BIOCELL2009, 33(2): 133-136

Briefnote

ISSN 0327 - 9545PRINTED IN ARGENTINA

Differences in intracellular localization of com stunt spiroplasmasin magnesium treated maize.

CLAUDIA NOME'*, PAULO CÉSAR MAGALHÃES2, EUZABETH OUVEIRA2, SERGIO NOME AND IRMA GRACIELA LA-

GUNAl

I. Instituto Nacional de Investigaciones Agropecuarias - Instituto de Fitopatología y Fisiología Vegetal (INTA-IFFIVE).Córdoba. Argentina

2. Embrapa Milho e Sorgo. Caixa Postal 151 - 35701-970, Sete Lagoas, MG, Brasil.

Key words: mollicutes, cytopathology, plant pathogen, magnesium deficiency

ABSTRACT: Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in amagnesium-deficient soil, and it has been shown that the spiroplasma alters the plants' magnesium absorp-tion. ln the current study we compared changes associated to either spiroplasma infection, two soil rnagne-sium levels and their combinations. Plant symptoms were recorded and correlated with transmission electronmicroscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterationsnor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopicalvein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. In-fected plants on a low magnesium treatrnent had an ageing aspect, ultrastructurally showed chloroplasts andmitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, andspiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magne-sium treatment showed similar symptoms and ultrastructural alterations as eíther non-infected plants on thelow magnesium treatment or ín ínfected plants 011 the low magnesium treatment, but differ from them ín thatthe spiroplasma cells were located ínsíde the cytoplasm. Results suggest that magnesium ís involved ín theplant-pathogen ínteractíon.

Plant pathogens include viruses, viroids, fungi,baeteria, and within the latter, the Mollieutes, a bacte-rial c1ass including the families Mycoplasmataeeae,Spiroplasmataceae and Acheloplasmataeeae. They arethe smallest known prokaryotes and they lack a cell wall.Their genome oseillates from 577 to 2220 kpb, and they

*Address correspondence to: Claudia Nome. Instituto Nacionalde Investigaciones Agropecuarias - Instituto de Fitopatología yFisiologia Vegetal (INTA-IFFIVE). Camino 60 cuadras Km 5 1/2. X5020lCA Córdoba, ARGENTINA.E-mail: cfnome@yahoo.com.arReceived: May 23, 2008. Revised version received: June 1,2009. Accepted: June 2, 2009.

are extremely demanding from the nutritional point ofview, therefore very difficult to grow in eulture medium(Bové, 1988).

The Spiroplasmataceae include the genus Spiro-plasma, whose name derives from the helical morphol-ogy and the motility of cells in liquid media. They havebeen recognized in diseased plants less than thirty yearsago, and they have been reeognized also as pathogensof warm-blooded animais and insects (Maramorosch,1981; Bastian et aI., 2007). They are diagnosed by se-rological and molecular methods, and by eleetron mi-croseopy. The genus narne Phytoplasma has not yet beenformally recognized, but it is currently at "candidatus

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status" and is used for [Spiroplasmataceae] bacteria thatcannot be cultured, and they belong to the monophyl-etic order Acholeplasmatales (Lee et al., 2000). Theyare only detected by molecular methods, electron mi-croscopy, and by the response of the host plant to anti-biotic treatment. Phytoplasmas exist both in plants andin insects feeding on phloem of infected plants. As forspiroplasmas, such insects have been described as thepathogen vectors. Inside the plants, both phytoplasmasand spiroplasmas are confined out of the cytoplasm ofthe sieve cells, while in insects they are distributed inseveral tissues (Conci et aI., 2001; Ammar andHogenhout, 2005).

Spiroplasma kunkelii is a maize pathogen transmit-ted by the Ieafhopper Dalbulus maydis (A+Y), produc-ing a disease named com stunt disease. It has been re-ported that the infected plants show a lower magnesiumabsorption, Oliveira et aI. (2002, 2005).

The symptoms of maize plants grown on magne-sium deficient soil (concentrations below 0,5 Cmol cdm") are leaf veins yellowing! whitening all along theirlength, and purple coloring on the tips of older leaves,Coelho and França (1995). Almost identical symptomsare the ones produced by the spiroplasma infection (Tsaiand Miller, 1995).

The aim of this work was to compare plant alter-ations associated with low soil magnesium levels andby the infection with S. kunkelii, and to assess the pos-sibility of spirop lasma-induced alterations were revertedby a high magnesium availability.

Zea mays plants, cv. Pop Zélia, were breed on soilfrom the Brazilian Cerrado (savanna) with natural1y showslow magnesium levels, to which magnesium was addedto obtain either 0.20 or 1.0 Cmol c dm? (they will bereferred to as the low and the high magnesium treatments).They were kept in greenhouse with stable conditions of27°C, 16 hours of lighting and 60% of humidity. Someplants were kept uninfected, while others were exposedto the leafhopper Dalbulus maydis for inoculation withS. kunkelii after seven days of plant emergence.

Sheath tissue samples of maize leaves (2 x 3 mm)sixty seven days old (sixty days after inoculation) werecollected and fixed in Karnovsky fluid, post fixed inosmium tetroxide 1%, contrasted in uranyl acetate 0,5%,dehydrated in acetone solution series, pre-embedded inacetone-epoxy resin, and embedded in low viscositySpurr resin (polymerized at 70°C for 2 days). Ultrathinsections were made with a diamond blade and sectionswere contrasted with lead citrate pH 12 and with uranylacetate 2%. Sections were observed with a transmis-sion electron microscope Jeol 1220 JEM EXII.

CLAUDIA NOME et ai.

Uninfected plants on the high magnesium treatmentshowed no abnormal symptoms, while maize plants onthe low magnesium treatment showed vein yellowing

FIGURE 1. Uninfected maize cells on the high mag-nesium treatment. Normal chloroplasts are seen in thesheath bundle and the parenchyma. Vascular bundleshows no splroplasmas. In this and in ali subsequentfigures, stars indicate chloroplasts; arrows indicatespiroplasmas; Cy is cytoplasm; CW, cell wall; SB, sievebundle; X, xylem; Ph, phloem; P, phloem parenchyma.Scale bar represents 7 11m.

FIGURE 2. Uninfected maize cells on the low magne-sium treatment.. Picture shows distorted chloroplastsof sheath bundle and normal parenchymal chloroplasts.Scale bar represents 211m.

CORN STUNT SPIROPLASMAS IN MAGNESIUM TREATED MAIZE

all along their leaves and some whitening down on thesheath. Also, infected plants cultivated on low magne-sium treated soil appeared olel, but showed a more in-tense purple coloring and sometimes even leaf apex ne-crosis. Infected plants on the high magnesium treatmentalso showed vein yelIowing alI along the sheath and purplecoloring on older leaves; such plants appeared olel,though _they were less damaged than spiroplasma infected plantson the low magnesium treatment.

FIGURE 3. Spiroplasma infected maize cells on the lowmagnesium treatment. Micrograph shows devastatedchloroplasts both in sheath bundle and the parenchyma.Scale bar represents 2 um.

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As expecteel, uninfected plants on high magnesiumtreatment showed no ultrastructural alterations (Fig. 1).However, uninfected plants on the low magnesium treat-ment exhibited chloroplast alterations, swollen chloro-plasts with disordered grana, in ali of the studied tis-sues (Fig. 2). Spiroplasma infected plants on the lowmagnesium treatment also showed disorganized chlo-roplasts, mainly in their veins, and the pathogen wasseen inside phloem sieve tubes, but not in the cytoplasm(Figs. 3,5). In contrast, infected plants on the high mag-nesium treatment, showed the spiroplasma both outsideand inside the cytoplasm ofphloem cells (Figs. 4, 6).

FIGURE 4. Sieve tube of infected maíze plant on the lowmagnesium treatment wíth spíroplasmas. Ordínary ímageof spíroplasmas spread loose out of the cytoplasm. Scalebar represents 500 nm.

FIGURE 5. Sieve tube of an ínfected maize plant on the hígh magnesíum treatment showingspiroplasmas within their cells. Notably, the pathogen is not only wíthin síeve tubes but alsoínsíde cytoplasm of young phloem cells and phloem parenchyma. Scale bar represents 1 um,

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It is known that low magncsium conccntrations in-duce maize plants to show leaf vein yellowing (INFOPOS,2001; Bull, 1993). Earlier studies have shown that S.kunkelii infection reduces magnesium concentration inmaize plants (Oliveira et al., 2002,2005; Ammar et aI.,2005). We may suggest, therefore, that the symptom ex-pression may be related to a competition for the cationbetween the plant and the pathogen.

lnfected plants on the low magnesium treatmentwere more damaged than infected plants on the highmagnesium treatment. It is notable, however, that thespiroplasma locates inside ofthe young phloem cells of

FIGURE 6. Same as in Fig. 5. Scale bar represents 1 um.

CLAUDIA NOME et ai.

infected plants on the high magnesium treatrnent. Thismay suggest that magnesium influences the way thatthe pathogen invades the plant, and that it allows thespiroplasma to introduce inside the young cells, and toremain alive, and to proliferate within their cytoplasm.

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