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Plant Species Biology (2012) ,

doi: 10.1111/j.1442-1984.2011.00358.x

Nematode-induced galls in Miconia albicans: effect of host plant density and correlations with performance
psbi_358 1..7

LEONARDO RODRIGO VIANA,* FERNANDO AUGUSTO OLIVEIRA SILVEIRA,* JEAN CARLOS SANTOS, LUIZ HENRIQUE ROSA, JUVENIL E. CARES, ADALBERTO CORRA CAF-FILHO and GERALDO WILSON FERNANDES* *Evolutionary Ecology and Biodiversity and Department of General Biology, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Minas Gerais, Institute of Biology, Universidade Federal de Uberlndia, 38400-901, Uberlndia, Minas Gerais, and Department of Phytopathology, Institute of Biological Sciences, Universidade de Braslia, 70910-900, Braslia, Distrito Federal, Brazil

Abstract
The study of nematodes parasitizing native plants plays a crucial role in understanding plantpathogen interactions. In the present study we describe the patterns of attack by an undescribed species of Ditylenchus occurring in Miconia albicans (Melastomataceae), a widespread, native shrub from the Brazilian cerrado. We also tested the hypothesis that nematode-induced leaf galls negatively correlate to host plant performance and that gall density is a function of host plant density. We collected paired healthy and attacked shoots from 28 individuals of M. albicans and estimated the leaf area lost to nematodeinduced galls in up to 10 leaves per shoot. We analyzed the relationships between leaf area lost to nematode galls and reproductive traits. Nematode attack levels were also compared to the spatial distribution of the host plant. Inorescence length and fruit production were signicantly reduced in attacked shoots compared with healthy shoots. Seeds from attacked shoots showed no signicant reduction in germinability or germination time when compared with seeds collected from healthy shoots. Gall density was positively correlated with host density. Despite being seldom studied in tropical ecosystems, nematodes may play an important role in plant tness and in structuring tropical communities.
Keywords: biocontrol, Brazil, cerrado, density-dependent parasitism, Ditylenchus, plant nematode interaction. Received 2 March 2011; revision received 6 June 2011; accepted 22 August 2011

Introduction
Plant-parasitic nematodes comprise a huge group of organisms that colonize plant tissues reducing host reproduction (Sasser & Freckman 1987; Dangl & Jones 2001). They can be the cause of devastating epidemics in crops leading to serious economic losses, attracting the attention of farmers, plant breeders and scientists (Plowright et al. 2002). Nematodes manipulate host physiology to optimize their parasitic lifestyles (Freckman & Caswell 1985), adversely affecting host photosynthesis, water and nutrient translocation, respiration, cell membrane permeability
Correspondence: Geraldo Wilson Fernandes Email: gwilson@icb.ufmg.br 2012 The Society for the Study of Species Biology

and the processes of transcription and translation (Agrios 2005). Nematodes can also directly or indirectly inuence nutrient uptake in plants by their negative effect on root health (Vaast et al. 1998). All these physiological disturbances can indirectly result in lower seed output. Some nematode species, however, may have a greater impact on host tness by colonizing reproductive structures (Sturhan & Brzeski 1991). As most plantnematode studies have been carried out on economically important crops (Plowright et al. 2002), virtually nothing is known about the patterns and processes of nematodes parasitizing native, Neotropical, noneconomically important plants. Although it is quite clear that plant cover inuences the diversity and community structure of soil nematodes in the Brazilian amazonian

L. R. VIANA ET AL. 1998). Miconia albicans appears to play an important role in the cerrado because of its widespread distribution and biotic interactions. Its greenish fruits are dispersed by birds, rodents and possibly ants (Magnusson & Sanaiotti 1987; Goldenberg & Shepherd 1998). Leaves are simple, opposite and elliptic, with a pubescent, whitish abaxial surface. Leaves are continuously produced year round so that expanding and mature leaves can be found in both dry and rainy seasons. The foliage is exposed to high levels of herbivory caused by insects (Paleari & Santos 1998) and casual observations indicate that at least one galling insect attacks its leaves and another species induces a stem gall. In the study area, owers are produced in a scorpioid panicle from June to October and mature fruits occur during the mid-rainy season (Bedetti et al. 2011). There are no reports of nematode galling on this species.

and cerrado vegetations (Huang & Cares 2006), the ecology of nematodes and their host plants is still unclear in the tropics, where few studies of plantnematode interactions have been conducted (Seixas et al. 2004a,b). Some aggressive plant-parasitic nematodes, with narrow host ranges have been regarded as potential agents for biological control of undesirable plant populations. The nematode Fergusobia sp., a galling pathogen of owering buds and leaves of myrtaceous plants in Australia, and its vector y (Fergusonina sp.) have been studied with the aim of establishing biological control programs for Melaleuca quinquenervia (Myrtaceae) (Cav.) S.T. Blake, an aggressive invader in Australia and Florida (Goolsby et al. 2000; Giblin-Davis et al. 2004). Miconia calvescens de Candolle, the velvet tree, was introduced as an ornamental in Hawaii and other Pacic islands, and is now growing out of control as an exotic species. In a search for organisms with the potential to biologically control M. calvescens, Seixas et al. (2004a,b) singled out Ditylenchus drepanocercus Goodey, 1953 because it is capable of causing angular leaf spot and because it has narrow host specicity. Therefore, studying the ecology of nematode-induced galls is of interest to gain insight into their potential as agents of biological control. In the present study, we report the attack by an undescribed Ditylenchus species on Miconia albicans (Sw.) Triana, a native shrub from the cerrado (Brazilian savanna). The patterns of pathogen attack on native plants and their impact on the reproductive success of their hosts is unknown. Thus, the role of pathogens in regulating plant population structure is still unclear. We tested the hypothesis that nematode-induced galls negatively correlate to host plant performance and that gall density is a function of host plant density.

Nematode extraction, identication, histopathology and pathogenicity test

For nematode extraction, samples of galled leaves of M. albicans were placed in a conventional blender with 500 mL of distilled water and blended for 20 s at the highest speed. The resulting leaf suspension was poured through two stacked metal sieves, a 60-mesh screen on top of a 400-mesh screen. The nematode suspension containing ne leaf residue recovered from the lower screen was claried by centrifugation according to the modied method of Jenkins (1964). Nematodes and plant residue were concentrated at the bottom of the centrifuge tubes by centrifugation for 5 min at 1006 g, the supernatant was discarded and the pellet suspended again in a sucrose solution (456 g in 1 L of water). Following a second centrifugation for 1 min at 112 g, the oating nematodes were recovered free from sugar in a 400 mm mesh screen. The same procedures were applied to samples of non-galled leaves. The nematodes were heat killed by immersion in water at 50C for 1 min, xed in 3% formalin (Flegg & Hooper 1970) and glycerin inltrated following the method of Seinhorst (1959). For identication, adults of both sexes were hand picked under a stereo microscope, mounted with anhydrous glycerin on glass slides and covered with coverslips. The slides were sealed with Canada balsam. The nematode specimens were identied based on morphology and morphometry with the aid of a compound light microscope according to Sturhan and Brzeski (1991). For histopathological observations, freehand sections through fresh leaf galls were cut with a razor blade and thin sections were stained with cotton blue and mounted on glass slides. The sectioned galls were examined with a light microscope. For the pathogenicity test, galled leaves were collected from the same study site (PES) and brought to the Plant
Plant Species Biology ,

Materials and methods

Study site and species

The present study was conducted at the Pirapitinga Ecological Station (PES) in Trs Marias, Minas Gerais, southeastern Brazil. The PES is a 1100 ha man-made island, created in 1965 as a result of the lling of the Trs Marias reservoir (1823S, 4520W), at an altitude of 560 m a.s.l. The average annual temperature in the region varies from 21C to 25C, and the average annual precipitation is 1200 mm (Bedetti et al. 2011). Miconia albicans is a widespread evergreen shrub of the cerrado vegetation of Brazil reaching up to 3 m tall. It occurs from southern Mexico to the Brazilian state of Paran (Goldenberg 2004). Miconia albicans is an obligatory apomictic species. The whitish, small and scentless owers are not visited by bees and produce pollen with null viability (Renner 1989; Goldenberg & Shepherd
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N E M AT O D E G A L L S I N A N E O T R O P I C A L S H R U B Pathology Laboratory of the University of Braslia. An assay was carried out on M. albicans specimens growing in native cerrado vegetation in the Biological Experimental Station of the University of Braslia. A suspension of living nematodes, obtained as described above, was diluted to approximately 15 nematodes per mL and applied by misting both leaf surfaces of healthy young leaves, after sunset to preserve leaf moisture. Healthy plant shoots in the same plants served as uninoculated controls.

Impact on plant traits

To evaluate the impact of nematodes on host-plant reproduction, we determined two categories based on symptoms of nematode gall abundance; one hereafter termed healthy (absence of nematodes) and the other, attacked (presence of nematodes). We collected one healthy and one attacked shoot from 28 individuals of M. albicans in the eld. A 500-m transect was established and adult plants up to 2 m either side of the transect were sampled. Samples were taken in December 2002 during the fruiting peak, allowing for a comparison of reproductive traits between shoots in both categories. The leaf area attacked by nematodes was visually estimated in the eld and classied into six categories: (0) undamaged; (1) 15%; (2) 612%; (3) 1325%; (4) 2650%; (5) 51100%. Up to 10 leaves per shoot were examined. The index of nematode damage per shoot (IP) was calculated according to the following formula (Garca-Guzmn & Dirzo 2001):

attacked shoots was compared. From the same 28 individuals we randomly sampled in December 2002, mature fruits were collected, washed and dried for 2 days. Seeds were placed in Petri dishes covered with two sheets of lter paper and moistened with Nistatin solution (2%), as necessary, to avoid the growth of contaminant fungi. The Petri dishes were placed in germination chambers under a constant temperature of 25C and a 12-h photoperiod for 30 days, a period of time in which non-dormant seeds tend to germinate. Germination was veried every 23 days and seeds were considered to have germinated when radicle protrusion was observed. From each shoot, four replicates of 10 randomly selected seeds were set per individual. As germinability was compared from seeds removed from healthy and attacked shoots from the same individual, eight replicates were tested (n = 56; 4 replicates 14 individuals). The germination percentage values were transformed as the square root arcsine to obtain data normalization and we compared the seed germinability of attacked and healthy shoots using a paired t-test (Zar 1996). We also calculated the mean germination time (MGT) using the following equation:

MGT = ( nd ) N
where n is the number of seeds that germinated between scoring intervals, d is the incubation period in days at that time point and N is the total number of seeds that germinated in the treatment (Tompsett & Pritchard 1998). The MGT values from attacked and healthy shoots were also compared using a paired t-test in an attempt to verify whether nematode attack decreased the speed of seed germination.

IP = [(Li )(i )] n
where Li is the number of leaves in each category of damage, i is the category of damage and n is the total number of leaves sampled per plant. We considered healthy shoots to be those that presented IP values in the categories 0 and 1 because damage at these levels was almost imperceptible to the naked eye and therefore less reliably attributable to nematode attack. We considered attacked shoots to be those in which the IP values were greater than or equal to 6%, corresponding to the remaining categories (25). These IP categories were also used to test nematode attack in relation to host spatial distribution. Using a ruler we measured shoot length (from internode to shoot apex) and inorescence length (from rachis base to its apex). We also counted the number of fruits in both attacked and healthy shoots to detect nematode effects on plant reproduction. Because of the non-normal nature of our data a Wilcoxon test was used to compare shoot length, inorescence length and fruit production between attacked and healthy shoots (Zar 1996). To test the impact of nematodes on seed germination, the germination of seeds removed from healthy and
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Host plant spatial distribution and gall abundance

In the study area, M. albicans individuals were easily visually recognized as occurring in isolated and clumped patches. To assess whether host density affects gall abundance we measured the distances of the ve closest individuals of M. albicans from 30 selected individuals along a 500-m transect. The 30 plants were visually separated a priori into two groups: 15 were considered to be isolated (average distance to the ve nearest neighbors = 341 41.2 cm) and 15 clumped (average distance to the ve nearest neighbors = 86.5 12.0 cm). The distance between isolated individuals was signicantly larger than that between clumped individuals (MannWhitney U-test; U = 223, P < 0.001). We evaluated 10 leaves from each M. albicans individual using the IP index. A linear regression analysis was used to compare the mean distance (log-transformed) between plants versus an index of nematode damage per plant (IP).
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L. R. VIANA ET AL. vascular tissues. The gall comprised more than one hollow chamber or locus (Fig. 1b) separated from one another by a parenchymatous septum. Laboratory analyses indicated the presence of only one species of nematode associated with M. albicans leaf galls. Juvenile, adult male and female nematodes were recovered from galled leaves, but no nematodes were recovered from ungalled leaves. Juvenile, female and male nematodes (Fig. 1c) were observed moving freely in each of the locus spaces; nematode eggs were also observed. The nematode was

Results

Gall etiology
The nematode-induced galls are amorphous and can be found isolated or coalescent. The greenish-brown spherical galls found mainly on the abaxial side of the leaf (with a few on the adaxial side) were densely covered with trichomes (Fig. 1a). Cross-sections of the galls showed a structure comprising tissues spanning from the epidermis through to the spongy mesophyll, including bundles of

Fig. 1 Galls induced by Ditylenchus sp. on Miconia albicans. (a) Abaxial view of a M. albicans leaf with galls, (b) leaf gall showing the hollow chambers or loci (arrows) in cross-section and (c) micrograph of nematode bodies (n) inside a locus of a leaf gall (n = each individual nematode). 2012 The Society for the Study of Species Biology Plant Species Biology ,

N E M AT O D E G A L L S I N A N E O T R O P I C A L S H R U B
Table 1 Impact of Ditylenchus sp. galls on Miconia albicans non-reproductive and reproductive traits assessed in December 2002 at Pirapitinga Ecological Station, Minas Gerais, Brazil Plant trait Shoot length (cm) Inorescence length (cm) No. fruits produced Seed germinability (%) MGT (days) Healthy shoots 15.7 2.2 (28) 15.9 2.3 (28) 42.8 4.6 (27) 58.1 26.0(56) 11.2 2.7 (56) Attacked shoots 13.5 1.6 (28) 5.6 0.5 (28) 16.0 7.2 (27) 54.4 28.2(56) 10.4 2.8 (56) W

232.5 ns 304* 337*** 0.47 ns 1.52 ns

Values are mean standard deviation and the numbers in parentheses represent sample size (n). Bold values are the t-values from paired t-tests. *P < 0.01 and ***P < 0.001.

identied as Ditylenchus sp. with characteristics of an undescribed amphimictic species. Leaves were observed daily for 2 weeks after inoculation. Unlike the symptoms observed from natural infections, articially inoculated shoots had galls almost equally dispersed on both sides of the leaf, stem and petioles. Four weeks after inoculation, eggs, juveniles and adult nematodes were recovered from galls extracted from inoculated leaves. Uninoculated shoots remained free of galls.

Impact on plant traits

Nematodes not only occurred on leaves (Fig. 1a), but they were also able to colonize owers and fruits. Gall abundance was unrelated to shoot length (U = 420, P = 0.646; Table 1). However, inorescence length (U = 575, P 0.001) was smaller on attacked shoots compared with healthy ones. The number of fruits produced per shoot on attacked shoots was nearly one-third of that produced per healthy shoot (U = 688, P < 0.001; Table 1). Furthermore, from the 28 attacked shoots, only 15 yielded healthy fruits. The fruits of the other 13 attacked shoots were severely damaged, bearing no seeds and having an overall clumped and necrotic appearance. In contrast, all but one healthy shoot yielded healthy fruits (n = 27). A decrease of 4% in seed germinability of attacked shoots was observed when compared with healthy ones, but this difference was not statistically signicant (Table 1). Moreover, the MGT of seeds collected from healthy and attacked shoots did not differ statistically (Table 1).

Fig. 2 Linear regression analysis between mean distance to the ve nearest neighbors of Miconia albicans conspecics and the index of pathogen damage (IP) of galls induced by Ditylenchus sp. y = 2.09 - 0.765x.

Host plant spatial distribution and gall abundance

Individuals of M. albicans at high densities supported higher abundance of nematode-induced galls than plants growing at low densities. Mean plant distance and IP were negatively related (F1.28 = 31.2; P < 0.05; r2 = 0.53; Fig. 2).

Discussion
Many phytopathogenic nematodes decrease host performance by altering host physiology (Hussey et al. 2002;
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Doyle & Lambert 2003; Lohar et al. 2004). Plant-parasitic species of Ditylenchus have been shown to induce a diversity of plant reactions, including tissue maceration on bulbs and tubers, and necrotic lesions and galls on stems, leaves and owering organs (Sturhan & Brzeski 1991). The presence of eggs and several active nematode stages moving freely inside the gall loci, in addition to the results of the pathogenicity test, unequivocally indicate that this nematode is responsible for the development of leaf galls on M. albicans. This is the rst report of Ditylenchus on M. albicans. The negative correlation between gall abundance and inorescence size and fruit production suggests a negative impact on plant reproduction attributed to nematode galls. Drainage of photosynthates is frequently observed in plants attacked by pathogens, including plant nematodes (Agrios 2005), particularly those that attack plant reproductive structures, such as Ditylenchus spp. (Sturhan & Brzeski 1991), Anguina spp. (Krall 1991) and Aphelenchoides spp. (Nickle & Hooper 1991). Because photosynthates would otherwise be allocated to reproductive structures, the whole process thereby potentially reduces plant tness (Gilbert 2002). However, because of the
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L. R. VIANA ET AL.
Bedetti C. S., Aguiar D. B., Jannuzzi M. C., Moura M. Z. D. & Silveira F. A. O. (2011) Abiotic factors modulate phenotypic plasticity in an apomictic shrub (Miconia albicans (SW.) Triana) along a soil fertility gradient in a Neotropical savanna. Australian Journal of Botany 59: 274282. Dangl J. L. & Jones J. D. G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826833. Doyle E. A. & Lambert K. N. (2003) Meloidogyne javanica chorismate mutase 1 alters plant cell development. Molecular Plant Microbe Interactions 16: 123131. Flegg J. J. M. & Hooper D. J. (1970) Extraction of free-living stages from soil. In: Southey J. F. (ed.). Laboratory Methods for Work with Plant and Soil Nematodes. Technology Bulletin. Commonwealth Agricultural Bureaux, London, pp. 522. Freckman D. W. & Caswell E. P. (1985) The ecology of nematodes in agroecosystems. Annual Review of Phytopatology 23: 275 295. Garca-Guzmn G. & Dirzo R. (2001) Patterns of leaf-pathogen infection in the understory of a Mexican rain forest: incidence, spatiotemporal variation, and mechanisms of infection. American Journal of Botany 88: 634645. Giblin-Davis R., Center B. J., Davies K. A., Purcell M. F., Scheffer S. J., Taylor G. S., Goolsby J. A. & Center T. D. (2004) Histological comparisons of Fergusobia/Fergusonia-induced galls on different myrtaceous hosts. Journal of Nematology 36: 249262. Gilbert G. S. (2002) Evolutionary ecology of plant diseases in natural ecosystems. Annual Review of Phytopathology 40: 1343. Goldenberg R. (2004) O gnero Miconia (Melastomataceae) no estado do Paran. Acta Botanica Brasilica 18: 927947. Goldenberg R. & Shepherd G. J. (1998) Studies on the reproductive biology of Melastomataceae in Cerrado vegetation. Plant Systematics and Evolution 211: 1329. Goolsby J. A., Makinson J. & Purcell M. (2000) Seasonal phenology of the gall-making y Fergusonina (Diptera: Fergusoninidae) and its implications for biological control of Melaleuca quinquenervia. Australian Journal of Entomology 39: 336343. Huang S. P. & Cares J. E. (2006) Nematode communities in soil under different land-use systems in Brazilian Amazon and savannah vegetation. In: Moreira F. M., Siqueira J. O. & Brussaad L. (eds). Soil Biodiversity in Amazonian and Other Brazilian Ecosystems. CABI Publishing International, Wallingford, pp. 163183. Hussey S. R., Davis E. L. & Baum T. J. (2002) Secrets in secretions: genes that control nematode parasitism of plants. Brazilian Journal Plant Physiology 14: 183194. Jenkins W. R. (1964) A rapid centrifugal-otation technique for separating nematodes from soil. Plant Disease Reporter 48: 692. Krall E. L. (1991) Wheat and grass nematodes: Anguina, Subanguina, and related nematodes. In: Nickle W. R. (ed.). Manual of Agricultural Nematology. Marcel Dekker, New York, pp. 721 760. Lohar D. P., Schaff J. E., Laskey J. G., Kieber J. J., Bilyeu K. D. & Bird D. M. (2004) Cytokinins play opposite roles in lateral root formation, and nematode and rhizobial symbioses. The Plant Journal 38: 203214. Magnusson W. E. & Sanaiotti T. M. (1987) Dispersal of Miconia seeds by the rat Bolomys lasiurus. Journal of Tropical Ecology 3: 277278. Nickle W. R. & Hooper D. J. (1991) The Aphelenchina: bud, leaf, and insect nematodes. In: Nickle W. R. (ed.). Manual of

ability of plants to tolerate the attack of natural enemies (Strauss & Agrawal 1999), there may be no overall consequences to plant tness. Therefore, these results should be viewed with caution and further experimental studies are required to establish a causal effect of galls on plant tness. Pathogen damage by Ditylenchus was negatively correlated to distance among neighboring M. albicans clusters, showing that plants at higher densities had lost more leaf area to nematode-induced galls than isolated plants. In other density-dependent systems, plant-pathogen attack also generally decreases as distance from the inoculum increases (Gilbert 2002). Little is known about nematode attack patterns in natural ecosystems. For nematode species that infect above-ground plant parts, one or more developmental stages must be able to move quickly within thin and often transient lms of water on foliar surfaces. However, most foliar-parasitic nematodes have one or more infective stages that occur in the soil. These stages typically must nd roots or stems emerging from the soil to ascend foliage in order to complete their life cycle and/or disperse (Robinson 2002). Therefore, density dependency is consistent with this mode of transmission. To our knowledge, this is the rst study to investigate the density-dependent effects on nematode-induced galls on native Neotropical plants. The relative contribution of plant-parasitic nematodes is certainly overlooked (Seixas et al. 2004a,b) and should be explored in future studies to further augment our knowledge regarding herbivory in cerrado plant communities. The results obtained in the present study indicate that future community studies in the cerrado should consider the inuence of nematodes to obtain a more comprehensive knowledge of the ecology and evolution of interactions in this neotropical savanna. Moreover, as Ditylenchus spp. appear to be host-range limited to some Miconia spp. (Seixas et al. 2004a), it is highlighted as a promising species for controlling invasive Miconia species. Our results suggest a low impact caused by nematode-induced galls; however, further studies may provide an ecological basis for biological control.

Acknowledgments
We thank the staff of the Pirapitinga Ecological Station for logistical support. This study was supported by grants provided by CNPq (30 3352/2010-8), FAPEMIG (CRA 583/03, APQ-04105-10, RDP-00048-10) and the US Fish and Wildlife Services.

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