts (Montanari et al. 2004b). The acquisition of benomyl tolerance did not affect the fitness of the fungus (data not shown). To prepare sporal suspension, Ta 312 B2 was grown in Petri dishes on PDA (39 g PDA l1 deionised water) for 5 days at 22 1C in the dark, and then it was incubated in the light at room temperature for two additional days to improve sporulation. Spores were suspended in sterile deionised water, gently scraped, sieved through cheese cloth and washed in deionised water by centrifugation at 6000g.
Organic products and enrichment with T. atroviride and Calcium-Lignosulphonate The following compost products were used: (i) spent mushroom (Agaricus bisporus) compost (Fungo Spergola, Bologna, Italy) produced from wheat straw-bedded horse manure at two maturity levels: taken after steaming at the end of the mushroom production process (Young Spent Mushroom Compost – YSMC), and taken 3 months after steaming (Mature Spent Mushroom Compost – MSMC); (ii) green compost (GC) derived from fruit, vegetable and garden wastes taken 1 month after heat peaking, (Nuova Geovis, Bologna, Italy); (iii) commercial peat moss (PM), (Compo GmbH, Munster, Germany) and potting soil (PS), (Compo Agricoltura, Milano, Italy) were used as controls. Some physical and chemical properties were determined at the laboratory of ‘‘Chimica del Suolo,’’ University of Bologna (Table 1).
ARTICLE IN PRESS Collembola in composts Table 1.
187
Physical and chemical parameters of products before enrichment with Trichoderma atroviride 312 B2 NH+4–N (g kg1)
NO 3 –N (g kg1)
Total C (g kg1)
C/N
8.1
0.05
0.3
173
21.4
53
7.4
0.04
0.03
128
17.3
40 o1 6.7
18.3 5.6 13.0
0.3 0.2 0.07
0.05 0.02 0.6
295 345 318
16.1 61.6 25.5
Product
pH
Electrical conductivity (mS cm1)
Ash (%)
Young spent mushroom compost Mature spent mushroom compost Green compost Peat moss Potting soil
7.8
2.9
41
7.4
2.9
7.7 5.6 5.0
1.3 o1 1.5
Total N (g kg1)
All products were sieved through a 5 mm mesh; one half of the organic products were enriched with a sporal suspension of Ta 312 B2 (YSMC+Ta, MSMC+Ta, GC+Ta, PM+Ta, PS+Ta) to obtain a final concentration of 8 105 conidia ml1 product. Sterile water without conidia was added to the other half of YSMC, MSMC, GC, PM and PS. All products were individually placed in black plastic bags (10 l) and maintained at 60% water content in the dark at 15–18 1C. A part of MSMC compost was amended with Ca-Ls (Bretax C, Burgo Cartiere, Tolmezzo, Italy) (MSMC+Ca-Ls) (1% v/w) and maintained in the dark at 15–18 1C.
Gut content
Survival tests
To investigate the viability of conidia of Ta 312 B2 after passage through the springtail gut, specimens of P. armata were fed on conidia of the fungus in glass jars containing clay. When the gut was filled with conidia (dark green), 10 adults or ten 2-day old juveniles were transferred into 90 mm diameter Petri dishes containing a selective agar medium for Ta 312 B2 (TSM B10; Montanari et al. 2004b). The test was repeated eight times for a total of 80 adults and 80 juveniles. The animals were removed about 6 h later, after faecal pellets were deposited and the guts were empty. The dishes were incubated at 23 1C in the dark for 4 days and examined under a stereomicroscope. The number of faecal pellets producing Ta 312 B2 mycelium out of the total number plated was counted for each dish. Some faecal pellets were squashed on slides, covered by a cover slip and examined under a light microscope.
To study the effect of the different products on survival of P. armata, 20 sexually mature animals of the same age starved for 48 h, were introduced into each glass jar, which contained 30 ml of each enriched or not-enriched organic product separately. The products were taken from plastic bags 3 months after inoculation with Ta 312 B2 spores. Five replicates were made for each treatment. All jars were kept moist by regularly adding distilled water, and they were maintained under controlled conditions at 20 1C in the dark for 2 months. Thereafter, animals were hand sorted from each substrate and counted. The effect of MSMC compost amended with Ca-Ls on P. armata survival was also studied. This compost was chosen because in a previous study it proved to be particularly suitable for Ta 312 B2 establishment (Montanari et al. 2004b). Also in this test, twenty sexually mature animals of the same age, starved for 48 h, were introduced into each glass jar containing 30 ml of MSMC compost alone or amended with Ca-Ls. Five replicates were made for each treatment. After 2 months animals were hand sorted and counted.
To examine the gut content, springtails hand sorted from the substrate, as reported above, were fixed in Gisin’s fluid and subsequently mounted on slides in Gisin’s mounting medium (Gisin 1970). Analysis of the gut content was performed for all specimens collected from jars filled with organic products enriched with Ta 312 B2. The slides were analysed using a Leitz Diaplan microscope under differential interference contrast (DIC).
Viability of conidia
Statistical analysis Statistical analysis was performed with Statgraphics Plus (1996). Two-way ANOVA was used for analysing P. armata survival (product presence/ absence of Ta 312 B2). Percentage data were arc sin
ARTICLE IN PRESS 188
M.A. Sabatini et al.
transformed before ANOVA. After ANOVA data were compared with the least significant differences (LSD) test at a significance level of P ¼ 0:05. The t-test was also performed to compare the survival data of each enriched and not-enriched product with the mycoparasitic fungus and the survival of springtails collected from jars containing MSMC amended or not with Ca-Ls.
numbers (percentages) of specimens from enriched and not-enriched product. Moreover, the mean numbers (percentages) of adult specimens collected from jars containing MSMC compost alone (95%72.2) or MSMC+Ca-Ls (96%73.1) were not significantly different (t-test). In addition to adults introduced into the jars at the beginning of the experiments, live juveniles were also observed in enriched and not-enriched products, but they were not studied quantitatively.
Results
Gut content
Survival tests
Analysis of the gut content of animals collected from products enriched with Ta 312 B2 (Fig. 1) showed that 13–36% animals had an empty gut and 13–46% had mineral particles and/or organic material of different origin such as plant debris, wood bits, animal exuviae and amorphous material but no fungal material in their guts. Independent of the product, 40–74% of the animals had propagules of different fungi mixed with mineral particles and/ or organic material, such as plant debris, wood bits and amorphous material, in their guts. The percentage of animals with fungal material in the gut is
After 2 months a high number of adult animals was still alive in jars containing composts, PM and PS with and without antagonistic fungus (YSMC+Ta: 81.7%74.4 and YSMC: 93.3%71.7; MSMC+Ta: 86.7% 73.3 and MSMC: 76.7%77.3; GC+Ta: 85.0%72.9 and GC: 81.7%78.3; PM+Ta: 91.7%76.0 and PM: 85%70.1; PS+Ta: 81.7%74.4 and PS: 95%72.9). No statistical differences were found among the mean numbers (percentages) of P. armata collected from different types of product or between the mean
Figure 1. Distribution of food items in the gut of Protaphorura armata collected from jars with products enriched with Trichoderma atroviride 312 B2.
ARTICLE IN PRESS Collembola in composts divided in two, indicating that animals that had in the gut some conidia belonging to the genus Trichoderma along with other fungi was only 5–29%.
Viability of conidia Fungal colonies developed from 100% of faecal pellets produced by adult and juveniles specimens of P. armata fed with conidia, when the faecal pellets were placed on selective agar medium. Moreover, these colonies produced conidia when kept under suitable conditions. Microscopic examinations of faecal pellets showed that the majority of conidia were undamaged, and only very few conidia lacked cytoplasmic content.
Discussion The results of this study indicate that the tested compost products, enriched or not-enriched with propagules of the mycoparasitic fungus T. atroviride or with Ca-Ls, did not affect P. armata survival and did not block reproduction or development. In the present work an absence of negative effects of Trichoderma on P. armata was observed in composts, PS and PM, organic materials commonly used in the field and in container media to sustain plant growth. This is in line with results of a bioassay carried out by introducing collembolans, and propagules of T. harzianum, a mycoparasitic fungus, in washed sterile sand (Innocenti et al. 2001). Moreover, the mean number of P. armata specimens with empty gut found in this study is comparable with data obtained under natural conditions (Newell 1984; Lupetti et al. 1989) indicating that the feeding behaviour of the springtails in the tested product might reflect those in the field. It was demonstrated that conidia of Trichoderma are fed by Collembola and that they are not toxic for animals. However conidia of Trichoderma are not a preferred food for P. armata in the presence of other food, not only on agar medium in Petri dishes (Innocenti et al. 1997) but also in composts and in other organic products. In fact, a few P. armata specimens collected from organic products enriched with T. atroviride conidia had a very low amount of conidia in their gut. The majority of these conidia were not damaged during transit through the gut and thus were viable and could potentially form colonies. On the contrary, transit through the springtail gut damaged the conidia of the pathogenic fungus F. culmorum (Sabatini et al.
189 2004). Current knowledge does not offer an explanation as to why transit through the gut damages some conidia, such as those of F. culmorum, which for the most part lacked cytoplasmic content after ingestion (Sabatini et al. 2004), whereas others such as those of T. atroviride were not damaged. Overall the results suggest that feeding activity of springtails reduces inoculum of pathogenic fungi and favours Trichoderma species. Our findings confirm the statements of Wiggins and Curl (1979) and Williams et al. (1998), who affirm that soil animals, including springtails may be important in the dispersal of biocontrol agents. In conclusion, compost can be enriched with Trichoderma and Lignosulphonate without affecting Collembola survival when the enriched compost comes in contact with these animals in the soil. Future work will aim to prove the biocontrol effect of compost enriched with Collembola and T. atroviride and/or Ca-Ls against plant diseases caused by soil borne fungi.
References Crouau, Y., Gisclard, C., Perotti, P., 2002. The use of Folsomia candida (Collembola, Isotomidae) in bioassays of waste. Appl. Soil Ecol. 19, 65–70. Curl, E.A., 1979. Effects of mycophagous Collembola on Rhizoctonia solani and cotton-seedling disease. In: Schippers, B., Gams, W. (Eds.), Soil-Borne Plant Pathogens. Academic Press, London, pp. 253–269. Gisin, H., 1952. Notes sur les Collemboles, avec de´membrement des espe `ces des Onychiurus armatus, ambulans, et fimetarius auctorum. Mitt. Schweiz. Entomol. Ges. 25, 1–22. Gisin, H., 1970. Liquides pour la fixation, l’e´tude, le montage et la conservation des Collemboles. Rev. E´col. Biol. Sol. 7, 45–49. Hoitink, H.A.J., 1990. Production of disease suppressive compost and container media, and microorganism culture for use therein. US patent 4, 960, 348. Hoitink, H.A.J., Fay, P.C., 1986. Basis for the control of soil-borne plant pathogens with composts. Annu. Rev. Phytopathol. 24, 93–114. Hoitink, H.A.J., Bohem, M.J., 1999. Biocontrol within the context of soil microbial communities: a substrate dependent phenomenon. Annu. Rev. Phytopathol. 37, 427–446. Hopkin, S.P., 1997. Biology of the Springtails (Insecta: Collembola). Oxford University Press, Oxford. Innocenti, G., Cappi, C., Sabatini, M.A., 1997. Interactions of Collembola with soil-borne cereal pathogen and biological control Fungi. In: Tvaruzek, L. (Ed.), Proceedings of the International Conference on ‘‘Protection of cereal crops against harmful organisms’’, Kromeriz, July 1–4, 1997, Agricultural
ARTICLE IN PRESS 190 Research Institute, Kromeriz, Czech Republic, pp. 72–75. Innocenti, G., Montanari, M., Sabatini, M.A., 2001. Trichoderma, Collembola, pathogenic fungus, plant interactions. Biological control of fungal and bacterial plant pathogens. IOBC/WPRS Bull. 24, 345–348. Lartey, R.T., Curl, E.A., Peterson, C.M., 1994. Interactions of mycophagous Collembola and biological control fungi in the suppression of Rhizoctonia solani. Soil Biol. Biochem. 26, 81–88. Lazarovitis, G., 2001. Management of soil borne plant pathogens with organic soil amendments: a disease control strategy salvaged from the past. Can. J. Plant Pathol. 23, 1–7. Lupetti, P., Malatesta, E., Dallai, R., 1989. Observations on soil fauna activity and on midgut content of Collembola. In: Dallai, R. (Ed.), Proceedings of the Third International Seminar on Apterygota, University of Siena, Siena, pp. 307–315. Meier, J.N., Fyles, J.W., Mackenzie, A.F., O’Halloran, I.P., 1993. Effects of lignosulphonate-fertilizer applications on soil respiration and nitrogen dynamics. Can. J. Plant Pathol. 73, 233–242. Montanari, M., Ventura, M., Innocenti, G., 2004a. Exploitation of spent mushroom compost in biological control against melon Fusarium wilt disease. IOBC/ WPRS Bull. 27, 247–250. Montanari, M., Ventura, M., Innocenti, G., Sabatini, M.A., 2004b. Compost as substrate for Trichoderma. IOBC/ WPRS Bull. 27, 187–190. Mueller, B.R., Roth, M., Rittner, P., 1993. Influence of compost and lime on population structure and element concentrations of forest soil invertebrates. Biol. Fertil. Soils 15, 165–173. Newell, K., 1984. Interaction between two decomposer basidiomycetes and a collembolan under Sitka spruce: distribution, abundance and selective grazing. Soil Biol. Biochem. 16, 227–233. Petersen, S.O., Henriksen, K., Mortensen, G.K., Krogh, P.H., Brandt, K.K., Sorensen, J., Madsen, T., Petersen,
M.A. Sabatini et al. J., Gron, C., 2003. Recycling of sewage sludge and household compost to arable land: fate and effects of organic contaminants, and impact on soil fertility. Soil Till. Res. 72, 139–152. Pfotzer, G.H., Schuler, C., 1997. Effects of different compost amendments on soil biotic and faunal feeding activity in an organic farming system. Biol. Agric. Hortic. 15, 177–183. Popkin, R., 1995. Good news for waste watchers: recycling, composting show results for the future. Environ. Prot. Agency J. 2, 18–19. Postma, J., Montanari, M., van den Boogert, P., 2003. Microbial enrichment to enhance the disease suppressive activity of compost. Eur. J. Soil Biol. 39, 157–163. Sabatini, M.A., Innocenti, G., 2001. Effects of Collembola on plant–pathogenic fungus interactions in simple experimental systems. Biol. Fertil. Soils 33, 62–66. Sabatini, M.A., Grazioso, P., Altomare, C., Innocenti, G., 2002. Interactions between Onychiurus armatus and Trichoderma harzianum in take-all disease suppression in a simple experimental system. Eur. J. Soil Biol. 38, 71–74. Sabatini, M.A., Ventura, M., Innocenti, G., 2004. Do Collembola affect the competitive relationships among soil-borne plant pathogenic fungi? Pedobiologia 48, 603–608. Soltani, N., Conn, K.L., Abbasi, P.A., Lazarovitis, G., 2002. Reduction of potato scab and Verticillium wilt with ammonium lignosulphonate soil amendment in four Ontario potato fields. Can. J. Plant Pathol. 24, 332–339. Statgraphics Plus, 1996. Statgraphics Plus, Version 2.1. Statistical Graphics, Rockville. Wiggins, E.A., Curl, E.A., 1979. Interactions of Collembola and microflora of cotton rhizosphere. Phytopathology 69, 244–249. Williams, R.H., Whipps, J.M., Cooke, R.C., 1998. Role of soil mesofauna in dispersal of Coniothyrium minitans: transmission to sclerotia of Sclerotinia sclerotiorum. Soil Biol. Biochem. 30, 1929–1935.
www.elsevier.de/pedobi
PROCEEDINGS OF THE XITH INTERNATIONAL COLLOQUIUM ON APTERYGOTA, ROUEN, FRANCE, 2004
Survival and feeding activity of Protaphorura armata in different composts Maria Agnese Sabatinia,, Gloria Innocentib, Matteo Montanarib, Sonia Ganassia a
Dipartimento di Biologia Animale, Universita ` degli Studi di Modena e Reggio Emilia, via Campi, 213/D; I-41100 Modena, Italy b Dipartimento di Protezione Valorizzazione Agroalimentare, Alma Mater Studiorum Universita ` degli Studi di Bologna, viale Fanin, 46, 1-40127 Bologna, Italy Received 10 May 2005; accepted 21 December 2005
KEYWORDS Springtail; Compost; Trichoderma atroviride; Conidia viability; Gut content analysis; Ca-Lignosulphonate
Summary Effects of compost products, enriched or not-enriched with a strain of the mycoparasitic fungus Trichoderma atroviride, on the survival of the collembolan Protaphorura armata and the viability of fungal conidia after the transit through the springtail gut were investigated. The effect of compost enriched with CaLignosulphonate (Ca-Ls), a low cost by-product of the acid sulphite pulping process, with lignin-like structure, on P. armata was also evaluated. All compost products enriched or not with the mycoparasitic fungus or Ca-Ls did not affect P. armata survival. No statistical differences were found in animal survival for different types of product or in enriched and not-enriched products. In addition to adults, live juveniles were also observed in all compost products. The gut content of animals, collected at the end of the survival test from compost enriched with T. atroviride, was examined under the light microscope, and in a few cases observations revealed the presence of some T. atroviride conidia. Subsequent tests carried out to study the viability of conidia after the transit through the springtail gut showed that colonies of the fungus developed from all faecal pellets produced by adult and juveniles specimens of P. armata previously fed on conidia of T. atroviride. These results suggest compatibility between Collembola and Trichoderma or Ca-Ls in the composts. & 2006 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: 0592055550; fax: 0592055548.
E-mail address: [email protected] (M.A. Sabatini).
Composting in part solves the problem of disposal and reuse of wastes, and the use of compost is important in modern sustainable agriculture,
0031-4056/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2005.12.005
ARTICLE IN PRESS 186 particularly in southern Europe where soils have a poor organic content as well as in areas continuously used for arable production where organic matter levels are decreasing. Compost is a hygienic humus-rich product used as an amendment to improve soil structure and promote plant growth (Popkin 1995). Compost can also provide biological control against plant diseases (Hoitink and Fay 1986); the suppressive effect seems to be related to indigenous microbial consortia (Hoitink 1990; Postma et al. 2003) and depends on the origin and quality of the compost. In particular, the nature of organic matter, maturity level and salinity strongly influence the activity of microorganisms (Hoitink and Bohem 1999). To enhance suppressiveness to plant diseases, compost can be enriched with selected strains of microbial antagonists or with different products such as Ca-Lignosulphonate (Ca-Ls), a by-product of the pulping process, available in large amounts at low cost. Ca-Ls is used to improve chemical–physical parameters and microbial biocontrol activity of soil to enhance plant growth and health (Meier et al. 1993; Lazarovitis 2001; Soltani et al. 2002). Enrichment of compost with Ca-Ls and the mycoparasitic fungus Trichoderma atroviride improved its suppressiveness against melon Fusarium wilt disease (Montanari et al. 2004a). In soils enriched or not-enriched composts come in contact with components of the soil community; however, the effects of composts on soil animals have been scarcely investigated (Mueller et al. 1993; Pfotzer and Schuler 1997; Crouau et al. 2002; Petersen et al. 2003). It has been shown in microcosm studies that the collembolan Protaphorura armata may significantly control disease caused by Gaeumannomyces graminis var. tritici and Fusarium culmorum, two of the most important soil borne pathogenic fungi of cereals (Sabatini and Innocenti 2001). Previous studies also demonstrated the absence of negative interactions between springtails and T. harzianum, a fungus controlling fungal plant diseases, in tests carried out in Petri dishes or under controlled conditions in the glasshouse (Curl 1979; Wiggins and Curl 1979; Lartey et al. 1994; Innocenti et al. 2001; Sabatini et al. 2002). The aim of this study was to investigate (i) the interactions between the collembolan P. armata, a species used also in ecotoxicological tests (Hopkin 1997), and compost products, enriched or not enriched with a strain of the mycoparasitic fungus T. atroviride or Ca-Ls and (ii) the effect of the feeding activity of this fungivorous collembolan species on the viability of conidia of T. atroviride.
M.A. Sabatini et al.
Materials and methods Test organisms Collembola: The Collembola used belonged to the species P. armata (Tullberg, 1869) sensu Gisin, 1952. Springtails, derived from specimens collected in a cultivated cereal field of the University of Bologna located in the Po Valley near Carpi (Modena, Italy), were reared for several generations in the laboratory. They were maintained in glass jars containing clay saturated with distilled water and kept in a thermostatic chamber at 20 1C. Animals were fed on brewer’s yeast. Fungi: The benomyl-tolerant strain of T. atroviride Karsten 312 B2 (Ta 312 B2) was used. The fungus is kept in the collection of the ‘‘Dipartimento di Protezione Valorizzazione Agroalimentare,’’ University of Bologna. Cultures were stored in tubes on PDA amended with 5 mg l1 of benomyl at 5 1C. This fungus is characterised by a high antagonistic activity (Montanari et al. 2004a), and it is suitable for establishment in composts (Montanari et al. 2004b). The acquisition of benomyl tolerance did not affect the fitness of the fungus (data not shown). To prepare sporal suspension, Ta 312 B2 was grown in Petri dishes on PDA (39 g PDA l1 deionised water) for 5 days at 22 1C in the dark, and then it was incubated in the light at room temperature for two additional days to improve sporulation. Spores were suspended in sterile deionised water, gently scraped, sieved through cheese cloth and washed in deionised water by centrifugation at 6000g.
Organic products and enrichment with T. atroviride and Calcium-Lignosulphonate The following compost products were used: (i) spent mushroom (Agaricus bisporus) compost (Fungo Spergola, Bologna, Italy) produced from wheat straw-bedded horse manure at two maturity levels: taken after steaming at the end of the mushroom production process (Young Spent Mushroom Compost – YSMC), and taken 3 months after steaming (Mature Spent Mushroom Compost – MSMC); (ii) green compost (GC) derived from fruit, vegetable and garden wastes taken 1 month after heat peaking, (Nuova Geovis, Bologna, Italy); (iii) commercial peat moss (PM), (Compo GmbH, Munster, Germany) and potting soil (PS), (Compo Agricoltura, Milano, Italy) were used as controls. Some physical and chemical properties were determined at the laboratory of ‘‘Chimica del Suolo,’’ University of Bologna (Table 1).
ARTICLE IN PRESS Collembola in composts Table 1.
187
Physical and chemical parameters of products before enrichment with Trichoderma atroviride 312 B2 NH+4–N (g kg1)
NO 3 –N (g kg1)
Total C (g kg1)
C/N
8.1
0.05
0.3
173
21.4
53
7.4
0.04
0.03
128
17.3
40 o1 6.7
18.3 5.6 13.0
0.3 0.2 0.07
0.05 0.02 0.6
295 345 318
16.1 61.6 25.5
Product
pH
Electrical conductivity (mS cm1)
Ash (%)
Young spent mushroom compost Mature spent mushroom compost Green compost Peat moss Potting soil
7.8
2.9
41
7.4
2.9
7.7 5.6 5.0
1.3 o1 1.5
Total N (g kg1)
All products were sieved through a 5 mm mesh; one half of the organic products were enriched with a sporal suspension of Ta 312 B2 (YSMC+Ta, MSMC+Ta, GC+Ta, PM+Ta, PS+Ta) to obtain a final concentration of 8 105 conidia ml1 product. Sterile water without conidia was added to the other half of YSMC, MSMC, GC, PM and PS. All products were individually placed in black plastic bags (10 l) and maintained at 60% water content in the dark at 15–18 1C. A part of MSMC compost was amended with Ca-Ls (Bretax C, Burgo Cartiere, Tolmezzo, Italy) (MSMC+Ca-Ls) (1% v/w) and maintained in the dark at 15–18 1C.
Gut content
Survival tests
To investigate the viability of conidia of Ta 312 B2 after passage through the springtail gut, specimens of P. armata were fed on conidia of the fungus in glass jars containing clay. When the gut was filled with conidia (dark green), 10 adults or ten 2-day old juveniles were transferred into 90 mm diameter Petri dishes containing a selective agar medium for Ta 312 B2 (TSM B10; Montanari et al. 2004b). The test was repeated eight times for a total of 80 adults and 80 juveniles. The animals were removed about 6 h later, after faecal pellets were deposited and the guts were empty. The dishes were incubated at 23 1C in the dark for 4 days and examined under a stereomicroscope. The number of faecal pellets producing Ta 312 B2 mycelium out of the total number plated was counted for each dish. Some faecal pellets were squashed on slides, covered by a cover slip and examined under a light microscope.
To study the effect of the different products on survival of P. armata, 20 sexually mature animals of the same age starved for 48 h, were introduced into each glass jar, which contained 30 ml of each enriched or not-enriched organic product separately. The products were taken from plastic bags 3 months after inoculation with Ta 312 B2 spores. Five replicates were made for each treatment. All jars were kept moist by regularly adding distilled water, and they were maintained under controlled conditions at 20 1C in the dark for 2 months. Thereafter, animals were hand sorted from each substrate and counted. The effect of MSMC compost amended with Ca-Ls on P. armata survival was also studied. This compost was chosen because in a previous study it proved to be particularly suitable for Ta 312 B2 establishment (Montanari et al. 2004b). Also in this test, twenty sexually mature animals of the same age, starved for 48 h, were introduced into each glass jar containing 30 ml of MSMC compost alone or amended with Ca-Ls. Five replicates were made for each treatment. After 2 months animals were hand sorted and counted.
To examine the gut content, springtails hand sorted from the substrate, as reported above, were fixed in Gisin’s fluid and subsequently mounted on slides in Gisin’s mounting medium (Gisin 1970). Analysis of the gut content was performed for all specimens collected from jars filled with organic products enriched with Ta 312 B2. The slides were analysed using a Leitz Diaplan microscope under differential interference contrast (DIC).
Viability of conidia
Statistical analysis Statistical analysis was performed with Statgraphics Plus (1996). Two-way ANOVA was used for analysing P. armata survival (product presence/ absence of Ta 312 B2). Percentage data were arc sin
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transformed before ANOVA. After ANOVA data were compared with the least significant differences (LSD) test at a significance level of P ¼ 0:05. The t-test was also performed to compare the survival data of each enriched and not-enriched product with the mycoparasitic fungus and the survival of springtails collected from jars containing MSMC amended or not with Ca-Ls.
numbers (percentages) of specimens from enriched and not-enriched product. Moreover, the mean numbers (percentages) of adult specimens collected from jars containing MSMC compost alone (95%72.2) or MSMC+Ca-Ls (96%73.1) were not significantly different (t-test). In addition to adults introduced into the jars at the beginning of the experiments, live juveniles were also observed in enriched and not-enriched products, but they were not studied quantitatively.
Results
Gut content
Survival tests
Analysis of the gut content of animals collected from products enriched with Ta 312 B2 (Fig. 1) showed that 13–36% animals had an empty gut and 13–46% had mineral particles and/or organic material of different origin such as plant debris, wood bits, animal exuviae and amorphous material but no fungal material in their guts. Independent of the product, 40–74% of the animals had propagules of different fungi mixed with mineral particles and/ or organic material, such as plant debris, wood bits and amorphous material, in their guts. The percentage of animals with fungal material in the gut is
After 2 months a high number of adult animals was still alive in jars containing composts, PM and PS with and without antagonistic fungus (YSMC+Ta: 81.7%74.4 and YSMC: 93.3%71.7; MSMC+Ta: 86.7% 73.3 and MSMC: 76.7%77.3; GC+Ta: 85.0%72.9 and GC: 81.7%78.3; PM+Ta: 91.7%76.0 and PM: 85%70.1; PS+Ta: 81.7%74.4 and PS: 95%72.9). No statistical differences were found among the mean numbers (percentages) of P. armata collected from different types of product or between the mean
Figure 1. Distribution of food items in the gut of Protaphorura armata collected from jars with products enriched with Trichoderma atroviride 312 B2.
ARTICLE IN PRESS Collembola in composts divided in two, indicating that animals that had in the gut some conidia belonging to the genus Trichoderma along with other fungi was only 5–29%.
Viability of conidia Fungal colonies developed from 100% of faecal pellets produced by adult and juveniles specimens of P. armata fed with conidia, when the faecal pellets were placed on selective agar medium. Moreover, these colonies produced conidia when kept under suitable conditions. Microscopic examinations of faecal pellets showed that the majority of conidia were undamaged, and only very few conidia lacked cytoplasmic content.
Discussion The results of this study indicate that the tested compost products, enriched or not-enriched with propagules of the mycoparasitic fungus T. atroviride or with Ca-Ls, did not affect P. armata survival and did not block reproduction or development. In the present work an absence of negative effects of Trichoderma on P. armata was observed in composts, PS and PM, organic materials commonly used in the field and in container media to sustain plant growth. This is in line with results of a bioassay carried out by introducing collembolans, and propagules of T. harzianum, a mycoparasitic fungus, in washed sterile sand (Innocenti et al. 2001). Moreover, the mean number of P. armata specimens with empty gut found in this study is comparable with data obtained under natural conditions (Newell 1984; Lupetti et al. 1989) indicating that the feeding behaviour of the springtails in the tested product might reflect those in the field. It was demonstrated that conidia of Trichoderma are fed by Collembola and that they are not toxic for animals. However conidia of Trichoderma are not a preferred food for P. armata in the presence of other food, not only on agar medium in Petri dishes (Innocenti et al. 1997) but also in composts and in other organic products. In fact, a few P. armata specimens collected from organic products enriched with T. atroviride conidia had a very low amount of conidia in their gut. The majority of these conidia were not damaged during transit through the gut and thus were viable and could potentially form colonies. On the contrary, transit through the springtail gut damaged the conidia of the pathogenic fungus F. culmorum (Sabatini et al.
189 2004). Current knowledge does not offer an explanation as to why transit through the gut damages some conidia, such as those of F. culmorum, which for the most part lacked cytoplasmic content after ingestion (Sabatini et al. 2004), whereas others such as those of T. atroviride were not damaged. Overall the results suggest that feeding activity of springtails reduces inoculum of pathogenic fungi and favours Trichoderma species. Our findings confirm the statements of Wiggins and Curl (1979) and Williams et al. (1998), who affirm that soil animals, including springtails may be important in the dispersal of biocontrol agents. In conclusion, compost can be enriched with Trichoderma and Lignosulphonate without affecting Collembola survival when the enriched compost comes in contact with these animals in the soil. Future work will aim to prove the biocontrol effect of compost enriched with Collembola and T. atroviride and/or Ca-Ls against plant diseases caused by soil borne fungi.
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