CROP PROTECTION

Histopathological and morphological alterations caused by Plasmodiophora brassicae in Brassica oleracea L.

Alteraciones histopatológicas y morfológicas causadas por Plasmodiophora brassicae en Brassica oleracea L.

Donald Riascos¹, Emiro Ortiz¹, Daimy Quintero², Lina Montoya², and Lilliana Hoyos-Carvajal^3,4

¹ Students M.Sc., Program in Agricultural Sciences with emphasis in Plant Pathology, Faculty of Agronomy, Universidad Nacional de Colombia. Bogota (Colombia).
² Students, Faculty of Agronomy, Universidad Nacional de Colombia. Bogota (Colombia).
³ Assistant professor, Crop Protection Department, Faculty of Agronomy, Universidad Nacional de Colombia. Bogota (Colombia).
⁴ Corresponding author: limhoyosca@unal.edu.co

Received for publication: 19 March, 2010. Accepted for publication: 2 February, 2011.

ABSTRACT

Plasmodiophora brassicae is a plant pathogen of the Brassicaceae family, which presents a remarkable ability to survive in soil and high capacity of infection, significantly reducing crop yields. The present histopathologycal study conducted with the aim of contributing to knowledge of the infection cycle of the pathogen, showed the presence of multinucleated plasmodia at cortex and periderm cells level in infected cabbage roots, as well as thickening and disruption of cell wall. As a result of this disarray was observed in diseased tissues, mainly in the cortex, compared with healthy tissues in uninfected plants. The inoculation cabbage seedlings with dormant spores of P. brassicae at concentrations of 1 · 10⁷ and 1 · 10⁸ spores mL^-1 by immersion and spray, induced a higher growth and less growth longitudinal lateral roots compared with uninoculated plants as well as presence of young plasmodia at 28 days after inoculation.

Key words: Plamodiophora brassicae, Brassica oleraceae L., histopathology, gall, plasmodia, inoculation, hypertrophy.

RESUMEN

Plasmodiophora brassicae es un patógeno de plantas de la familia Brassicaceae que presenta notoria habilidad de sobrevivencia en suelo y alta capacidad de infección, lo cual reduce considerablemente el rendimiento de los cultivos. El presente estudio se realizó para contribuir al conocimiento del proceso infeccioso de este patógeno en el repollo, una especie de interés económico para Colombia, pues la información disponible es incipiente y desactualizada. A fin de determinar el daño a nivel celular se practicaron cortes histológicos en plantas con infecciones naturales; adicionalmente, se hicieron pruebas de patogenicidad con el fin de hacer un seguimiento de los síntomas iniciales de la enfermedad bajo condiciones controladas. Los resultados indicaron la presencia de plasmodios multinucleados en las células del cortex y la peridermis, así como engrosamiento y rotura de la pared celular en raíces de repollo infectadas, además de pérdida en la diferenciación de los tejidos, principalmente del cortex, comparado con tejidos sanos de plantas no infectadas. La inoculación de plántulas de repollo con esporas latentes de P. brassicae en concentraciones de 1 · 10⁷ y 1 · 10⁸ esporas mL^-1, mediante inmersión y adición de inóculo en el sustrato, indujo mayor crecimiento lateral y menor crecimiento longitudinal de las raíces comparado con plantas no inoculadas, además de la presencia de plasmodios jóvenes a los 28 días de la inoculación.

Palabras clave: Plamodiophora brassicae, Brassica oleraceae L., histopatología, agallas, plasmodios, inoculación, hipertrofia.

Introduction

The club root disease in crucifers is caused by Plasmodiophora brassicae Woronin. In Colombia it has been reported on the main crucifer growing areas in the departments of Cundinamarca, Antioquia and Caldas, in crops of broccoli, cabbage, cauliflower, Brussels sprouts, radishes and Chinese cabbage among others, reducing yields of 20 - 50 %, becoming a major constraint to the production of these vegetables (Jaramillo and Diaz, 2006).

P. brassicae is an inhabitant of the soil, pathogenic on plants of the Brassicaceae family (Karling, 1968; Voorrips, 1995), which cannot grow in any state saprophytic life cycle, and this is divided in two stages, a primary phase occurs in root hairs and the secondary phase in the cells of root cortex, associated with cellular hyperplasia and hypertrophy, consequently inducing abnormal root growth and formation of club root o hernia in susceptible hosts (Cao et al., 2007; Friberg et al., 2008; Karling, 1968; Voorrips, 1995). The disease cycle begins with the germination of dormant spores (Friberg et al., 2005), which can survive in soil for more than 15 years in the absence of a host plant (Friberg et al., 2008). It is assumed that the germination of dormant spores is stimulated due to the presence of host plants who release specific substances from the roots, the same way, increases in moisture and temperature promote the germination of dormant spores which affect host root hairs during primary infection phase (Ingram and Tommerup, 1972; Manzanares et al., 2001).

In the secondary phase infection extends into the root cortex, where plasmodium, develops a multinucleated sctructure that is fractionated (Karling, 1968; Suwabe et al., 2003). Each piece is surrounded by a membrane and develops within zoosporangia, however, some, produces dormant spores zoosporangia (Vidhyasekaran, 2004), and consequently causing cellular hyperplasia and hypertrophy, reflected it in the galls on the roots of the plant. Subsequently, dormant spores are released into the soil when galls formed by host tissue are disintegrated (Ingram and Tommerup, 1972). The dormant spores are highly resistant structures, formed approximately 25% chitin, 2.5% other carbohydrates, 34% protein and 18% lipids (Moxham and Buczacki, 1983), which allows survives and remain viable in soil for long periods of time (Karling, 1968; Voorrips, 1995).

The aims of this study were observe and compare different histopathological changes produced by P. brassicae in cabbag, Brassica oleracea var. Capitata and a wild host (turnip, Brassicarapa) and determinate initial morphological changes in cabbage plants inoculated with the pathogen.

Materials and methods

Plant material

Mature plants of turnip (B. rapa) and cabbage (B. oleracea var. Capitata), healthy and with the presence of club root were collected in the greenhouses of the Faculty of Agronomy of the Universidad Nacional de Colombia, Bogotá, in order for studies histology changes. For patogenicity tests were used green cabbage seeds B. oleracea L. cv. Früher Dithmarscher.

Processing of tissues for histological observations

The root samples of turnip (B. rapa) and cabbage (B. oleracea var. Capitata), with natural infections were rinsed with water to remove soil residues. Subsequent, were cutted of 1 cm and fixed in 20 mL of FAA (10% formalin - acetic acid 5%, 85% - alcohol 70%) for 24 h. Then, slices were dehydrated through successive steps in alcohol at 70, 80, 90, 96 and 96%, each with 24 hours. Tissues were cleared in xylene for 24 hours and then melted paraffin, imbibes from 56 to 58ºC for 24 h. Finally, were prepared paraffin blocks, cutting with rotating type Minot microtome model 820 Spencer (American Optical, Delhi) and staining with safranin and fast green. The observations and imaging were performed in light microscopy (Olympus 31X).

Preparation P. brassicae of suspension

Roots of cabbage plants with the presence of mature galls induced by P. brassicae dormant spores were removed. Spore suspension was prepared mashing galls in deionized sterile water (1:10 w/v). The homogenate was filtered as described by Narisawa and Hashiba (1998). The filtrate was centrifuged at 300 rpm for 20 minutes according to Suzuki et al. (1992). The button was removed and supernatant was centrifuged twice, obtaining a spore suspension, from this, was verified the presence of dormant spores. The final concentration of suspension was adjusted to two levels: 1 · 10⁷ spores mL^-1 and 1 · 10⁸ spores mL^-1, by counting on hemacytometer.

Inoculation of cabbage seedlings

Sterile peat was Pro Mix was used as substrate for growing seeds of cabbage seeds B. oleracea L. cv. Früher Dithmarscher. For the inoculation of cabbage seedlings were used two methods, first consisted in immersion of plant in the spore suspension and then were sown in the nursery, others were planted directly, and then added the suspension of dormant spores to the substrate. Controls of seedlings were immersed in distilled water and then planted, resulting in the following treatments: T1) direct addition of 2 mL of inoculum in the soil at a concentration of 1 · 10⁷ spores mL^-1; T2) roots immersed in 2 mL of inoculum at a concentration of 1 · 10⁷ spores mL^-1; T3) direct addition of 2 mL of inoculum in the soil at a concentration of 1 · 10⁸ spores mL^-1; T4) roots immersed in 2 mL of inoculum at a concentration 1 · 10⁸ spores mL^-1; T5) uninoculated control. The seedlings were maintained for 30 days a temperature 18°C and ambient brightness. Frequent irrigation was provided to field capacity and fertigated to 15 and 21 days after inoculation (dpi) with 20 mL of Hogland solution.

Morphological observations of infection with P. brassicae in cabbage

On plants of cabbage B. oleracea L. cv. Früher Dithmarscher, were carry out three destructive sampling roots at 15 dpi (1^st sampling), 21 dpi (2^nd sampling) and 28 dpi (3^rd sampling). Tissues were washed with water to remove residual peat and morphological observations were made by direct observation under a stereoscope and for pathogen structures assess tissues were immersed in 10% KOH for 8 h, later in 12% KOH for 2 h-bath. Then, those were stained with trypan blue solution for 2 h, and washed with water. Finally the roots were spread on glass slides and observed by light microscopy optics.

Results and discussion

Histopathology of cabbage root galls P. brassicae

In the plates with tissue sections of roots affected by P. brassicae, plasmodia were observed in different stages of development in cabbage (Fig. 1A) and turnip roots (Fig. 1B).

The observed structures are part of the life cycle of primary and secondary disease (Suwabe et al., 2003). It can be seen elongation and/or increased cell size associated with hypertrophy and rupture processes of the cells containing the pathogen (Fig. 2A), which is consistent with what was mentioned by Donald et al. (2008) and Kobelt et al. (2000), this being a mechanism of spread of the pathogen within the host tissue.

Symptoms of cortical invasion by P. brassicae included disruption of the cell wall, presence of vesicles or inclusion bodies inside the cell wall, cell wall thickness in combination with plasmodesmata and elongation and/or disorganization host nuclei. Ameboidal shape of pathogen has been reported in root cortical cells and, it is often found in association with disruption of cell walls, and is presumed that it is the structure of the parasite that penetrates and invades the cortical cells (Donald et al., 2008; Kobelt et al., 2000). The observations carried out can be seen in the cell wall thickening (Figure 2B).

At the level of epidermis, periderm and cortex become visible a loss of tissue differentiation (Figs. 3 A, B).

Kobelt et al. (2000) described compatible interaction: Cvi ecotype of Arabidopsis and P. brasicae founding that sporeforming protozoan parasite are located in large cells of the cortex near the periderm, while young secondary plasmodia are found mainly in small cells next to central cylinder, which corroborates the histological differences found in this study.

Observations of artificial infection of P. brassicae in cabbage seedlings

At 15 dpi, plants inoculated at a concentration of 1 · 10⁸ spores mL^-1 had a lower longitudinal growth of the root system (4 - 6.7 cm), compared with plants inoculated at a concentration of 1 · 10⁷ spores mL^-1 (3 - 8.5 cm) and control (5.5 - 8.5 cm). Regardless of inoculation method, there was increased lateral growth (1 - 1.5 cm) as shown in Figure 4, which could be related to an imbalance of growth regulators in host-pathogen interaction, such as auxins and cytokinins, (Ludwig-Müller and Schuller, 2008). Epicotyl length was higher in control plants compared to inoculated plants, suggesting that P. brassicae affects the growth and development of infected plants.a

Samples taken 21 dpi revealed that plants inoculated at a concentration of 1 · 10⁸ spores mL^-1 by direct addition and immersion (T3 and T4) showed abundant lateral growth compared with control plants (T5), with values of 0.4 cm, 0.5 cm and 0.3 cm, respectively. As seen in the stereoscope was evident in the axis tissue loss and darkening of the main root in the inoculated treatments, mainly in T4 (Figure 5). At this time young plasmodia were found in treatments of immersion.

The results at 28 dpi were consistent with those obtained for the first and second sampling, for showing lower growth and higher longitudinal lateral growth for treatments T3 and T4. According to Devos et al. (2005) and Siemens et al. (2006), infection with P. brassicae results in the formation of adventitious roots, resulting in swelling, due to the involvement of auxins, cytokinins and xiloglucan enzyme transglycosylase/hydrolase (XTH).

According to Voorrips and Kanne (1997) symptoms in infected plants may have variations from small swellings with ovoid or globular in the roots to the formation of large galls on the entire root system, which is consistent with that observed in this work. The growth rate of infected plants is delayed and chlorosis occurs because the presence of large galls usually causes a reduction in nutrient uptake and water. As a result, infected plants grow abnormally and production is reduced (Karling, 1968; Voorrips, 1995).

Discolored roots showed the presence of young plasmodia the beginning of infection associated with loss of main root axis and increased lateral root growth, being more noticeable at a concentration of 1 · 10⁸ spores mL^-1 by immersion. In advanced stages was observed a higher number of multinucleated plasmodia with different sizes and shapes. It is noteworthy that other treatments except the controls were also positive for the presence of pathogen structures.

Methods developed in this study allow us to describe damage induced by P. brassicae at histologic level and its relationship to physiological alterations in susceptible hosts. Based on the results of pathogenicity tests, morphological changes registered are an indicator of the presence of the pathogen in early phenological stages, therefore, this information is important for those who produce plant material, it allows recognition of initial signs of the disease.

Acknowledgements

To Mr. Wadith de León and Mr. Jesús León technical assistants Plant Laboratory of the Faculty of Agronomy, Universidad Nacional in Bogota, for support in carrying out this work.

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