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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi s. II a. Biologie vegetală, 2016, 62, 2: 19-28

http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html ISSN: 1223-6578, E-ISSN: 2247-2711

HISTOCHEMICAL ASPECTS OF HELIANTHUS ANNUUS L. – OROBANCHE CUMANA WALLR. PATHOSYSTEM

Maria DUCA1, Olesea TABARA1*

Abstract: Broomrape is a root holoparasitic angiosperm that causes severe losses in sunflower yield. During evolution, simultaneous with the emergence of new more virulent races of broomrape, Helianthus annuus has developed a broad spectrum of defensive mechanisms against the parasite. A general response to pathogen attack is represented by modifications of composition and properties of host's cell walls, especially accumulation of lignin and callose at the attachment zone, which prevent broomrape penetration and connection to the sunflower vascular system. Thus, the aim of the present work was to investigate the histological aspects of the resistance in host-parasite system Helianthus annuus L.- Orobanche cumana Wallr. An abundant accumulation of lignin and callose in the roots of resistant genotypes cultivated in soil infested with broomrape, compared to the control, has been established. These results indicated that the phenylpropanoid pathway was activated, synthesis of lignin was increases and cell wall was fortified. Keywords: sunflower, broomrape, defense mechanism, resistance, lignin, callose.

Introduction

Plant defensive mechanisms are similar for a wide range of biotic factors, from

bacteria to flowering parasitic plants (Joel and Portnoy, 1990), demonstrating the universal nature of imune reaction and the importance of nonspecific resistance.

To perform successfully the infection process, the pathogen should penetrate many defense lines, as a series of specific and non-specific resistance mechanisms are activated. Among them, there are the constructive mechanisms of passive defense which represent the morphological and structural barriers (the leaf wax layer, cell wall). At the same time, a number of compounds (phenolic compounds, phytoalexins) act as cytotoxic ones and inhibite the seeds germination (Montesinos, 2000). In a few minutes of contract between the pathogenic agent and host, reactive oxygen species (ROS) are formed (Perez and Brown, 2014), which activate the defense gene expression in adjacent cells, through the accumulation of reduction-oxidation enzymes, initiation of hypersensitive response (HR) or induction of the metabolites – antagonists to the invader. HR is a form of programmed cell death as part of hypersensitive response in site of parasite attachment or in place of haustorium penetration for limiting the parasites development to establish host vascular continuity (xylem and phloem connections) (Coll et al., 2011). Pathogen localisation can launch the systemic response – systemic acquired response (SAR), involving signaling through phytohormones in particular salicylic acid, jasmonic acid, ethylene, changing the balance between the opposing signaling pathways (Niu et al., 2011), the expression of pathogenesis-related proteins (Durrant and Dong, 2004), increasing the activity of specific enzymes - for example peroxidase. Another resistance mechanism is presented by the

1 University of the Academy of Sciences of Moldova, 3/2 Academiei Street, MD-2028, Chisinau, Republic of Moldova *Corresponding author. E-mail address: [email protected]

Duca, M., Tabara, O. 2016/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 62, 2: 19-28

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specific resistance genes R of a host, corresponding to the pathogenicity gene Arv of parasite. This mechanism ”gene-for-gene” only operates against a single race of a pathogen.

All these mechanisms have been identified as defensive reactions against the sunflower broomrape or similar parasite species of Orobanche, Striga, Triphysaria genus.

The aim of the present work was to identify the histo-anatomical changes of sunflower grown in the background infected by Orobanche cumana Wallr. It was of special interest to examine the pattern of lignin and callose deposition in the compartments of compatible and incompatible pathosystems.

Materials and methods

Plant material. For this purpose, six genotypes with known type of specific resistance were selected (Table 1).

Table 1. Characteristics of plant material

Genotype Resistance to race Company, source Favorit, F1 Race F, (with Or6 gene) INCDA Fundulea, România (Pricop and Cristea, 2012) LC-1093A Race F, (with Or6 gene) INCDA Fundulea, România (Păcureanu-Joiţa et al., 2007) PR64 LE20, F1 Race G AMG-Agroselect Comerț, Moldova LG-5542, F1 Race G INCDA Fundulea, România Performer, F1 Susceptible INCDA Fundulea, România (Pricop and Cristea, 2012) LG5525, F1 Susceptible Î.C.S. Limagrain Moldova S.R.L.

Broomrape population was collected from the central region of Republic of

Moldova (Singera location), where the sunflower cultivated field are characterized by higher frequency, intensity and degree of attack (Duca et al., 2013b; Duca et al., 2016a).

Plants cultivation. Sunflower plants were grown in 4 l vegetation pots with soil and sand mixture (1/1) exposed to open field conditions. Soil was infected with 30 mg broomrape seeds in 200 g soil mixture.

Control samples were grown in uninfected mixture and were placed at a distance of 20 m from the infected ones. Plants were irrigated according to the average temperatures in May-July and the optimal conditions for the parasites development.

Collection of plant material. The biologic material was collected in temporal dynamics during the four development phases of pathosystem, within 67 days. Sunflower roots were collected in different vegetation periods depending on the development stages of the parasite (Duca et al., 2013a; Louarm et al., 2016).

Sections preparation. Helianthus root cross section were cut by hand. Images were taken at the optical microscope (XSZ-206T, Ningbo Wason Optical Instrument Co., Ltd.) equipped with CCD camera (MEM1300, Future Optics Sci. & Tech. Co., Ltd) connected to the computer.

Lignin identification. Root sections were placed in Petri dishes with 0.1% Safranin O solution over a period of 1 min, then were washed. Lignin deposits in cell walls stained red (Voigt, 2014).

Callose identification. Root sections were placed in Petri dishes with 0.005% Aniline Blue solution over a period of an hour, then washed with diluted glycerin (1:3), we placed on a slide and taking pictures. Cell walls with callose show a deep blue colour (Bordallo et al., 2002).


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Results and discussions

Resistance mechanisms against rhizoparasites can be classified into three groups: pre-attachment, post-attachment and post-haustorial resistance (Louarn et al., 2016).

The pre-attachment defensive mechanisms act in endodermis or cortex of a potential host plant to avoid or prevent parasite attachment, including the absence or reduced production of germination stimulant (strigolactone) and germination inhibition (Fernandez-Aparicio et al., 2014). Recent studies (Duca et al., 2016b) demonstrated that high enough level of affinity of population collected from central region of Moldova to the host plant with higher stimulation of germination with exudates obtained from sensitive sunflower genotypes and that obtained from differential resistant lines to physiological latest races.

Our studies were focused on two types of resistance mechanisms: post-attachment and post-haustorial and included the hystochemical analysis of two basic components – lignin and callose, 142 sections being examined on different phases of pathosystem development.

To detect exactly the developmental phases, the preventive anticipated tests were performed with the sensible and resistant genotypes. The number of days related to each phase, was different form those established in previous studies of parasite’s ontogenetic development (Rotarenco, 2010), being influenced by the climatic conditions, cultivated genotype and broomrape population. Thus, the first connection attachments in ours investigations were established at 18-21 days. Once the close contact with host plant is accomplished, it allows the parasite to adsorb the mineral and organic substances and the thickening in the shape of tubercle is formed. These are tubercles detected in 35 days after sowing. Tubercles are further transformed into the undeground shoots, discovered after the 40 post-infection days, which then are prolonged and penetrating the soil, come to the surface, thus, forming the flowering shoots of parasite. On the soil surface the overhead shoots appeared after 48-50 cultivation days (Rotarenco, 2010) but in our experimental pots at 53, 67 days it was observed development of the underground and overhead shoots.

Post-attachement resistance, characterized by the inhibition of haustorium formation, the reduction of haustorium growth such as thickened host root cell walls, broomrape cellular disorganization or tubercule necrosis (Labrousse et al., 2001, Echevarria- Zomeno et al., 2006), included the analysis of sections obtained at 18 and 21 days after sowing.

Penetration process analysis in central cylinder and formation of mechanical barriers have showed different densities of red colour of lignin content between healthy and infected genotypes (Figs. 1, 2). On the other hand the accumulation of callose was found in the cell wall of the epidermis in resistant genotypes grown on the background of the infection (Fig. 4).

The post-haustorial resistance is activated when the haustorium has formed and the parasite attempts to penetrate the central cylinder and connect to the vascular system (Lozano-Baena et al., 2007; Scholes and Press, 2008) and different constitutive or induced incompatibility or host resistance mechanisms are initiated (Timko and Scholes, 2013).

The resistance genotypes, included in this reaserch were characterized by a small number of parasitic attachments or total absence at 35, 53 and 67 days. In susceptible


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genotypes was observed necrosis of some bulbs after activation of resistance mechanism. From incompatible interactions with a parasite can occur at discrete times during parasite ingress and are either rapid or delayed.

Histochemical research of presence and accumulation of studied compounds in sunflower genotypes artificially infected with broomrape have revealed the activation of defensive mechanisms dependent on development stage, genotype and studied compound.

The lignin presence was not varied significantly in sections of control group in resistant and sensible genotypes on different stages of development (Fig. 1).

In case of plant-host interaction (Fig. 2) the highest lignin accumulation was attested in LC-1093 genotype and its hybrid - Favorit which have Or6 broomrape resistance gene. The activation of the lignin biosynthesis and pericycle deposition in cells of Favorit occurs starting with 35 days. In case of LC-1093, the highest level of this compound was detected at 53 cultivation days. On the background of infection, for the PR64LE20 genotype the formation of physical barriers and reinforcement of the cell walls as a result of lignification was established at 63 days.

No changes were observed for LG-5542 (resistant genotype) and LG-5525 (susceptible genotype), grown in the infected soil.

The Performer hybrid, late reaction the infected genotypes showed differences in lignin accumulation level in the forming underground stems and flowering shoots (53 days and respectively 63 days).

Similar data were observed in resistant vetch (Vicia spp.) infected with Phelipanche aegyptiaca by the increase of phenolics and lignin concentrations and greater peroxidase activity in the apoplastic space between host and parasite cells (Goldwasser et al., 2000). The lignified and/or suberized cells were considered as physical barriers installed by strengthening cell walls in other host-parasite systems such as Lycopersicon esculentum parasitized by Cuscuta reflexa (Sahm et al., 1995), Vigna unguiculata infected with Striga gesnerioides (Moore et al., 1995), Pisum sativum - Orobanche sp. (Hassan et al., 2004). Sections prepared in penetration of pathogen infection in susceptible roots showed a more intense lignin accumulation when connecting haustorium.

Study of callose accumulation in the sections of resistant genotypes revealed increase deposition in genotype Favorit after 35 days after sowing (Fig. 3).

In comparison with all studied genotypes sections of resistant forms unlike those of susceptible were shaped callose accumulation in root epidermis when the first forming attachments (Fig. 4). Sections obtained from infected with broomrape susceptible genotypes showed various aspects of callose accumulation. Performer hybrid demonstrated depositions of this compound in the stage of root pathogen shoot forming (Fig. 5).

Section of all genotypes grown in the control condition showed no difference in callose accumulation degree at the level of the epidermis as well as the central cylinder.

In another effort to compare a resistance mechanisms to a series of sunflower genotypes on O. cumana, callose deposits LR1 was attested in xylem in immediate contact with the parasite through and HaGSLI gene supraexpression (encoding the enzyme glucan synthase) (Letousey et al., 2007). Gindro et al. (2006) were studied callose storage in Vitis vinifera infected with Plasmopara viticola. This phenomenom was detected only on resistant genotypes and stops pathogen penetration (Gindro et al. 2006). Callose deposition was reported at the point of parasite penetration in cowpea (Vigna unguiculata) infected


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with Striga gesnerioides (Botanga and Timko, 2005) and various vegetables infected with O. crenata (Pérez-de-Luque et al., 2008).

Conclusions

The increase in lignin accumulation for the resistant genotypes has reached the highest level in case of Favorit hybrid, starting with the active defense phase of the plant (35 days). Being lower for LC-1093A, this compound concentration was detected after 53 cultivation days of after outset infection, and for PR64LE20 – after 63 days. Genotype LG-5542 cultivated on the background of infection showed no modifications in callose and lignin accumulation.

Infected susceptible genotypes have manifested the differences with regard to the lignification level of central cylinder, in Performer hybrid – on the stages of underground and overhead shoots (53 and 63 days respectively).

Cultivation on the background of infection of the resistant genotypes have revealed the abundant callose accumulation in the central cylinder of Favorit hybrid at the phase of 35 days after cultivation.

Comparing the sections of all the studied genotypes, for the resistant forms, unlike the sensible ones, the callose accumulation in the root epidermis was detected at the moment of first attachments formation.

The sections obtained from the susceptible forms infected with broomrape, revealed the accumulation of this compound on the formation stage of root pathogen air sprouts.

In control forms of all genotypes at, no difference was observed with regard to callose accumulation degree, as well as lignin on the root level.

Acknowledgements I would like to thank Dr. Sestacova T., PhD. Acciu A. and Ursu V. for suggestions

in made the experience and proposed aid in collecting plant material and carrying sections. The research was achieved inside the project "Resistance of sunflower (Helianthus annuus L.) to broomrape (Orobanche cumana Wallr.): genetic-molecular and physiological mechanisms" (15.817.05.03F).

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Explanation of the plates

Plate I Figure 1. Sections obtained from healthy sunflower root (160-times magnified) Plate II Figure 2. Sections obtained from sunflower root grown in the background of infection (160-times magnified) Plate III Figure 3. Callose accumulation in the sections of resistant genotype, Favorit (160-times magnified) Figure 4. Sections at 18 days phase (formation of attachments) (160-times magnified) Figure 5. Callose accumulation in the sections of susceptible genotype, Performer (160-times magnified)


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Plate I

Figure 1


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Plate II

Figure 2


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Plate III

Figure 3

Figure 4

Development phase of the pathogen 18 days 21 days 35 days 53 days 67 days 67 days

Section at the connecting region between hausto-rium and host root

Figure 5

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