Ditylenchus Sp en Miconia Albicans

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Nematode-induced galls inMiconia albicans: effect ofhost plant density and correlations with performancepsbi_358 1..7

LEONARDO RODRIGO VIANA,* FERNANDO AUGUSTO OLIVEIRA SILVEIRA,* JEAN CARLOS SANTOS,LUIZ HENRIQUE ROSA, JUVENIL E. CARES, ADALBERTO CORRA CAF-FILHO andGERALDO WILSON FERNANDES**Evolutionary Ecology and Biodiversity andDepartment 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,andDepartment of Phytopathology, Institute of Biological Sciences, Universidade de Braslia, 70910-900, Braslia, DistritoFederal, 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), awidespread, 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. albicansand estimated the leaf area lost to nematode-

induced 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. Inflorescence length and fruit

production were significantly reduced in attacked shoots compared with healthy shoots.

Seeds from attacked shoots showed no significant reduction in germinability or germi-

nation time when compared with seeds collected from healthy shoots. Gall density was

positively correlated with host density. Despite being seldom studied in tropical ecosys-

tems, nematodes may play an important role in plant fitness and in structuring tropicalcommunities.

Keywords: biocontrol, Brazil, cerrado, density-dependent parasitism, Ditylenchus, plantnematode interaction.

Received 2 March 2011; revision received 6 June 2011; accepted 22 August 2011

Introduction

Plant-parasitic nematodes comprise a huge group oforganisms that colonize plant tissues reducing host repro-duction (Sasser & Freckman 1987; Dangl & Jones 2001).They can be the cause of devastating epidemics in cropsleading to serious economic losses, attracting the attentionof farmers, plant breeders and scientists (Plowrightet al.2002). Nematodes manipulate host physiology to opti-mize their parasitic lifestyles (Freckman & Caswell 1985),adversely affecting host photosynthesis, water and nutri-ent translocation, respiration, cell membrane permeability

and the processes of transcription and translation (Agrios2005). Nematodes can also directly or indirectly influencenutrient uptake in plants by their negative effect on roothealth (Vaast et al. 1998). All these physiological distur-

bances can indirectly result in lower seed output. Somenematode species, however, may have a greater impacton host fitness by colonizing reproductive structures(Sturhan & Brzeski 1991).

As most plantnematode studies have been carried outon economically important crops (Plowright et al. 2002),virtually nothing is known about the patterns and pro-cesses of nematodes parasitizing native, Neotropical, non-economically important plants. Although it is quite clearthat plant cover influences the diversity and communitystructure of soil nematodes in the Brazilian amazonian

Correspondence: Geraldo Wilson FernandesEmail: [email protected]

Plant Species Biology (2012) , doi: 10.1111/j.1442-1984.2011.00358.x

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and cerrado vegetations (Huang & Cares 2006), theecology of nematodes and their host plants is still unclearin the tropics, where few studies of plantnematode inter-actions have been conducted (Seixas et al. 2004a,b). Someaggressive plant-parasitic nematodes, with narrow hostranges have been regarded as potential agents for biologi-

cal control of undesirable plant populations. The nema-todeFergusobiasp., a galling pathogen of flowering budsand leaves of myrtaceous plants in Australia, and itsvector fly (Fergusonina sp.) have been studied with the aimof establishing biological control programs for Melaleucaquinquenervia(Myrtaceae) (Cav.) S.T. Blake, an aggressiveinvader 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 Hawaiiand other Pacific islands, and is now growing out ofcontrol as an exotic species. In a search for organisms withthe potential to biologically control M. calvescens, Seixas

et al. (2004a,b) singled out Ditylenchus drepanocercusGoodey, 1953 because it is capable of causing angular leafspot and because it has narrow host specificity. Therefore,studying the ecology of nematode-induced galls is ofinterest to gain insight into their potential as agents ofbiological control.

In the present study, we report the attack by anundescribed Ditylenchus species on Miconia albicans(Sw.) Triana, a native shrub from the cerrado (Braziliansavanna). The patterns of pathogen attack on native plantsand their impact on the reproductive success of their hostsis unknown. Thus, the role of pathogens in regulatingplant population structure is still unclear. We tested the

hypothesis that nematode-induced galls negatively corre-late to host plant performance and that gall density is afunction of host plant density.

Materials and methods

Study site and species

The present study was conducted at the Pirapitinga Eco-logical Station (PES) in Trs Marias, Minas Gerais, south-eastern Brazil. The PES is a 1100 ha man-made island,created in 1965 as a result of the filling of the Trs Marias

reservoir (1823S, 4520W), at an altitude of 560 m a.s.l.The average annual temperature in the region varies from21C to 25C, and the average annual precipitation is1200 mm (Bedettiet al. 2011).

Miconia albicans is a widespread evergreen shrub ofthe cerrado vegetation of Brazil reaching up to 3 m tall.It occurs from southern Mexico to the Brazilian state ofParan (Goldenberg 2004). Miconia albicans is an obliga-tory apomictic species. The whitish, small and scentlessflowers are not visited by bees and produce pollen withnull viability (Renner 1989; Goldenberg & Shepherd

1998).Miconia albicans appears to play an important role inthe cerrado because of its widespread distribution andbiotic interactions. Its greenish fruits are dispersed bybirds, rodents and possibly ants (Magnusson & Sanaiotti1987; Goldenberg & Shepherd 1998). Leaves are simple,opposite and elliptic, with a pubescent, whitish abaxial

surface. Leaves are continuously produced year round sothat expanding and mature leaves can be found in bothdry and rainy seasons. The foliage is exposed to highlevels of herbivory caused by insects (Paleari & Santos1998) and casual observations indicate that at least onegalling insect attacks its leaves and another speciesinduces a stem gall. In the study area, flowers are pro-duced in a scorpioid panicle from June to October andmature fruits occur during the mid-rainy season (Bedettiet al. 2011). There are no reports of nematode galling onthis species.

Nematode extraction, identification, histopathology andpathogenicity test

Fornematodeextraction, samples of galled leavesofM. al-bicanswere placed in a conventional blender with 500 mLof distilled water and blended for 20 s at the highestspeed. The resulting leaf suspension was poured throughtwo stacked metal sieves, a 60-mesh screen on top of a400-mesh screen. The nematode suspension containingfine leaf residue recovered from the lower screen wasclarified by centrifugation according to the modifiedmethod of Jenkins (1964). Nematodes and plant residuewere concentrated at the bottom of the centrifuge tubes by

centrifugation for 5 min at 1006g, the supernatant wasdiscarded and the pellet suspended again in a sucrosesolution (456 g in 1 L of water). Following a second cen-trifugation for 1 min at 112g, the floating nematodes wererecovered free from sugar in a 400 mm mesh screen. Thesame procedures were applied to samples of non-galledleaves. The nematodes were heat killed by immersionin water at 50C for 1 min, fixed in 3% formalin (Flegg &Hooper 1970) and glycerin infiltrated following themethod of Seinhorst (1959). For identification, adults ofboth sexes were hand picked under a stereo microscope,mounted with anhydrous glycerin on glass slides and

covered with coverslips. The slides were sealed withCanada balsam. The nematode specimens were identifiedbased on morphology and morphometry with the aid ofa compound light microscope according to Sturhan andBrzeski (1991). For histopathological observations, free-hand sections through fresh leaf galls were cut with arazor blade and thin sections were stained with cottonblue and mounted on glass slides. The sectioned gallswere examined with a light microscope.

For the pathogenicity test, galled leaves were collectedfrom the same study site (PES) and brought to the Plant

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Pathology Laboratory of the University of Braslia. Anassay was carried out on M. albicansspecimens growingin native cerrado vegetation in the Biological Experimen-tal Station of the University of Braslia. A suspension ofliving nematodes, obtained as described above, wasdiluted to approximately 15 nematodes per mL and

applied by misting both leaf surfaces of healthy youngleaves, after sunset to preserve leaf moisture. Healthyplant shoots in the same plants served as uninoculatedcontrols.

Impact on plant traits

To evaluate the impact of nematodes on host-plant repro-duction, we determined two categories based on symp-toms of nematode gall abundance; one hereafter termedhealthy (absence of nematodes) and the other, attacked(presence of nematodes). We collected one healthy andone attacked shoot from 28 individuals ofM. albicans inthe field. A 500-m transect was established and adultplants up to 2 m either side of the transect were sampled.Samples were taken in December 2002 during the fruitingpeak, allowing for a comparison of reproductive traitsbetween shoots in both categories.

The leaf area attacked by nematodes was visually esti-mated in the field and classified 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 calcu-lated according to the following formula (Garca-Guzmn& Dirzo 2001):

IP L= ( )( )[ ] i i n

where Li is the number of leaves in each category ofdamage, i is the category of damage and n is the totalnumber of leaves sampled per plant.

We considered healthy shoots to be those that pre-sented IP values in the categories 0 and 1 because damageat these levels was almost imperceptible to the naked eyeand therefore less reliably attributable to nematode attack.We considered attacked shoots to be those in which the IPvalues were greater than or equal to 6%, corresponding tothe remaining categories (25). These IP categories were

also used to test nematode attack in relation to host spatialdistribution. Using a ruler we measured shoot length(from internode to shoot apex) and inflorescence length(from rachis base to its apex). We also counted the numberof fruits in both attacked and healthy shoots to detectnematode effects on plant reproduction. Because of thenon-normal nature of our data a Wilcoxon test was used tocompare shoot length, inflorescence length and fruit pro-duction between attacked and healthy shoots (Zar 1996).

To test the impact of nematodes on seed germination,the germination of seeds removed from healthy and

attacked shoots was compared. From the same 28 indi-viduals we randomly sampled in December 2002, maturefruits were collected, washed and dried for 2 days. Seedswere placed in Petri dishes covered with two sheets offilter paper and moistened with Nistatin solution (2%), asnecessary, to avoid the growth of contaminant fungi. The

Petri dishes were placed in germination chambers undera constant temperature of 25C and a 12-h photoperiodfor 30 days, a period of time in which non-dormantseeds tend to germinate. Germination was verified every23 days and seeds were considered to have germinatedwhen radicle protrusion was observed.

From each shoot, four replicates of 10 randomlyselected seeds were set per individual. As germinabilitywas compared from seeds removed from healthy andattacked shoots from the same individual, eight replicateswere tested (n = 56; 4 replicates 14 individuals). Thegermination percentage values were transformed as the

square root arcsine to obtain data normalization and wecompared the seed germinability of attacked and healthyshoots using a pairedt-test (Zar 1996). We also calculatedthe mean germination time (MGT) using the followingequation:

MGT nd N= ( )

where n is the number of seeds that germinated betweenscoring intervals, d is the incubation period in days at thattime point and N is the total number of seeds that germi-nated in the treatment (Tompsett & Pritchard 1998). TheMGT values from attacked and healthy shoots werealso compared using a pairedt-test in an attempt to verifywhether nematode attack decreased the speed of seedgermination.

Host plant spatial distribution and gall abundance

In the study area,M. albicansindividuals were easily visu-ally recognized as occurring in isolated and clumpedpatches. To assess whether host density affects gall abun-dance we measured the distances of the five closest indi-viduals ofM. albicansfrom 30 selected individuals alonga 500-m transect. The 30 plants were visually separated

a priori into two groups: 15 were considered to be iso-lated (average distance to the five nearest neighbors =341 41.2 cm) and 15 clumped (average distance to thefive nearest neighbors=86.5 12.0 cm). The distancebetween isolated individuals was significantly largerthan that between clumped individuals (MannWhitneyU-test; U=223, P


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Results

Gall etiology

The nematode-induced galls are amorphous and can befound isolated or coalescent. The greenish-brown spheri-cal galls found mainly on the abaxial side of the leaf (with

a few on the adaxial side) were densely covered withtrichomes (Fig. 1a). Cross-sections of the galls showed astructure comprising tissues spanning from the epidermisthrough to the spongy mesophyll, including bundles of

vascular tissues. The gall comprised more than onehollow chamber or locus (Fig. 1b) separated from oneanother by a parenchymatous septum. Laboratory analy-ses indicated the presence of only onespecies of nematodeassociated withM. albicansleaf galls. Juvenile, adult maleand female nematodes were recovered from galled leaves,

but no nematodes were recovered from ungalled leaves.Juvenile, female and male nematodes (Fig. 1c) wereobserved moving freely in each of the locus spaces;nematode eggs were also observed. The nematode was

Fig. 1 Galls induced by Ditylenchus sp. onMiconia albicans.(a)AbaxialviewofaM. albicans leaf with galls, (b) leaf gall showing thehollowchambers or loci (arrows) in cross-section and (c) micrograph of nematode bodies (n) inside a locus of a leaf gall (n =each individualnematode).

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identified as Ditylenchus sp. with characteristics of anundescribed amphimictic species.

Leaves were observed daily for 2 weeks after inocula-tion. Unlike the symptoms observed from natural infec-tions, artificially inoculated shoots had galls almostequally dispersed on both sides of the leaf, stem and peti-oles. Four weeks after inoculation, eggs, juveniles andadult nematodes were recovered from galls extracted

from inoculated leaves. Uninoculated shoots remainedfree of galls.

Impact on plant traits

Nematodes not only occurred on leaves (Fig. 1a), butthey were also able to colonize flowers and fruits. Gallabundance was unrelated to shoot length (U=420,P =0.646; Table 1). However, inflorescence length (U=575,P 0.001) was smaller on attacked shoots comparedwith healthy ones. The number of fruits produced pershoot on attacked shoots was nearly one-third of that pro-

duced per healthy shoot (U=

688,P

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Ditylenchus Sp en Miconia Albicans