EFFECTS OF FUNGICIDES ON ENDOPHYTIC FUNGI AND PHOTOSYNTHESIS IN SEEDLINGS OF A TROPICAL TREE, Guarea guidonia (Meliaceae)
El efecto de fungicidas sobre los hongos endófitos y la fotosíntesis en plántulas de un árbol tropical, Guarea guidonia (Meliaceae)
MIGUEL A. GAMBOA GAITÁN1, SHIYUN WEN2, NED FETCHER3,PAUL BAYMAN
Department of Biology, University of Puerto Rico, San Juan Puerto Rico, USA.
1Departamento de Biología, Facultad de Ciencias, Universidad Nacional de Colombia, Sede Bogotá.
2Department of Computer Science and Engineering, University of Puerto Rico, Mayagüez, USA.
3Department of Biology, University of Scranton, Scranton, USA.
Presentado febrero 21 de 2005, aceptado abril 18 de 2005, correcciones mayo 25 de 2005.
]]> ABSTRACT
Endophytes are microorganisms that live within healthy plant tissues, and include fungi and bacteria. They can be mutualists, comensals or even latent pathogens. Presence of these endosymbionts may affect host physiology, for example by consuming products of photosynthesis (endophytes are heterotrophs) or producing toxic metabolites. In this work two fungicides were used to eliminate fungal endophytes from seedlings of Guarea guidonia. Lightsaturated photosynthesis (Amax) was measured in endophytefree plants and compared with control plants. Each fungicide killed different fungal endosymbionts. Phomopsis was more susceptible to benomyl while Colletotrichum was more susceptible to propiconazole. Although suggestive, values of Amax were not significantly different for each treatment compared with control plants. No prediction can be made at this point about the final outcome of a given plantendophytic fungi interaction.
Key words: endophytes, tropics, photosynthesis, fungicides, Guarea.
RESUMEN
Numerosos microorganismos viven asintomáticamente en las plantas, hallándose en todos los órganos y tejidos en los que han sido buscados. Estos microbios reciben el nombre de endófitos, y pueden ser bacterias y hongos. Su papel es ampliamente discutido en la actualidad, y se ha encontrado evidencia a favor de una relación mutualista entre el hospedero y el huésped, aunque en otros casos ésta no se ha podido demostrar. El efecto de los endófitos en su hospedero está siendo recientemente estudiado, y algunos casos muestran que pueden alterar algunas funciones de la planta hospedera. En este trabajo se estudió el efecto de la presencia de endófitos sobre la fotosíntesis en plántulas de Guarea guidonia, en las cuales se habían eliminado sus endosimbiontes naturales usando dos fungicidas comerciales. Cada fungicida tuvo efecto selectivo sobre diversos grupos de hongos y hubo diferencias en la tasa neta de fotosíntesis en ambos casos al compararlos con el grupo control. Sin embargo, los datos aquí presentados y los previamente publicados muestran que no es posible aún hacer una predicción exacta acerca del efecto de un grupo de hongos endófitos sobre la fisiología de su hospedero natural.
Palabras clave: hongos endófitos, trópico, fungicidas, fotosíntesis, Guarea.
INTRODUCTION
The exact role and the net outcome of endophytic fungi on their hosts has not been clearly established, and is a matter of current debate. To date, very few attempts have been made to measure the effects of endophytes on plant physiology, and most of these have worked with C3 grasses in temperate climates. This can be addressed by developing experiments in which endophytic microorganisms are eliminated from hosts, and compare physiological performance with naturally infected hosts. The purpose of this work is to study the effect of naturally ocurring endophytic fungi on photosynthetic rates in seedlings of a tropical timber tree. Specifically we want to test if the saturated rate of photosynthesis (Amax) is lower in infected plants than in endophytefree plants.
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MATERIALS AND METHODSGrowth and treatment of plants. Seedlings of Guarea guidonia were collected from a secondary forest at the El Verde Research Station (EVRS), Luquillo Experimental Forest, Puerto Rico. The EVRS is 400 masl, located at 18º 19’ N, 65º 49’ W, with an average annual temperatute of 21-15°C, and an average annual precipitation of 3,450 mm (Brown et al., 1983). Only healthy seedlings at the two leaf stage were chosen. Seedlings were inidually potted in 4:1 soil:sand and grown for six months in a small clearing in the forest with G. guidonia trees nearby. This was done to allow plants to become naturally infected with endophytes. Plants were then moved to a shadehouse at EVRS and fertilized monthly with 17-6-10 Sierra® fertilizer. After seven months the seedlings were moved to a shadehouse at the University of Puerto Rico, Río Piedras campus. Light levels in the shadehouse were 500 µmol m-1 s-1, roughly one third of incident light as measured late in the morning. Plants were allowed to acclimatize six month before photosynthesis measurements were taken. To remove fungi plants were sprayed with a solution of propiconazole (Tilt®, 1{[2(2,4dichlorophenyl)4propyl1,3dioxolan2yl] methyl}1h1,2,4triazole; 3.8 mL/L water), a solution of benomyl (Benlate®, methyl 1(butylcarbamoyl)2benzimidazolecarbamate; 0.4 g/L), or water alone (for control plants). Plants were sprayed every four days for three weeks. Ten plants were used for each treatment.
Photosynthesis. Light saturated rate of photosynthesis (Amax) was measured with the Li6400 photosynthesis system (LICOR Inc., Lincoln, NE, USA). Three iniduals were selected from each treatment. Measurements were done on the youngest fully expanded, healthy leaf (previously tagged) on each seedling. Measurement light level was set to PFD of 1500 µmol m-1s-1, which was provided by a LED light source. Air temperature in the leaf chamber was controlled at 26 ± 0.1 ºC and relative humidity was more than 60%. The incoming air CO2 concentration in the leaf chamber was set at 360 ppm. The leaf was placed in the leaf chamber and allowed to acclimatize to the conditions in the chamber. The readings were taken after photosynthesis and stomata had stabilized and the rates remained constant. Net photosynthesis readings were taken on two days, April 12 and April 26, 1999.
Isolation and identification of fungi. Fungi were isolated from leaves to determine extent and type of endophyte infection, and ensure that propiconazole and benomyl were effective at eliminating endophytes. The same leaves used for photosynthesis measurements were used. Four 3x5 mm pieces were cut from each leaf. Leaf pieces were surface sterilized by washing in water with Tween 20 surfactant, followed by 70% ethanol for 1 min, 2.6% sodium hypoclorite (50% Clorox®) for 3 min, and 70% ethanol for 0.5 min. After surface sterilization, leaf pieces were plated on potato dextrose agar (12 g Bacto® potato dextrose broth and 20 g agar/L, with 50 ppm each streptomycin, penicillin and tetracycline added after autoclaving), and incubated for at least two weeks at 2022 ºC. Fungi were identified by colony and spore morphology. One of the most common fungi could not be induced to sporulate, and was identified by sequencing the ITS region of nuclear ribosomal DNA using PCR primers ITS1 and ITS4 (White et al., 1990). To determine statistical significance of differences in isolation frequency, number of leaf pieces with and without fungi (and with and without particular fungi) were compared with control leaves using Fisher’s exact tests.
RESULTS
Photosynthesis. On both days, net photosynthesis readings were highest for benomyltreated plants and lowest for propiconazoletreated plants (Table 1). However, differences were not significant at the 0.05 level (p = 0.615, p = 0.098, respectively). Readings on April 26 were higher than on April 12.
| April 12 | April 26 | |
| Propiconizole | 3.98 +/- 0.34 | ]]> 4.90 +/- 0.15 |
| Benomyl | 4.80 +/- 0.43 | 7.43 +/- 0.24 |
| Control | 4.72 +/- 0.45 | 6.05 +/- 0.18 |
| Nested ANOVA | F 2.25 - 0.50 | F 2.27 - 2.53 |
| p - 0.615 | p - 0.098 |
Table 1. Mean photosynthesis of Guarea guidonia seedlings treated with fungicides (in µmol m-1s-1). ]]>
Isolation of fungi. The most commonly isolated fungi from control leaves were Phomopsis (in 40% of leaf pieces), Phyllosticta (33%), and Colletotrichum (14%). No other fungal genera were isolated. Nine percent of leaves yielded two or more fungi, and some leaf pieces also yielded unidentified bacteria. Leaf pieces from fungicidetreated leaves yielded significantly fewer fungi than control leaves (Table 2; p = 0.007 for propiconazole, p < 0.001 for benomyl). Benomyltreated leaves yielded significantly fewer fungi than propiconazoletreated leaves (p = 0.002).| Benomyl | Propiconizole | Control | |
| Phomopsis | 5 | 42 | 40 |
| Colletotrichum | 2 | 0 | ]]> 14 |
| Phyllosticta | 7 | 6 | 33 |
| Two fungi | 2 | 0 | 9 |
Table 2. Frequency of endophytic fungi in fungicide-treated leaves of Guarea guidonia (in % of leaf pieces infected).
Phomopsis was significantly less common in benomyltreated leaves than in control leaves (p < 0.001). However, frequency of Phomopsis was not significantly affected by propiconazole (p = 0.67). On the other hand, propiconazole significantly reduced frequency of Colletotrichum (p = 0.03) whereas benomyl did not (p = 0.06). Both fungicides significantly reduced frequency of Phyllosticta (p = 0.01; p < 0.01). When the two fungicides were compared to each other, benomyl was significantly better than propiconazole at eliminating Phomopsis (p < 0.001) but differences in Colletotrichum and Phyllosticta were not significant (p = 1; p = 1).
DISCUSSION
Grasses are the most studied group in terms of plantendophyte interactions. The Poaceae has shown to be broadly infected by endophytes (White, 1987), and introduced grasses are protected by endophytic fungi against herbivory, although in natural communities the scenario seems more variable (Saikkonen et al., 1999). The presence of endophytes affects plant hosts from cellular to community level, and contrasting results have been found at different levels including plant community ersity (Spyreas et al., 2001). Recently, soil fertility was demonstrated to be a key factor in determining the final outcome of plantendophyte interaction (Lehtonen et al., 2005). Taken as a whole, there is not a clear scenario in which a unique status can be attributed to the plantendosimbiont relationship. It appears as if each plantendophytic fungi interaction should be studied separately in order to establish the nature of its relationship. Fungi isolated in this study coincide with most abundant taxa found in mature G. guidonia trees (Gamboa and Bayman, 2001), which supports the idea that they are real endophytes. Although some of these genera (Phomopsis and Colletotrichum) include pathogens of many plant species, no disease caused by them on G. guidonia has been reported in the literature, nor was any detected since the beginning of our research in 1995.
Benomyl and propiconazole are broad spectrum fungicides currently employed in agriculture, and many studies on the interaction between fungi and vascular plants (for example mycorrhizae) use benomyl as a systemic fungicide in order to get uninfected plants (Fitter and Nichols, 1988; Carey et al., 1992; Merryweather and Fitter, 1996). One of the main properties of this fungicide is the almost total lack of collateral effects (see Fitter and Nichols, 1988). Propiconazole has cytokininlike properties which could affect physiological aspects of the plants, and although we observed changes in the shape of leaves from plants treated with it, our data show that its effect on photosynthesis was negligible. Both fungicides effectively diminished the load of endophytic fungi, but we observed a differential effect of fungicides on endophytes, since Phomopsis was more susceptible to benomyl while Colletotrichum was more susceptible to propiconazole. This selective effect was previously reported in barley leaves (Riesen and Close, 1987), where propiconazole diminished populations of only certain endophytes. The meaning of this differential effect of fungicides on endophytes is unclear, and should be addressed carefully.
Carroll (1991) postulated that the boundaries between mutualism and pathogenesis are very thin, and we think that is useful and necessary to set experiments for clarifying the specific role of each species in fungal endophytic assamblages. This could be done by selectively eliminating competitors with fungicides as was done here. Since it is possible that some species are benefical while others are detrimental to the host, the net outcome of this symbiosis will be the summatory of benefits and costs for each of the endosymbionts involved (Bayman et al., 2002). In Theobroma cacao (cacao), a neotropical rainforest tree like Guarea guidonia, endophyte-free plants developed more lesions than endophyteinfected plants when inoculated with a fungal pathogen (Phytophtora sp., Arnold et al., 2003). Effects of endophytes on photosynthesis were not considered in this study. But if an endophyte simultaneously protects its host against pathogens and reduces photosynthesis, its net effect on the host will depend whether light availability or disease resistance is more critical at any given time. In conclusion, no clear generalizations can be made about the effects of endophytes on plants, since available data show a continuum of interactions ranging from mutualism to pathogenicity. More experiments dealing with both domesticated and wild plants are necessary, as well as studies of the net effect of endophytes on host fitness.
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