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Do Collembola affect the competitive relationships among soil-borne plant pathogenic fungi?
ARTICLE IN PRESS Pedobiologia 48 (2004) 603—608
www.elsevier.de/pedobi
6th INTERNATIONAL SEMINAR ON APTERYGOTA, SIENA, ITALY, 2002
Do Collembola affect the competitive relationships among soil-borne plant pathogenic fungi? Maria Agnese Sabatinia,, Maurizio Venturab, Gloria Innocentib 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 e Valorizzazione Agroalimentare, Alma Mater Studiorum—Universita ` degli Studi di Bologna, viale Fanin 46, I-40127 Bologna, Italy Received 17 September 2002; accepted 13 July 2004
KEYWORDS Protaphorura armata; Fusarium culmorum; Gaeumannomyces graminis var. tritici; Competition; Fungal viability; Food preference
Summary The feeding preference of the collembolan Protaphorura armata in the presence of Fusarium culmorum and Gaeumannomyces graminis var. tritici, two soil-borne fungi pathogenic for winter cereals, was studied in a simplified experimental system including wheat seedlings. Analysis of gut content of all animals from microcosms containing inoculum of both fungi showed that F. culmorum was clearly preferred but that G. graminis var. tritici was also fed. At microscopic examination the majority of F. culmorum conidia present in the gut lacked cytoplasmic content, and only few conidial cells were intact. The feeding preference of P. armata favoured G. graminis var. tritici over F. culmorum in the competition for infection sites on wheat plants; in fact, the former resulted the prevalent cause of plant disease. The viability of fungal propagules after passage through the gut of P. armata was also studied. No colonies of G. graminis var. tritici and only a few colonies of F. culmorum developed from faecal pellets set on agar medium. Fungal propagules dispersed by springtails were not sufficient to induce disease, as demonstrated by introducing animals, previously fed on fungal cultures separately, into microcosms containing a sterile substrate where wheat kernels were seeded. & 2004 Elsevier GmbH. All rights reserved.
Introduction In agricultural soils many fungal species, some potentially pathogenic for plants, occur together and compete with each other for nutrients on crop
residues and for infection sites on host plants (Widden, 1997). The effects of pathogen mixtures on disease depend on a complex network of interactions influenced by several biotic factors including mycoparasitic fungi and fungivorous
Corresponding author. Tel.: +0592055550; fax: +0592055548.
E-mail address: [email protected] (M.A. Sabatini). 0031-4056/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2004.07.003
ARTICLE IN PRESS 604 animals. Some of these factors affect the competition among soil-borne fungi during the saprotrophic phase on crop residues, thus affecting the shift to the parasitic phase (Cook and Baker, 1983). Garrett (1970) hypothesized that for any particular soilborne root infecting fungus the share of a potential substrate will be determined (i) directly by the competitive saprotrophic ability of the fungus, (ii) directly by its inoculum potential on the substrate and (iii) inversely by the inoculum density of its competitors. Collembola are one of the factors potentially affecting fungal competition on organic substrates and thus modulating disease; previous in vitro studies by Sabatini and Innocenti (1995, 2000a, b) demonstrated that Gaeumannomyces graminis var. tritici and Fusarium culmorum, two of the most important soil-borne fungi causing foot and root rot disease of winter cereals, were palatable for different species of Collembola. Moreover, when colonies of both fungi were grown in the same Petri dish on agarized medium, the collembolan Protaphorura armata preferred F. culmorum, but also grazed on G. graminis var. tritici (Sabatini and Innocenti, 2000b). Eventually, P. armata significantly decreased the severity of disease caused by these fungi on wheat seedlings in tests carried out in microcosms, where the two fungi were used separately (Sabatini and Innocenti, 2001). The aim of this study was to investigate in microcosms (i) the feeding preference of P. armata in the presence of both pathogenic fungi and wheat plants under conditions that more closely resemble field conditions than those of in vitro tests and (ii) the effect of Collembola on the competition for infection sites between F. culmorum and G. graminis var. tritici and thus on disease.
Materials and methods
M.A. Sabatini et al. Fungi. The virulent fungal isolates considered were Gaeumannomyces graminis (Sacc.) von Arx et Olivier var. tritici Walker (LM 14.95) and Fusarium culmorum (W.G. Smith) Sacc. (LM 20.91). Codes of isolates (LM) are the accession numbers of cultures from the collection of the ‘‘Dipartimento di Protezione Valorizzazione Agroalimentare,’’ University of Bologna. They were isolated from wheat plants of cereal fields in Italy. Cultures were maintained in tubes on potato dextrose agar (PDA, Difco, Sparks, MD, USA) under mineral oil at 5 1C in the dark. Each fungus was transferred from stored cultures onto PDA plates and cultured at 23 1C in the dark to obtain colonies 3–4 cm diameter. Mycelial plugs, 6 mm diameter, were cut from the edge of these cultures and used for preparation of inoculum. The fungi used are morphologically different and are easily distinguished at microscopic examination. F. culmorum produces yellow–red pigmented hyphae and 3–5 septate macroconidia, with a typical foot-shaped basal cell and curved ventral and dorsal surfaces, giving a reddish colour in mass. G. graminis var. tritici produces thick-walled, darkly pigmented hyphae, and no conidia. These fungi produce different symptoms of disease on wheat seedlings. Plants attacked by G. graminis var. tritici show blackened seminal roots; blackening of the roots often spreads upwards to the basal area of the stem. Seedlings infected by F. culmorum present brownish lesions at the base of the stems. Plants. Creso durum wheat (Triticum durum Desf.) a cultivar susceptible to G. graminis var. tritici and to F. culmorum was used in the experiments. Kernels were inspected under a stereomicroscope, and damaged ones were discarded. Seedlings were grown until growth stage 14 (four leaves unfolded, Zadoks et al., 1974).
Test organisms
Fungal inoculum preparation
Collembola. The Collembola used belonged to the species Protaphorura armata (Tullberg 1869). 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. Under these conditions the first oviposition occurs on average 20–21 days after hatching, and eggs hatch on average 12 days after oviposition.
The inoculum was prepared using a method modified from Simon and Sivasithamparam (1988). Twenty grams of a 1:1 (v:v) mixture of millet and wheat kernels steeped in tap water for 12 h and then drained by pressing with gauze, were dispensed into each of ten 250 ml Erlenmeyer flasks and autoclaved for 1 h at 120 1C on two consecutive days. Each of five flasks was then inoculated with five discs of agar (6 mm diameter) cut from the edge of an actively growing colony of G. graminis var. tritici; each of the remaining flasks was inoculated with five discs of agar cut from the edge of an actively growing colony of F. culmorum.
ARTICLE IN PRESS Do Collembola affect relationship among fungi After three weeks at 20 1C (72 1C) in the dark, the kernels colonized by G. graminis var. tritici or F. culmorum were extracted from the flasks, air-dried and stored at 5 1C. They were used as inoculum in the following experiments.
Effect of springtails on plant biomass in the presence or absence of fungal mixture Glass tubes, 35 mm diameter by 300 mm height, were used as experimental containers. To study the effect of P. armata on plant biomass in the presence of inoculum of both pathogenic fungi, each of 16 tubes was filled with a substrate consisting of 150 g of sterile sand mixed with 0.5% (w:w) of G. graminis var. tritici+0.5% of F. culmorum inoculum obtained as described above. Afterwards three kernels of Creso durum wheat, previously surface-sterilized with 1% sodium hypochlorite for 10 min, then rinsed in sterile water and dried at room temperature on sterile filter paper, were sown in each tube. Immediately after planting, 50 sexually mature specimens of P. armata of the same age starved for 48 h were added to each of eight out of the 16 prepared tubes. The other eight tubes lacking Collembola were used as controls. The final animal density was equivalent to about 40,000 ind. m 2 (13 cm depth, as the height of the substrate in the tube assay). This number of Collembola reflects the population density found in wheat fields in the Po Valley (Italy) (Sabatini et al., 1997). To study the effect of springtails on plant health, tubes containing sandy substrate, kernels of Creso durum wheat and springtails or lacking animals were also prepared. Eight replicates for each combination were prepared for a total of 16 tubes. The lower part of all tubes was covered with black plastic wrap to keep roots in the dark; the top of the tube was covered with a cloth to block movement of animals between tubes. Tube assays were placed upright in racks in a randomized complete block design and incubated in a growth chamber at 20–25 1C under 12 h day/night photoperiod and relative humidity of 70–80%. Each tube was watered with 5 ml of tap water at the start of the trial and weekly thereafter. Four weeks after planting the substrate, plants and springtails were carefully extracted from each tube. Wheat plants in each tube were cleaned of adhering substrate and observed for symptoms of disease, then dried at 80 1C for 24 h before being weighed.
605
Analysis of springtail gut contents Springtails hand sorted from the substrate at the end of the experiment were fixed in Gisin’s fluid and subsequently mounted on slides in Gisin’s mounting medium (Gisin, 1970). Analysis of gut content was performed for all specimens collected from microcosms where both fungi were present. The slides were analysed using a Leitz Diaplan microscope under differential interference contrast (DIC).
Effect of springtails on fungal propagule dispersal To study the effect of animals on inoculum diffusion, first the viability of G. graminis var. tritici and F. culmorum propagules after the passage through the gut was tested. Specimens of P. armata fed separately on pure cultures of the two fungi as previously described by Sabatini and Innocenti (2000a) were observed under a stereomicroscope to select animals with a reddish or dark brown gut, indicative respectively of an abundant presence of F. culmorum or G. graminis var. tritici propagules. Then ten adults or ten 2-day-old juveniles were introduced into each 90 mm diameter Petri dish containing PDA+streptomycin sulphate (50 mg l 1, Sigma, USA). The test was repeated eight times for a total of 80 adults and 80 juveniles for each fungus. The animals were removed about 6 h later, and the faecal pellets were placed in new PDA+streptomycin sulphate Petri dishes. The dishes were incubated at 23 1C in the dark for 4 days and examined for fungal colonies. Some faecal pellets collected from Petri dishes immediately after removal of animals were squashed on slides, covered by a cover slip and examined under a light microscope. To verify whether Collembola can transport on their body or in their gut sufficient inoculum to give rise to plant infection and cause disease, adult specimens of P. armata were allowed to feed for ten days on pure cultures of the two fungi separately. Fungal cultures were renewed daily. The animals were then introduced into microcosms prepared as described above containing sterile sand previously seeded with three surface-sterilized kernels of Creso durum wheat. Fifty specimens were introduced into each tube; eight replicates were run for each fungus. Controls consisted of tubes lacking Collembola. Five weeks after planting, wheat seedlings were cleaned of adhering substrate and observed for disease symptoms, then
ARTICLE IN PRESS 606 dried at 80 1C for 24 h before plant weight per tube was determined.
Statistical analysis Plant dry weights after logarithmic (log10) transformation were (a) compared with Student’s t-test in experiments on the effect of springtails on plant biomass in the presence or absence of fungal mixture, (b) analysed by one-way ANOVA and compared with Student–Newman–Keul test in the experiment on the effect of springtails on fungal propagule dispersal. Percentage values of gut content were arc–sin transformed (Snedecor and Cochran, 1980) and analysed by one-way ANOVA and compared with Student–Newman–Keul test.
Results Effect of springtails on plant biomass in the presence or absence of fungal mixture The total dry mass of 4-week-old wheat seedlings grown in substrate containing inoculum of both pathogenic fungi and P. armata specimens was significantly higher (Po0.05) than the biomass of plants grown in the absence of animals (49.378.9 mg and 39.5711.1 mg, respectively). There was no significant difference between the biomass of plants grown in substrate with or without Collembola in absence of pathogenic fungi (84.3719.6 mg and 80.4722.7 mg, respectively).
M.A. Sabatini et al. Analysis of infected plants showed that G. graminis var. tritici was the prevalent organism causing the disease when Collembola were present, whereas both pathogenic fungi heavily infected the plants in the absence of the animals.
Springtail gut contents All springtails collected from the substrate at the end of the experiment were alive. In addition to adults introduced into the tubes at the beginning of the experiment juveniles were present. From tubes containing inoculum of both fungi, 671 specimens (adults+juveniles) had been collected. Analysis of the gut content of all these animals showed that 37.44% had an empty gut and 21.65% had mineral particles and exuviae in the gut, 13.01% had only conidia of F. culmorum (Fig. 1A), and 4.11% had fragments of G. graminis var. tritici hyphae and mineral particles in their gut. The other animals contained propagules of both fungi only or mixed with mineral particles and/or organic material (Fig. 1B). Of these animals 19.74% had predominantly F. culmorum conidia and 4.04% predominantly G. graminis var. tritici hyphae in their gut (Fig. 1B). The number of animals containing only or predominantly F. culmorum in their gut was significantly higher (Po0.01) than the number of specimens containing only or predominantly G. graminis var. tritici. Microscopic examination of the gut contents showed that the majority of F. culmorum conidia lacked cytoplasmic content; only very few conidial cells were undamaged (Fig. 1A).
Figure 1. Gut contents of P. armata specimens collected from microcosms where inoculum of both F. culmorum and G. graminis var. tritici were present: (A) conidia of F. culmorum; arrow indicates an intact conidial cell (B) fragments of G. graminis var. tritici hyphae, conidia of F. culmorum (arrows) and mineral particles. Bars=25 mm.
ARTICLE IN PRESS Do Collembola affect relationship among fungi
Effect of springtails on fungal propagule dispersal Microscopic examination of faecal pellets produced by animals that had been fed on F. culmorum or G. graminis var. tritici colonies, revealed the presence of conidia of F. culmorum for the most part damaged, and/or hyphal fragments of both fungi. No colonies of G. graminis var. tritici developed on PDA medium from faecal pellets produced by springtails fed on this fungus; whereas few colonies of F. culmorum developed from the faecal pellets of both adults and juveniles fed on this fungus. No symptoms of disease were observed on wheat seedlings grown with P. armata specimens previously fed on fungal propagules of G. graminis var. tritici or F. culmorum and no pathogenic fungi were isolated from their tissues. The total dry biomass of wheat seedlings grown with springtails previously fed on fungal propagules of G. graminis var. tritici (97.273.5 mg) or F. culmorum (114.676.9 mg) was not significantly different from the dry weight of plants developed in the absence of animals (104.474.7 mg).
Discussion The results showed that wheat seedlings grown in the presence of P. armata and inoculum of G. graminis var. tritici and F. culmorum, two important pathogenic fungi of cereals, were mainly attacked by the former fungus, which is less palatable for P. armata. In the absence of Collembola, F. culmorum also heavily infected the plants. The study confirmed under conditions more closely resembling field than in vitro tests the previously observed feeding preference of P. armata for F. culmorum over G. graminis var. tritici (Sabatini and Innocenti, 2000b). Indeed, in the present study animals grazed on fungal propagules that colonized organic matter as occurs in agricultural soils. Moreover, the number of P. armata specimens with an empty gut found was similar to the number of Collembola with an empty gut collected in the field (Newell, 1984; Lupetti et al., 1989). This indicates that the feeding behaviour of the springtails in the microcosms resembled that in the field. Our results demonstrate that the transit through the gut damages fungal propagules; in fact, after ingestion, most conidia and hyphae lacked cytoplasmic content. Trehalase activity in the gut of P. armata is high (Urba ´ˇsek and Rusek, 1994) and this likely explains the digestion of fungal cell content, containing mainly trehalose (Siepel and Ruiter-
607 Dijkman, 1993). Therefore, the reduced viability of inoculum caused by ingestion and feeding of the animals on fungal propagules could explain why P. armata did not spread the disease caused by G. graminis var. tritici or F. culmorum. Similar results for the same and other pathogenic fungi and different species of Collembola have been reported before (Wiggins and Curl, 1979; Nakamura et al., 1992; Dromph and Borgen, 2001; Sabatini and Innocenti, 2001). The amount of viable propagules transported by springtails in their gut or cuticle probably was insufficient to induce disease. Results of this and other studies suggest that Collembola, including P. armata, prefer certain soil-borne fungi responsible of foot and root diseases of cereals (Sabatini and Innocenti, 1995, 2000a, b). Therefore, Collembola might affect competition among fungi pathogenic for the same crop, thus modulating plant diseases.
References Cook, R.J., Baker, K.F., 1983. The Nature and Practice of Biological Control of Plant Pathogens. APS Press, St. Paul, MN, USA. Dromph, K.M., Borgen, A., 2001. Reduction of viability of soil borne inoculum of common bunt (Tilletia tritici) by collembolans. Soil Biol. Biochem. 33, 1791–1795. Garrett, S.D., 1970. Pathogenic Root-infecting Fungi. Cambridge University Press, Cambridge. Gisin, H., 1970. Liquides pour la fixation, l’e´tude, le montage et la conservation des Collemboles. Rev. `col. Biol. Sol 7, 45–49. E Lupetti, P., Malatesta, E., Dallai, R., 1989. Observations on soil fauna activity and on midgut content of Collembola. In: Dallai, R. (Ed.), Third International Seminar on Apterygota. University of Siena, Siena, pp. 307–315. Nakamura, Y., Matsuzaki, I., Itakura, J., 1992. Effect of grazing by Sinella curviseta (Collembola) on Fusarium oxysporum f. sp. cucumerinum causing cucumber disease. Pedobiologia 36, 168–171. 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. Sabatini, M.A., Innocenti, G., 1995. Interactions between Collembola and soil-borne cereal pathogen fungi. Pol. Pismo Entomol. 64, 191–197. Sabatini, M.A., Innocenti, G., 2000a. Functional relationships between Collembola and plant pathogenic fungi of agricultural soils. Pedobiologia 44, 467–475. Sabatini, M.A., Innocenti, G., 2000b. Soil-borne plant pathogenic fungi in relation to some collembolan species under laboratory conditions. Mycol. Res. 104, 1197–1201.
ARTICLE IN PRESS 608 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., Rebecchi, L., Cappi, C., Bertolani, R., Fratello, B., 1997. Long-term effects of three different continuous tillage practices on collembolan populations. Pedobiologia 41, 185–193. Siepel, H., Ruiter-Dijkman, E.M., 1993. Feeding guilds of oribatid mites based on their carbohydrase activities. Soil Biol. Biochem. 26, 1491–1497. Simon, A., Sivasithamparam, K., 1988. Interactions among Gaeumannomyces graminis var. tritici and soil bacteria. Can. J. Microbiol. 34, 871–876.
M.A. Sabatini et al. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods. Iowa State University Press, Ames. Urba ´ˇsek, F., Rusek, J., 1994. Activity of digestive enzymes in seven species of Collembola (Insecta: Entognatha). Pedobiologia 38, 400–406. Widden, P., 1997. Competition and the fungal community. In: Wicklow, D.T., So ¨derstro ¨m, B. (Eds.), Environmental and Microbial Relationships. The Mycota IV. Springer, Berlin, pp. 135–147. Wiggins, E.A., Curl, E.A., 1979. Interaction of Collembola and microflora of cotton rhizosphere. Phytopathology 69, 244–249. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421.
www.elsevier.de/pedobi
6th INTERNATIONAL SEMINAR ON APTERYGOTA, SIENA, ITALY, 2002
Do Collembola affect the competitive relationships among soil-borne plant pathogenic fungi? Maria Agnese Sabatinia,, Maurizio Venturab, Gloria Innocentib 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 e Valorizzazione Agroalimentare, Alma Mater Studiorum—Universita ` degli Studi di Bologna, viale Fanin 46, I-40127 Bologna, Italy Received 17 September 2002; accepted 13 July 2004
KEYWORDS Protaphorura armata; Fusarium culmorum; Gaeumannomyces graminis var. tritici; Competition; Fungal viability; Food preference
Summary The feeding preference of the collembolan Protaphorura armata in the presence of Fusarium culmorum and Gaeumannomyces graminis var. tritici, two soil-borne fungi pathogenic for winter cereals, was studied in a simplified experimental system including wheat seedlings. Analysis of gut content of all animals from microcosms containing inoculum of both fungi showed that F. culmorum was clearly preferred but that G. graminis var. tritici was also fed. At microscopic examination the majority of F. culmorum conidia present in the gut lacked cytoplasmic content, and only few conidial cells were intact. The feeding preference of P. armata favoured G. graminis var. tritici over F. culmorum in the competition for infection sites on wheat plants; in fact, the former resulted the prevalent cause of plant disease. The viability of fungal propagules after passage through the gut of P. armata was also studied. No colonies of G. graminis var. tritici and only a few colonies of F. culmorum developed from faecal pellets set on agar medium. Fungal propagules dispersed by springtails were not sufficient to induce disease, as demonstrated by introducing animals, previously fed on fungal cultures separately, into microcosms containing a sterile substrate where wheat kernels were seeded. & 2004 Elsevier GmbH. All rights reserved.
Introduction In agricultural soils many fungal species, some potentially pathogenic for plants, occur together and compete with each other for nutrients on crop
residues and for infection sites on host plants (Widden, 1997). The effects of pathogen mixtures on disease depend on a complex network of interactions influenced by several biotic factors including mycoparasitic fungi and fungivorous
Corresponding author. Tel.: +0592055550; fax: +0592055548.
E-mail address: [email protected] (M.A. Sabatini). 0031-4056/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2004.07.003
ARTICLE IN PRESS 604 animals. Some of these factors affect the competition among soil-borne fungi during the saprotrophic phase on crop residues, thus affecting the shift to the parasitic phase (Cook and Baker, 1983). Garrett (1970) hypothesized that for any particular soilborne root infecting fungus the share of a potential substrate will be determined (i) directly by the competitive saprotrophic ability of the fungus, (ii) directly by its inoculum potential on the substrate and (iii) inversely by the inoculum density of its competitors. Collembola are one of the factors potentially affecting fungal competition on organic substrates and thus modulating disease; previous in vitro studies by Sabatini and Innocenti (1995, 2000a, b) demonstrated that Gaeumannomyces graminis var. tritici and Fusarium culmorum, two of the most important soil-borne fungi causing foot and root rot disease of winter cereals, were palatable for different species of Collembola. Moreover, when colonies of both fungi were grown in the same Petri dish on agarized medium, the collembolan Protaphorura armata preferred F. culmorum, but also grazed on G. graminis var. tritici (Sabatini and Innocenti, 2000b). Eventually, P. armata significantly decreased the severity of disease caused by these fungi on wheat seedlings in tests carried out in microcosms, where the two fungi were used separately (Sabatini and Innocenti, 2001). The aim of this study was to investigate in microcosms (i) the feeding preference of P. armata in the presence of both pathogenic fungi and wheat plants under conditions that more closely resemble field conditions than those of in vitro tests and (ii) the effect of Collembola on the competition for infection sites between F. culmorum and G. graminis var. tritici and thus on disease.
Materials and methods
M.A. Sabatini et al. Fungi. The virulent fungal isolates considered were Gaeumannomyces graminis (Sacc.) von Arx et Olivier var. tritici Walker (LM 14.95) and Fusarium culmorum (W.G. Smith) Sacc. (LM 20.91). Codes of isolates (LM) are the accession numbers of cultures from the collection of the ‘‘Dipartimento di Protezione Valorizzazione Agroalimentare,’’ University of Bologna. They were isolated from wheat plants of cereal fields in Italy. Cultures were maintained in tubes on potato dextrose agar (PDA, Difco, Sparks, MD, USA) under mineral oil at 5 1C in the dark. Each fungus was transferred from stored cultures onto PDA plates and cultured at 23 1C in the dark to obtain colonies 3–4 cm diameter. Mycelial plugs, 6 mm diameter, were cut from the edge of these cultures and used for preparation of inoculum. The fungi used are morphologically different and are easily distinguished at microscopic examination. F. culmorum produces yellow–red pigmented hyphae and 3–5 septate macroconidia, with a typical foot-shaped basal cell and curved ventral and dorsal surfaces, giving a reddish colour in mass. G. graminis var. tritici produces thick-walled, darkly pigmented hyphae, and no conidia. These fungi produce different symptoms of disease on wheat seedlings. Plants attacked by G. graminis var. tritici show blackened seminal roots; blackening of the roots often spreads upwards to the basal area of the stem. Seedlings infected by F. culmorum present brownish lesions at the base of the stems. Plants. Creso durum wheat (Triticum durum Desf.) a cultivar susceptible to G. graminis var. tritici and to F. culmorum was used in the experiments. Kernels were inspected under a stereomicroscope, and damaged ones were discarded. Seedlings were grown until growth stage 14 (four leaves unfolded, Zadoks et al., 1974).
Test organisms
Fungal inoculum preparation
Collembola. The Collembola used belonged to the species Protaphorura armata (Tullberg 1869). 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. Under these conditions the first oviposition occurs on average 20–21 days after hatching, and eggs hatch on average 12 days after oviposition.
The inoculum was prepared using a method modified from Simon and Sivasithamparam (1988). Twenty grams of a 1:1 (v:v) mixture of millet and wheat kernels steeped in tap water for 12 h and then drained by pressing with gauze, were dispensed into each of ten 250 ml Erlenmeyer flasks and autoclaved for 1 h at 120 1C on two consecutive days. Each of five flasks was then inoculated with five discs of agar (6 mm diameter) cut from the edge of an actively growing colony of G. graminis var. tritici; each of the remaining flasks was inoculated with five discs of agar cut from the edge of an actively growing colony of F. culmorum.
ARTICLE IN PRESS Do Collembola affect relationship among fungi After three weeks at 20 1C (72 1C) in the dark, the kernels colonized by G. graminis var. tritici or F. culmorum were extracted from the flasks, air-dried and stored at 5 1C. They were used as inoculum in the following experiments.
Effect of springtails on plant biomass in the presence or absence of fungal mixture Glass tubes, 35 mm diameter by 300 mm height, were used as experimental containers. To study the effect of P. armata on plant biomass in the presence of inoculum of both pathogenic fungi, each of 16 tubes was filled with a substrate consisting of 150 g of sterile sand mixed with 0.5% (w:w) of G. graminis var. tritici+0.5% of F. culmorum inoculum obtained as described above. Afterwards three kernels of Creso durum wheat, previously surface-sterilized with 1% sodium hypochlorite for 10 min, then rinsed in sterile water and dried at room temperature on sterile filter paper, were sown in each tube. Immediately after planting, 50 sexually mature specimens of P. armata of the same age starved for 48 h were added to each of eight out of the 16 prepared tubes. The other eight tubes lacking Collembola were used as controls. The final animal density was equivalent to about 40,000 ind. m 2 (13 cm depth, as the height of the substrate in the tube assay). This number of Collembola reflects the population density found in wheat fields in the Po Valley (Italy) (Sabatini et al., 1997). To study the effect of springtails on plant health, tubes containing sandy substrate, kernels of Creso durum wheat and springtails or lacking animals were also prepared. Eight replicates for each combination were prepared for a total of 16 tubes. The lower part of all tubes was covered with black plastic wrap to keep roots in the dark; the top of the tube was covered with a cloth to block movement of animals between tubes. Tube assays were placed upright in racks in a randomized complete block design and incubated in a growth chamber at 20–25 1C under 12 h day/night photoperiod and relative humidity of 70–80%. Each tube was watered with 5 ml of tap water at the start of the trial and weekly thereafter. Four weeks after planting the substrate, plants and springtails were carefully extracted from each tube. Wheat plants in each tube were cleaned of adhering substrate and observed for symptoms of disease, then dried at 80 1C for 24 h before being weighed.
605
Analysis of springtail gut contents Springtails hand sorted from the substrate at the end of the experiment were fixed in Gisin’s fluid and subsequently mounted on slides in Gisin’s mounting medium (Gisin, 1970). Analysis of gut content was performed for all specimens collected from microcosms where both fungi were present. The slides were analysed using a Leitz Diaplan microscope under differential interference contrast (DIC).
Effect of springtails on fungal propagule dispersal To study the effect of animals on inoculum diffusion, first the viability of G. graminis var. tritici and F. culmorum propagules after the passage through the gut was tested. Specimens of P. armata fed separately on pure cultures of the two fungi as previously described by Sabatini and Innocenti (2000a) were observed under a stereomicroscope to select animals with a reddish or dark brown gut, indicative respectively of an abundant presence of F. culmorum or G. graminis var. tritici propagules. Then ten adults or ten 2-day-old juveniles were introduced into each 90 mm diameter Petri dish containing PDA+streptomycin sulphate (50 mg l 1, Sigma, USA). The test was repeated eight times for a total of 80 adults and 80 juveniles for each fungus. The animals were removed about 6 h later, and the faecal pellets were placed in new PDA+streptomycin sulphate Petri dishes. The dishes were incubated at 23 1C in the dark for 4 days and examined for fungal colonies. Some faecal pellets collected from Petri dishes immediately after removal of animals were squashed on slides, covered by a cover slip and examined under a light microscope. To verify whether Collembola can transport on their body or in their gut sufficient inoculum to give rise to plant infection and cause disease, adult specimens of P. armata were allowed to feed for ten days on pure cultures of the two fungi separately. Fungal cultures were renewed daily. The animals were then introduced into microcosms prepared as described above containing sterile sand previously seeded with three surface-sterilized kernels of Creso durum wheat. Fifty specimens were introduced into each tube; eight replicates were run for each fungus. Controls consisted of tubes lacking Collembola. Five weeks after planting, wheat seedlings were cleaned of adhering substrate and observed for disease symptoms, then
ARTICLE IN PRESS 606 dried at 80 1C for 24 h before plant weight per tube was determined.
Statistical analysis Plant dry weights after logarithmic (log10) transformation were (a) compared with Student’s t-test in experiments on the effect of springtails on plant biomass in the presence or absence of fungal mixture, (b) analysed by one-way ANOVA and compared with Student–Newman–Keul test in the experiment on the effect of springtails on fungal propagule dispersal. Percentage values of gut content were arc–sin transformed (Snedecor and Cochran, 1980) and analysed by one-way ANOVA and compared with Student–Newman–Keul test.
Results Effect of springtails on plant biomass in the presence or absence of fungal mixture The total dry mass of 4-week-old wheat seedlings grown in substrate containing inoculum of both pathogenic fungi and P. armata specimens was significantly higher (Po0.05) than the biomass of plants grown in the absence of animals (49.378.9 mg and 39.5711.1 mg, respectively). There was no significant difference between the biomass of plants grown in substrate with or without Collembola in absence of pathogenic fungi (84.3719.6 mg and 80.4722.7 mg, respectively).
M.A. Sabatini et al. Analysis of infected plants showed that G. graminis var. tritici was the prevalent organism causing the disease when Collembola were present, whereas both pathogenic fungi heavily infected the plants in the absence of the animals.
Springtail gut contents All springtails collected from the substrate at the end of the experiment were alive. In addition to adults introduced into the tubes at the beginning of the experiment juveniles were present. From tubes containing inoculum of both fungi, 671 specimens (adults+juveniles) had been collected. Analysis of the gut content of all these animals showed that 37.44% had an empty gut and 21.65% had mineral particles and exuviae in the gut, 13.01% had only conidia of F. culmorum (Fig. 1A), and 4.11% had fragments of G. graminis var. tritici hyphae and mineral particles in their gut. The other animals contained propagules of both fungi only or mixed with mineral particles and/or organic material (Fig. 1B). Of these animals 19.74% had predominantly F. culmorum conidia and 4.04% predominantly G. graminis var. tritici hyphae in their gut (Fig. 1B). The number of animals containing only or predominantly F. culmorum in their gut was significantly higher (Po0.01) than the number of specimens containing only or predominantly G. graminis var. tritici. Microscopic examination of the gut contents showed that the majority of F. culmorum conidia lacked cytoplasmic content; only very few conidial cells were undamaged (Fig. 1A).
Figure 1. Gut contents of P. armata specimens collected from microcosms where inoculum of both F. culmorum and G. graminis var. tritici were present: (A) conidia of F. culmorum; arrow indicates an intact conidial cell (B) fragments of G. graminis var. tritici hyphae, conidia of F. culmorum (arrows) and mineral particles. Bars=25 mm.
ARTICLE IN PRESS Do Collembola affect relationship among fungi
Effect of springtails on fungal propagule dispersal Microscopic examination of faecal pellets produced by animals that had been fed on F. culmorum or G. graminis var. tritici colonies, revealed the presence of conidia of F. culmorum for the most part damaged, and/or hyphal fragments of both fungi. No colonies of G. graminis var. tritici developed on PDA medium from faecal pellets produced by springtails fed on this fungus; whereas few colonies of F. culmorum developed from the faecal pellets of both adults and juveniles fed on this fungus. No symptoms of disease were observed on wheat seedlings grown with P. armata specimens previously fed on fungal propagules of G. graminis var. tritici or F. culmorum and no pathogenic fungi were isolated from their tissues. The total dry biomass of wheat seedlings grown with springtails previously fed on fungal propagules of G. graminis var. tritici (97.273.5 mg) or F. culmorum (114.676.9 mg) was not significantly different from the dry weight of plants developed in the absence of animals (104.474.7 mg).
Discussion The results showed that wheat seedlings grown in the presence of P. armata and inoculum of G. graminis var. tritici and F. culmorum, two important pathogenic fungi of cereals, were mainly attacked by the former fungus, which is less palatable for P. armata. In the absence of Collembola, F. culmorum also heavily infected the plants. The study confirmed under conditions more closely resembling field than in vitro tests the previously observed feeding preference of P. armata for F. culmorum over G. graminis var. tritici (Sabatini and Innocenti, 2000b). Indeed, in the present study animals grazed on fungal propagules that colonized organic matter as occurs in agricultural soils. Moreover, the number of P. armata specimens with an empty gut found was similar to the number of Collembola with an empty gut collected in the field (Newell, 1984; Lupetti et al., 1989). This indicates that the feeding behaviour of the springtails in the microcosms resembled that in the field. Our results demonstrate that the transit through the gut damages fungal propagules; in fact, after ingestion, most conidia and hyphae lacked cytoplasmic content. Trehalase activity in the gut of P. armata is high (Urba ´ˇsek and Rusek, 1994) and this likely explains the digestion of fungal cell content, containing mainly trehalose (Siepel and Ruiter-
607 Dijkman, 1993). Therefore, the reduced viability of inoculum caused by ingestion and feeding of the animals on fungal propagules could explain why P. armata did not spread the disease caused by G. graminis var. tritici or F. culmorum. Similar results for the same and other pathogenic fungi and different species of Collembola have been reported before (Wiggins and Curl, 1979; Nakamura et al., 1992; Dromph and Borgen, 2001; Sabatini and Innocenti, 2001). The amount of viable propagules transported by springtails in their gut or cuticle probably was insufficient to induce disease. Results of this and other studies suggest that Collembola, including P. armata, prefer certain soil-borne fungi responsible of foot and root diseases of cereals (Sabatini and Innocenti, 1995, 2000a, b). Therefore, Collembola might affect competition among fungi pathogenic for the same crop, thus modulating plant diseases.
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