International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(1) pp. 010-015 , January, 2011 Available online http://www.interesjournals.org/IRJPS Copyright © 2011 International Research Journals
Full Length Research Paper
Pattern of root colonization by arbuscular mycorrhizal fungi in Verbesina virgata and their effects on plant growth and leaf physical attributes Rocío Vega-Frutis1, 2,*, Irene Sánchez-Gallen1, Patricia Guadarrama3, Irene Sandoval González 1, Silvia Castillo-Argüero1 1
Facultad de Ciencias, Departamento de Ecología y Recursos Naturales, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria 04510, México, DF. 2 Current address: Departamento de Biología Evolutiva, Instituto de Ecología, A.C. Carretera Antigua a Coatepec No. 351, El Haya, C.P 91070, Xalapa; Veracruz. México. 3 Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de Abrigo s/n, C.P. 97355, Sisal; Yucatán, México. Accepted 5 January, 2011
Verbesina virgata (Asteraceae) is the stable food of generalist herbivores in xerophytic vegetation of southern Mexico City where nitrogen and phosphorous are limiting soil resources for plant growth and therefore it is likely that arbuscular mycorrhizal fungi play an important role in the biology of V. virgata. We characterized root colonization by arbuscular mycorrhizal fungi of V. virgata under field conditions and explored the effects of arbuscular mycorrhizal fungi on plant attributes in a nursery experiment. Root colonization of V. virgata under field conditions varied seasonally. Our experimental data suggest that mycorrhizal interaction is costly for V. virgata. Arbuscular mycorrhizal interaction increased the photosynthetic capabilities of V. virgata but plants grown with arbuscular mycorrhizal fungi were smaller than control plants. In addition, plants colonized by arbuscular mycorrhizal fungi had larger but thinner leaves than control ones, suggesting that the former may be more acceptable for herbivores. Key words: Arbuscular mycorrhizal fungi, Leaf area ratio, Net assimilation rate, Pedregal de San Ángel, Specific leaf area INTRODUCTION Verbesina virgata Cav. (Asteraceae) is considered a key species in remnants of xerophytic vegetation in the south of Mexico City (Castillo-Argüero et al., 2004). This perennial species contributes with about 15.1% the overall net aboveground primary productivity in the Ecological Reserve El Pedregal de San Ángel and supports a diverse and abundant community of herbivores and pollinators (Cano-Santana, 1994; Mendoza and Tovar, 1996; Figueroa-Castro and CanoSantana, 2004). Like in many other xerophytic environments nitrogen, phosphorous and water are limiting soil resources for plant growth (Schmitter, 1994). *Correspondence author: E-mail:
[email protected]; Telephone (52) (228) 8 42 18 00 ext 3005 Fax number (52) (228) 8 18 60 09
Therefore, it is likely that arbuscular mycorrhizal (AM) fungi play an important role in the biology of V. virgata by increasing its root surface area and therefore improving its nutritional and water status. AM fungi occur in ca. 74% of angiosperm (Brundrett, 2009). In general, AM fungi facilitate plant soil nutrient uptake (specially phosphorus) via extra-radical hyphae that translocate soil nutrient from some distance away of the root-depleted rhizosphere to the root cortex alleviating plant nutrition (George, 2000; Smith and Read, 2008). Although the AM fungi can consume from 4% - up to 30% of the plants’ photosynthates (Jakobsen and Rosendahl, 1990; Finlay and Söderström, 1992) the benefit is usually greater. Mycorrhizal plants are often more competitive and better suited to deal with environmental stresses (e.g. heavy metal, soil compaction, salinity and drought; Miransari,
Vega-Frutis et al. 011 2010) and pathogens of the root (Larsen and Bødker, 2001) that non-mycorrhizal plants. Considerable amount of work has been done on the effects of AM fungi on the growth of plants in different habitats (Smith and Read, 2008), but there is little information about morphology leaf. Krishna et al. (1981) observed that the size of the midrib vein, major veins, minor veins and the last vein in the leaves of mycorrhizal Elusine coracana plants were bigger than leaves of plants without mycorrhizal. The AM fungi could promote plant growth by increasing the leaf area per unit of plant biomass (Bass and Lambers, 1988; Kavanová et al., 2006). Additionally, maximum colonization by AM fungi during summer (when plants resprout vegetatively or have flowers/fruits) and less colonization during winter and early spring has been observed in many studies (e.g. Sigüenza et al., 1996; Allen et al., 1998; Lugo et al., 2003; Núñez-Castillo and Álvarez-Sánchez, 2003). In this study, we characterized root colonization by AM fungi of V. virgata under field conditions and explored in a nursery experiment the effects of AM fungi on growth and leaf attributes of V. virgata. We aimed to answer three questions: is V. virgata a mycotrophic species? Does the average percentage of root colonized by AM fungi of V. virgata under field conditions vary seasonally? Is there any differential effect of AM fungi interaction on the growth and leaf physical attributes of V. virgata? Since in our study area V. virgata is the stable food for generalist herbivores we are particular interested in the effects of AM fungi on leaf attributes such as leaf area (Krishna et al., 1981; Augé et al., 1995; Grimoldi et al., 2005; Kavanová et al., 2006), leaf area ratio (Grimoldi et al., 2005), and the specific leaf area (Grimoldi et al., 2005) that could potentially alter the plant – herbivore interaction of V. virgata. MATERIAL AND METHODS Study area The study was carried out in the Ecological Reserve of El Pedregal de San Ángel in southern Mexico City (19º 19’ N, 99º 11’ W) at 2300 m above sea level. The protected area is maintained by the Universidad Nacional Autónoma de México (UNAM, 2005) and covers an area of 237 ha. The predominant vegetation of this area is xerophytic shrubland, established upon consolidated lava flows (Castillo-Argüero et al., 2004). Precipitation distribution along the year marks two seasons, dry season from November to May and rainy season from June to October. Mean annual precipitation is 835 mm and mean annual temperature is 15.5 ºC (Castillo-Argüero et al., 2004). The soil is rich in organic matter with high contents of potassium and calcium but nitrogen and phosphorous are limiting resources (Schmitter, 1994), the pH is 5.4 ± 0.7 as a consequence of acid rains, common in Mexico City (Castellanos, 2001). Five genera (Glomus, Acaulospora, Entrophospora, Gigaspora and Scutellospora) and 25 species of AM fungi have been isolated from the study area (Hernández et al., 2003). Plant species Verbesina virgata (Asteraceae) a perennial species is the most
abundant plant and the second species in biomass production per year (108 g/m2) in the study area (Cano-Santana, 1994). It is a food source to leaf, nectar and pollen consumers, especially Sphenarium purpurascens (Acrididae), the main herbivore in the Reserve (CanoSantana, 1994; Anaya, 1999). Verbesina virgata flower from August to December as most of the whole species in this shrubland, and fruit production occurs from December to February. V. virgata is an evergreen species (Rzedowski and Rzedowski, 2001), and is dispersed by insects and wind (Meave et al., 1994). One species of this genus, V. encelioides, has been previously reported as mycotrophic (Koske et al., 1992). Patterns of mycorrhizal colonization We collected fine roots from eight adult plants of V. virgata in the field during the dry and rainy seasons. The roots were processed and stained accordingly to Koske and Gemma (1989). Colonization percentage by AM fungi was estimated following magnified intersection method (McGonigle et al., 1990). We prepared 25 root segments (15 mm long) per plant; each root segment was examined in three equally spaced sections (75 observation field per plant) under a light microscope. Arbuscular mycorrhizal colonization and growth parameters We collected seeds from randomly selected individuals in the Reserve. All seeds were surface-washed with a solution of chlorine (0.4 % v/v with water) for five minutes and then rinsed with abundant tap water. Seeds were germinated in a mixture of sterilized soil (from the study area) and vermiculite mixture (1:1 in volume). Forty-five days after germination 70 seedlings were randomly allocated in three groups. Thirty seedlings were planted in pots with sterilized soil (-M) and another 30 seedlings were potted in unsterilized soil (+M), whereas the remaining 10 seedlings were oven dried at 70° C during three days and their mass was used as an overall estimate of the initial size of potted plants. The soil for the experimental pots was collected from the study area during the dry season around the base of the plants of V. virgata. We quantified 819 spores in 100 g dry soil; the total number of spores of AM fungi was determined by wet-sieving and sucrose density centrifugation (Daniels and Skipper, 1982). We mixed (1:1 in volume) this soil with sterilized red volcanic rock, and later we steam sterilized it together with all other components of soil mixture (120 ºC) for one hour during three consecutive days (Azcón and Barea, 1997). The plants were kept in a nursery (25 °C and 80% RH); each pot (20 cm high and 11 cm diameter) contained one plant and about 750 g of soil. Additionally, the pots with sterilized soil received 1 mL of a soil microbial suspension filtered through a 5.0 µm nitrocellulose millipore filter to partially return the microorganisms to the sterilized soil. The plants were watered and randomly redistributed within the nursery every other day for 120 days. Fifteen days after initiating the experiment, we marked the leaves of 15 plants in each treatment and recorded the survival of leaves each 15 days; we made sure that marks did not cause any leaf damage. At the end of the experiment all plants were harvested and oven dried to estimate separately leaf biomass (LM), shoot biomass (SM), and root biomass (RM), the three components of the total dry biomass (TDM). The root:shoot ratio was estimated as
R:S=
RM . (LM + SM )
Before the leaves were dried we
estimated leaf area (LA) in an automatic image analyzer and following Hunt (1982) we calculated leaf area
Int. Res. J. Plant Sci. 012
ratio LAR =
growth
LA TDM
, specific leaf area SLA =
rate RGR =
LA LM
ln (TDM t 2 ) − ln (TDM t1 ) , t 2 − t1
, relative
and
net
assimilation rate
NAR =
(TDM t 2 − TDM t1 ) × [ln ( AFt 2 ) − ln ( AFt1 )] , (t 2 − t1) × ( AFt 2 − AFt1 )
where, t1 refers to the data of the 10 plants harvested at the beginning of the experiment, t2 refers to the data of the final harvest, t2 – t1 = time elapsed between harvests (120 days). In addition, we collected root samples from six plants from both treatments at the end of the experiment to estimate the percentage root colonization by AM fungi as described above. Statistical analysis We used t-test to compare colonization percentage by AM fungi in V. virgata plant adults between seasons. Colonization percentages were arc-sin transformed before the analyses. To explore mycorrhizal dependent responses of V. virgata we used t-test and for RGR and NAR we compared the difference between the treatments following a bootstrap procedure with 5000 random interactions. For leaf demography, we utilized a censored survival analysis. All analyses were performed with the statistical language R (R Development Core Team 2007). Data are presented as means ± standard error.
fungi colonized plants (4.89 ± 0.33 g) was significantly (t56 = 2.6, P < 0.05) lower than average TDM of control plants, those grown in the absence of symbionts (6.26 ± 0.39 g), but RGR did not differ between treatments (Pbootstrap = 0.24). In the same way control plants (2.26 ± 0.17 g) allocated significantly (t56 = 5.4, P < 0.001) more resource to RM (Figure 1b) than did plants grown with AM fungi (1.39 ± 0.12 g). Consistent with these observations, R:S (Figure 1c) and NAR (Figure 1d) were significantly higher (t56 = 5.4, P < 0.001 and Pbootstrap < 0.0001, respectively) in control plants (0.56 ± 0.02 g and 0.40 ± 0.02 g mm2 day-1, respectively) than in colonized plants (0.37 ± 0.02 g and 0.30 ± 0.02 g mm2 day-1, respectively). Root colonization by AM fungi also had significant effects on leaf attributes (Figures 1e-g) such as LA (t56 = 2.8, P < 0.05), SLA (t56 = 3.9, P < 0.001) and LAR (t56 = 5.5, P < 0.001). Those plants grown with AM fungi had on average a larger LA (555.2 ± 35.6 mm2), SLA (434.8 ± 21.6 mm2 g1 ) and LAR (126.15 ± 9.2 mm2 g-1) than control plants (435.7 ± 23.8 mm2, 330.5 ± 14.2 mm2 g-1, 72.41 ± 3 mm2 -1 g , respectively) but we detected no effect of AM fungi on the LM (t56 = 1.5, P > 0.05). Further, leaves survival analysis showed that leaves in plants grown with AM fungi lasted significantly longer (χ2 1= 16.9, P < 0.001) than those of control plants (Figure 1h), namely in the treatment without AM fungi the survival of leaves were lower than with AM fungi.
RESULTS Patterns of mycorrhizal colonization Total percentage of root colonized by AM fungi in wild reproductive plants of V. virgata was on average 41 ± 4.3% and there was a strong seasonal pattern. The total percentage of root colonized by AM fungi in the dry season (30.3 ± 2.8%) was significantly lower (t14 = 3.7, P < 0.001) than in the rainy season (53.1 ± 5.4%). In contrast, the incidence of vesicles was significantly higher (t14 = 3.4, P < 0.001) in the dry season (6.1 ± 1.3 %) than in the rainy season (1.5 ± 0.3%) whereas arbuscules and coils were rarely observed (< 0.4 % in either season). Total root colonization by AM fungi in plants grown in the nursery (52.2 ± 9.8%) was similar to that observed in the rainy season in wild plants but the incidence of vesicles was over 10 times greater than that observed in wild plants (15.5 ± 4.8 %), although arbuscules were not observed. Also we found that the procedure of soil sterilization was not hundred percent effective since the plants grown in the autoclaved substrate had on average 12.6 ± 2.2% of their root length colonized by hyphae of AM fungi. Arbuscular mycorrhizal colonization and growth parameters The experimental plants showed differential effects of AM fungi interaction. The average TDM (Figure 1a) of AM
DISCUSSION Verbesina virgata is a mycotrophic species in the Ecological Reserve El Pedregal de San Ángel, previously Koske et al. (1992) reported that plants of V. encelioides growing on coastal strand in Hawaii are mycotrophic. In the field, we observed a higher incidence of hyphae in the rainy season and higher incidence of vesicles in the dry season in the roots of wild V. virgata is in agreement with the general pattern of root colonization reported in other vegetation types (Sigüenza et al., 1996; Allen et al., 1998; Núñez-Castillo and Álvarez-Sánchez, 2003). In general, arbuscules production tends to increase during the flowering period when the demand of soil nutrients increases, whereas the incidence vesicles tend to be more frequently during post flowering period (Allen et al., 1998; Núñez-Castillo and Álvarez-Sánchez, 2003). Consistent with this general pattern the incidence of arbuscules was higher in V. virgata during flowering period (August – December) (although the overall percentage of arbuscules was low), whereas vesicles were more frequently observed in the dry season. Similar patterns have been reported for other Asteraceae such as Aster tripolium and Inula crithmoides (Carvalho et al., 2001). The percentages of root colonization in the experimental plants grown in the presence of AM fungi were comparable to those observed during the rainy
Vega-Frutis et al. 013
Figure 1. Growth responses (mean ± standard error) of Verbesina virgata. a) Total dry biomass (TDM), b) root biomass (RM), c) root:shoot ratio (R:S), d) net assimilation rate (NAR), e) leaf area (LA), f) specific leaf area (SLA), g) leaf area ratio (LAR) and h) leaf survivorship. M+, plants grown with arbuscular mycorrhizal fungi and M-, plants grown in the absence of the symbionts.
season in adult plants. Therefore, this evidence suggests that V. virgata is a mycotrophic species in all stages of development. However, we found no effect of root colonization on RGR. Plants grown with AM fungi had less biomass than control plants, which contrasts with a vast number of examples in which AM fungal was associated with an increased plant biomass (e.g. Corkidi and Rincón, 1997; Guadarrama et al., 2004; Pezzani et al., 2006). Nevertheless, some studies have showed decreased or no effect on plant biomass when the plants are colonized by AM fungi. Philip et al. (2001) observed in Lythrum salicaria that the colonization by AM fungi decreased plant biomass both aboveground and belowground. In the same way, Botham et al. (2009) observed in Fragaria virginiana that inoculated plants with AM fungi grew at rates similar that control plants. The absence of a positive effect of mycorrhizal colonization on growth has been attributed to light limitation of photosynthesis (Dunham et al., 2003) or availability of soil nutrients (Johnson, 2010). However, in this study is not the case because in the nursery the plants grew with
light and although we did not estimate the abundance of soil nutrients, in the study area the nitrogen and phosphorous are limiting resources (Schmitter, 1994). We found, however, a change in biomass allocation between roots and shoots in AM fungi plants, which is in agreement with several studies (e.g. Cuenca et al., 1998; Camargo-Ricalde et al. 2010). In absence of AM fungi, V. virgata allocated more resources to the roots (increase R:S ratio) than to the shoots, possibility increasing their absorptive surface for uptake nutrients and water, while plants with AM fungi allocated less resources to the roots (decrease R:S ratio) because could be obtaining mineral nutrients from both extra-radical hyphae and soil solution. Accordingly, we also observed that AM plants had a larger LA, LAR and SLA compared with control plants, which is in agreement with findings of other studies with different plants species (Waschkies et al., 1994; Caglar and Bayram, 2006). In addition, survival of leaves were longer in AM plants than the non-mycorrhizal control plants. Generally, NAR positively correlates with LA and SLA as large values for these variables imply a greater
Int. Res. J. Plant Sci. 014
photosynthetic capability of plants (Lambers et al., 1998). Nevertheless, in spite of significantly higher LA, SLA and LAR in AM plants, the NAR was low compared with control plants. Lambers et al. (1998) suggested that low NAR values could reflect a high demand of carbon for root respiration. Therefore, it seems that AM costs surpass benefits in V. virgata and in spite of increase in foliar attributes; these plants were smaller than control plants at the end of the experiment. It is worth to consider that plants colonized by AM fungi had larger and thinner leaves than control ones, suggesting that the former plants may be more acceptable for herbivores (Cates and Rhoades, 1977; Rhoades, 1979) but herbivores did not contribute to low dry biomass of plants colonized by AM fungi since herbivory was absent under experimental conditions. Our results must be treated with caution, since only the soil of the non-mycorrhizal treatment was sterilized. Several authors have discussed that the process of steam sterilization alter the structure and physicochemical properties of the soil (Jones and Smith, 2004; Pezzani et al., 2006) but others authors have found no changes in soil nutrients before and after sterilization (Smith and Smith, 1981; Kula et al., 2005). Since our results are consistent with a vast literature we are inclined to support the idea that the observed changes are associated to the presence of AM fungi rather than be merely a consequence of potential changes in the chemical composition of the soil considering than nitrogen and phosphorous are intrinsically limiting resource for plant growth in the study area. In conclusion, reproductive plants of V. virgata showed a strong temporal variation in root colonization by AM fungi and seedlings showed a positive effect of AM fungi in leaf attributes such as LA, SLA and LAR whereas for TDM there was a negative effect. Because it is likely that leaf attributes were modified by root colonization by AM fungi we speculate that such changes could in turn modify other interactions such as herbivory (Gange et al., 2002; Klironomos et al., 2004; Kula et al., 2005; Bennett et al., 2006). The great relevance of the study area is that V. virgata is the stable food for the grasshopper Sphenarium purpurascens (Cano-Santana, 1994; Mendoza and Tovar, 1996; Anaya, 1999), and various other herbivores in El Pedregal de San Ángel. In the context, our data suggest that V. virgata is a species that can tolerate herbivory despite its thin leaves (high LA and high SLA) (Coley, 1983) and these characteristics may be at least partially molded by AM interactions. Future work is needed to generate patterns between triple-interaction (plant – AM fungi – herbivory) for V. virgata. ACKNOWLEDGMENTS We thank Yuriana Martínez-Orea, Oswaldo NúñezCastillo and Ricardo Vargas for their field and nursery
assistance. We also thank Patricia Olguín for keeping the nursery in perfect conditions during our experiment. Special thanks to Roger Guevara for his advice and comments that improved the manuscript. Financial assistance by Programa de Apoyo a Proyectos Institucionales de Mejoramiento de la Enseñanza (PAPIME; Project No. DO203398) is gratefully acknowledged. REFERENCES Allen EB, Rincón E, Allen MF, Pérez-Jimenez A, Huante P (1998). Disturbance and seasonal dynamics of mycorrhizae in a tropical deciduous forest in México. Biotropica. 30: 261-274. Anaya MC (1999). Variación temporal de los niveles de herbivoria de las compositae de la Reserva del Pedregal de San Ángel (México). Bachelor Thesis, Universidad Nacional Autónoma de México, México. Augé RM, Stodola AJ, Ebel RC, Duan X (1995). Leaf elongation and water relations of mycorrhizal sorghum in response to partial soil drying: two Glomus species at varying phosphorus fertilization. J. Exp. Bot. 46: 297-307. Azcón R, Barea J (1997). Mycorrhizal dependency of a representative plant species in Mediterranean shrublands (Lavandula spica L.) as a key factor to its use for revegetation strategies in desertificationthreatened areas. Appl. Soil Ecol. 7: 83-92. Bass R, Lambers H (1988). Effects of vesicular-arbuscular mycorrhizal infection and phosphate on Plantago major ssp. pleiosperma in relation to the internal phosphate concentration. Physiol plantarum. 74: 701-707. Bennett AJ, Alers-Garcia J, Bever JD (2006). Mechanisms of tritrophic interactions between plants, herbivores and AM fungi. Am. Nat. 167: 141-152. Botham R, Collin CL, Ashman T-L (2009). Plant-mycorrhizal fungus interactions affect the expression of inbreeding depression in wild strawberry. Int J Plant Sci. 170: 143-150. Brundrett MC (2009). Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil. 320: 37-77. Caglar S, Bayram A (2006). Effects of vesicular-arbuscular mycorrhizal (VAM) fungi on the leaf nutritional status of four grapevine rootstocks. Europ. J. Hort .Sci. 71: 109-113. Camargo-Ricalde SL, Montaño NM, Reyes-Jaramillo I, JiménezGonzález C, Dhillion SS (2010). Effect of mycorrhizae on seedlings of six endemic Mimosa L. species (Leguminosae-Mimosoidae) from the semi-arid Tehuacán-Cuicatlán Valley, Mexico. Trees. 24: 67-78. Cano-Santana Z (1994). Flujo de energía a través de Sphenarium purpurascens (orthoptera: Acrididae) y productividad primaria neta aérea en una comunidad xerófita. Doctorate Thesis, Universidad Nacional Autónoma de México, México. Carvalho LM, Caçador I, Martins-Loução MA (2001). Temporal and spatial variation of arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal). Mycorrhiza. 11: 303-309. Castillo-Argüero S, Montes-Cartas G, Romero-Romero MA, MartínezOrea Y, Guadarrama P, Sánchez-Gallen I, Núñez-Castillo O (2004). Dinámica y conservación de la flora del matorral xerófilo de la Reserva Ecológica del Pedregal de San Ángel (D.F. México). Bol. Soc. Bot. Mex. 74: 51-61. Castellanos VI (2001). Ecología de la oviposición de Sphenarium purpurascens (Orthoptera: Pyragomorphidae) en la Reserva del Pedregal de San Ángel, México, D.F. Bachelor Thesis, Universidad Nacional Autónoma de México, México. Cates RG, Rhoades DF (1977). Prosopis leaves as a resource for insects. In: Simpson BB (ed) Mesquite. Dowden, Hurchinson and Ross, Pennsylvania, pp. 61-83. Coley PD (1983). Herbivory and defensive characteristics of tree species in a lowland tropical forest. Ecol. Monogr. 53: 209-223. Corkidi L, Rincón E (1997). Arbuscular mycorrhizae in a tropical sand
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