Genotype and NaCl salinity influence Pythium ultimum damping-off in safflower

Communicated by Grzegorz Żurek

M. Pahlevani*, H. Bagmohamadi, M. Ghaderi, S.E. Razavi

Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran; *corresponding author: hpahlavani@yahoo.com

GENOTYPE AND NACL SALINITY INFLUENCE PYTHIUM ULTIMUM DAMPING-OFF IN SAFFLOWER

ABSTRACT

Increased seed germination and seedling growth in soils infected with damping-off pathogen, Pythium

ultimum, especially under saline conditions, are important goals in safflower breeding programs. Seeds of four

safflower varieties were cultured on germination media moistened with solutions of NaCl containing 105 zoospores of the pathogen per ml. NaCl concentrations were adjusted to produce salinities 0, -10, -14 and -18 bar. Analysis of variance indicated that the interactive effects of salinity, cultivar and pathogen significantly altered seed germination and seedling dry weight. In the absence of P. ultimum, increasing salinity signifi-cantly reduced seedling dry weight, but in pathogen-inoculated media, different values were observed. In the absence of salinity, the germination rate of inoculated seeds was 16.3% lower than that of non-inoculated seeds, whereas in a saline bed, the pathogen increased seed germination about 3.1 %. This finding clearly indicates that sodium chloride can reduce the pathogenesis of P. ultimum on safflower seedlings. The results showed that the presence of NaCl in the environment prevented rotting effects of the pathogen on seeds but intensified the mortality effect on seedlings. The simultaneous effects of salinity and pathogen reduced the usual adverse effects of either factor when applied separately. Thus, depending on amount of NaCl and/or Pythium infection present, different varieties are recommended to achieve an acceptable establishment and production.

Key words: germination, interaction, resistance, seed, zoospore,

INTRODUCTION

Salinity is the most widespread abiotic stress that seriously affects crop production in arid and semi arid regions. Almost 800 million hectares of farmland, more than 6% of total area of the world, suffer soil and/or water salinity problems (Munns, 2005). Although salinity stress has adverse

sequences on all growth stages, the extent and type of reactions exhibited by plants against the stress in germination processes is greater than in the next growth stages (Siddiqi et al., 2007). Like other osmotic stresses, salin-ity prevents or delays germination of seed, the most sensitive stage of the growth, decreases standing of seedlings in the soil, and finally reduces pro-duction (Balwin et al., 1996).

Safflower (Carthamus tinctorius L.) is a member of composite family that is used mainly in producing edible oils, feeding livestock and dyeing industries. Safflower is considered a moderately salt-tolerant crop that is also exceptionally deep rooted (Weiss, 1971). An abundance of biotic and abiotic stresses in most of the safflower production areas motivated breed-ers to evaluate the crop’s resistance to salinity and fungi diseases. The ef-fect of different levels of sodium chloride salinity on germination and seed-ling growth in safflower was examined by Hajghani et al., (2008). Their results indicated that safflower cultivars responded to salinity stress during germination and seedling growth processes completely differently and that seed germination is the most sensitive phase in the presence of sodium chloride. Varying reductions in seed germination and wet and dry seedling weight due to salinity stress were observed by Saddiqi et al., (2007) in a study of ten safflower genotypes. Although safflower has been over con-sidered because of its adaptability to important abiotic stresses like salinity, drought and low fertility, biotic stresses such as pathogens and pests are the main restriction factors for growing the crop (Zeinali, 1999). Some patho-gens of the genus Pythium and Pythophthora that cause seed rotting, pre and post-emergence damping-off, root and hypocotyl decay have been re-ported as the most important pathogenic limitations of the crop (Pahlavani

et al., 2006).

Generally damping-off is a fungal disease that leads to sudden seedling death; however, it can kill germinating seeds too. Pathogen Pythium

ulti-mum var ultiulti-mum Trow. has been identified as the causal agent of seed

rot-ting and seedling damping-off in the US, Australia, Canada and Iran (Huang et al., 1992). This pathogen invades germinating seeds and hypo-cotyls tissues of young seedlings and finally causes death (Kolte, 1985). Studies have proved that safflower shows a great weakness in facing P.

ulti-mum, so that in experimental infection, just 8 to 16% of seedlings survived

(Pahlavani et al., 2009; Huang et al., 1992). Ahmadi et al. (2008) also showed that seeding in P. ultimum infected plots killed up to 43% of seeds and damped-off up to 37% of seedlings of safflower genotypes. Members of

Pythium are considered pathogenic agents that spread in arable soils across

the world. More than 500 plant species have been identified so far as host for this genus.

Although the bilateral relations of safflower and P. ultimum have been largely formulated, once other stressful factors, which affect host,

patho-gens and their relations, are put into the field, a new window is opened for researchers. For example, it has been proved that salinity leads to increased capacity for infection in Phytophthora capsici susceptible pepper plants (Sanogo, 2004). Pistachio seedlings that were exposed to NaCl salinity be-fore inoculation with Verticillium dahliae showed more susceptibility to Verticillium wilting, especially in salinity susceptible stocks (Mohammadi and Banihashemi, 2007). Also, the simultaneous effect of NaCl and V. dahliae reduced dry weight and raised concentration of ions Na, K and Cl in shoot and root organs in relation to their separate effects. As a result of syn-ergistic effects between salinity and salinity-resistant seedlings in cucum-ber, mortality caused by Pythium aphanidermatum increased with the in-crease of salinity (Alsadi et al., 2010). A study on the effects of soil salinity on production of sporangium and zoospore of Pythophthora isolates in soy-bean indicated that resistance of different isolates against high amounts of salt was not identical (Blanker and McDonald, 1985). Improving germina-tion and growth under condigermina-tions of an abiotic stress like salinity and also a biotic stress such as fungi is possible but only with a good understanding of the interaction effects of the plant, salinity and pathogen. Because no work has been published describing the interactive effect of salinity, culti-var and P. ultimum on early growth of safflower, this present study was conducted to determine whether salinity promotes damping-off and whether seedlings lose dry weight in different genotypes of safflower. Another im-portant question is whether the response of safflower to the simultaneous presence of NaCl and P. ultimum is different from its response to either when applied separately.

MATERIALS AND METHODS

This study was performed at the plant breeding lab of Gorgan University of Agricultural Sciences and Natural Recourses (GUSNAR), Gorgan, Iran in 2009. The experiment comprised three factors: genotype (Aceteria, Dinger, LRV-5151 and Arak2811), salinity (0 as control, -10, -14 and -18 bar), and infection of P. ultimum (pathogen-inoculated and pathogen-free). These safflower genotypes had considerable variation for resistance to the disease and also had some important agronomic characteristics as revealed in a previous study (Ahmadi et al., 2008).

The fungus was cultured on corn-meal-agar (CMA) medium, after which square fragments of the culture were poured into autoclaved water and then kept for four days under fluorescent light to activate zoospores. Then the number of zoospores was counted by a hemocytometer, and the suspension was prepared with a final concentration of 105 zoospores in one milliliter. To blend different levels of salinity and pathogen, the required amounts of

NaCl were weighed and added to the pathogen suspension to obtain exact salinity concentration.

Fifty inoculated seeds of each genotype, after having been disinfected with 5% bleach (sodium hypochlorite) for three minutes, were arranged on a 50 × 50 cm2 paper towel soaked with the saline pathogen suspension (Kozik et al., 1991; Govindappa et al., 2005). Another paper towel of the same size was put over each towel to cover the seeds. The paper towels containing seeds were rolled and put in a plastic bag to prevent loss of wa-ter. All towels containing seeds were then placed in a dark incubator at 25±2 degree centigrade for seven days; water was added as needed to keep towels moist. Autoclaved distilled water was used for control. The experi-mental units were observed daily at a specified time and the number of ger-minated seeds and seedlings showing disease symptoms was recorded. Meanwhile, random samples of infected seedlings and rotted seeds were cultured to verify the presence of the pathogen (Van der Plaats-Niterink, 1981).

By the seventh day, the dry weight of all seedlings in each unit was measured with an exact balance after 48 hours drying in at 70°C oven. Evaluated characteristics included the seed germination (%), rate of seed germination, ratio of damping-off to germination, rate of damping-off and seedling dry weight. The formula below was used to calculated rate of ger-mination or damping-off (RGD) (Magiure, 1962):

where a is: number of germinated seeds in counting day; b: sum of ger-minated seeds in previous days; N: total number of seeds in each unit and

D: days after seeding.

The data of seed germination, rate of seed germination, and seedling dry weight was analyzed as a factorial fashion in a format of completely ran-domized design (CRD) with four replications. To obtain the ratio of damp-ing-off to germination and rate of dampdamp-ing-off, data was analyzed as facto-rial of genotype by salinity just for records in pathogen-inoculated units. Coefficient of variation (CV%) was calculated to visualize how salinity or pathogen varied the characteristics of the cultivars.. Data analysis was per-formed by SAS software (SAS, 1996) and Excel was used to draw graphs. A least significant difference (LSD) at 5% statistical level was done for means comparison. Prior to analysis, square root transformation was ap-plied for ratio of damping-off to germination and rate of damping-off.

RESULTS AND DISCUSSION

Analysis of variance showed that the effects of cultivar and salinity and interaction effects of cultivar×infection, salinity × infection and cultivar × salinity × infection were significant on seed germination (Table 1). For rate of germination, effects of cultivar, infection and salinity and also interac-tion effects of cultivar × salinity and salinity × infecinterac-tion were also signifi-cant. The results also indicated that cultivar, infection, and salinity, along with the interactions of salinity × infection and cultivar × salinity × infec-tion, significantly changed dry weight of safflower seedlings (Table 1).

Table 1 Analysis of variance for seed germination of safflower affected by cultivar, salinity

and infection to P. ultimum.

Analysis of variance also revealed that the factors cultivar and salinity and their two-way interactions had significant effects on the ratio of damp-ing-off to germination and the rate of dampdamp-ing-off (Table 2). Given the sig-nificant effects of two-and three-way interactions on the studied variables, LSD comparison means was done separately for each of the salinity levels within the pathogen-inoculated and pathogen-free environments and the results were presented in Figs 1 to 8. Salinity stress and infection with

P. ultimum reduced seed germination in safflower (Table 3).

Source of variation DF Mean of squares

Seed germination [%] Rate of germination Seedling dry weight

Cultivar 3 2844.62** 0.195** 0.866** Salinity 3 8537.45** 1.099** 0.898** Infection 1 16.53 0.013** 0.180** Cultivar× Salinity 9 91.23 0.031** 0.041* Cultivar× Infection 3 235.03** 0.016** 0.045* Salinity× Infection 3 460.86** 0.022** 0.346**

Cultivar× Salinity× Infection 9 123.03* 0.002 0.055**

Error 96 64.01 0.001 0.020

Table 2 Analysis of variance for damping off in safflower at P. ultimum infected media

affected by cultivar and salinity

Table 3 Mean trait of safflower at Pythium-free and Pythium-inoculated (in italic)

environments over different salinity levels

†: Data was recorded just at Pythium-inoculated environments

In both Pythium-free and -infected beds, germination of seeds was in in-verse proportion to the salinity level (Figs 1 and 2). At salinity 0 and -14 bar, the pathogen reduced average seed germination of the cultivars when compared to pathogen-free environments (Table 3). Average seed germina-tion of the cultivars decreased from 82.75% at salinity 0 to 42.50 at -18 bar in the absence of Pythium and decreased from 76.00% at salinity 0 to 53.25 at -18 bar in Pathogen-inoculated environments (Table 3). Reduction of seed germination under the influence of salinity has already been reported in safflower (Pahlavani et al., 2006). Mafton and Sepaskhah (1978) previ-ously studied the effects of NaCl salinity on two safflower cultivars and

Source of variation DF

Mean of squares Ratio of damping off

to germination Rate of damping off

Cultivar 3 0.099** 0.027* Salinity 3 0.852** 0.128** Cultivar× Salinity 9 0.024** 0.003** Error 48 0.008 0.001 CV (%) 20.44 22.05 Salinity level

[bar] Seed germination [%] Rate of germina-tion Seedling dry weight off to germination Ratio of damping damping off Rate of

0 82.750 0.594 1.157 —† — 76.000 0.497 0.795 0.637 0.086 -10 71.031 0.292 0.940 — — 73.375 0.296 0.960 0.087 0.011 -14 67.750 0.217 0.909 — — 64.625 0.211 0.821 0.130 0.015 -18 42.500 0.101 0.548 — — 53.250 0.122 0.671 0.075 0.013

found that when osmotic pressure increased from 0 to -25.1 atm, germina-tion declined from 83% to 0.

Fig. 1. Effect of salinity level without Pythium ultimum inoculation on seed germination of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity level, means followed

by the same letter are not significantly different according to LSD test (P < 0.05)

Fig. 2. Effect of salinity dosage with Pythium ultimum inoculation on seed germination of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity level, means followed

At all salinity levels, whether in the sterile or in the infected environ-ment, seed germination of cultivar Arak2811 was superior to other cultivars (Figs 1 and 2). In these conditions, the lowest germination was allocated to Dinger. The seed germination of Aceteria was intermediate in all experi-mental conditions (Fig. 1 and 2). In canola (Brassica napus L.), it has been shown that properties like percent and rate of seed germination, length and fresh weight of radicle and leaflet decreased significantly with increasing salinity (Bybordi and Tabatabaei, 2009). Other study showed that root rot severity of tomato seedlings infected with Fusarium oxysporum was in-creased significantly when seedlings were irrigated with saline water (Triky -Dotan et al., 2005). In the first experiment, disease severity in saline-irrigated (EC=3.2±0.1 ds × m-1) and non-saline-irrigated (EC=0.4±0.1 ds × m-1) plots was 75 and 38 %, respectively. In the second experiment, final incidence of the disease in plants irrigated with saline and nonsaline was observed at 12 and 4 %, respectively. Irrigation of tomato seedlings with 20 mM NaCl so-lution had no effect on plant growth but higher concentrations of NaCl had an inhibiting impact. The results of these researches also show that plants irrigated with saline water from the beginning have more susceptibility to disease than those irrigated with non-saline water from the beginning (Triky-Dotan et al., 2005). In both sterile and pathogen-infected environ-ments, coefficient of variation (CV%) of cultivars showed changes in seed germination (Table 4). In salinity -18 bar, infecting with P. ultimum de-creased CV% from 27.56 to 16.63% (Table 4). In this salinity, seed germi-nation of LRV5151 was increased from 46 to 53% because of infectithe pathogen (Fig. 1 and 2).

Table 4 Coefficient of variation (CV%) for safflower cultivars at Pythium-free and Pythium-

inoculated (in italic) environments over different salinity levels

†: Data was recorded just at Pythium-inoculated environments Salinity level [bar] Seed germination [%] Rate of germina-tion Seedling dry weight Ratio of damping off to germination Rate of damping off 0 12.53 29.47 21.95 —† — 15.03 32.27 26.78 33.18 47.63 -10 17.45 30.38 22.14 — — 14.21 21.87 19.91 44.74 57.03 -14 15.08 27.32 18.50 — — 16.61 27.57 24.21 48.67 67.55 -18 27.56 33.11 30.10 — — 16.63 25.75 20.86 56.27 88.72

Salinity and infection with P. ultimum reduced the rate of seed germination of the cultivars (Table 3). In both the pathogen-free and infected environments, average seed germination of cultivars dropped with an increase of salinity, al-though the decrease was less in the presence of the pathogen (Table 3). In the lack of salinity (0 bar), the pathogen reduced the rate of seed germination, but it had no strong effect in saline environments (-10, -14 and -18 bar). This finding clearly indicates that sodium chloride can reduce the pathogenesis of P. ultimum on safflower seedlings. The composition of the microbial community may be affected by salinity since the microbial genotypes differ in their tolerance of a low osmotic potential (Pankhurst et al., 2001; Gros et al., 2003). In fungi, a low osmotic potential decreases spore germination and the growth of hyphae and changes the morphology and gene expression, resulting in the formation of spores with thick walls (Mandeel, 2006). There is also a significant reduction in the total fungal count in soils salinized with different concentrations of sodium chloride (Mandeel, 2006; Llamas et al., 2008). Van Bruggen and Semenov (2000) reported that, over a longer term, there is a decrease in the genetic diver-sity of fungi as a result of stress.

Fig. 3. Effect of salinity level without Pythium ultimum inoculation on rate of seed germination of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity

level, means followed by the same letter are not significantly different according to LSD test (P < 0.05)

Increasing salinity from 0 to -18 bar decreased the rate of seed germina-tion from 0.594 to 0.101 in the pathogen-free environments and from 0.497 to 0.122 in the pathogen-inoculated environments (Table 3). Rate of seed germination shows how fast the seeds of a genotype complete their germi-nation. The fastest germinating genotype in the pathogen-free saline condi-tions was LRV5151, while in the pathogen-inoculated saline environments,

Arak2811 had the highest rate of seed germination (Fig. 3 and 4). The lowest rate of germination over all salinity levels in pathogenfree and -inoculated conditions belonged to the cultivar Dinger. The rate of seed ger-mination was intermediate for Aceteria in all salinity and infection condi-tions (Figs 3 and 4). Infection with P. ultimum increased CV% for rate of germination in the non-saline condition (0 bar) and decreased it in the high-est salinity level (-18 bar). However, there was no difference between pathogen-free and -inoculated conditions over the intermediate salinities (-10 and -14 bar) for CV% of the rate of seed germination (Table 4).

Fig. 4. Effect of salinity level with Pythium ultimum inoculation on rate of seed germination of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity

level, means followed by the same letter are not significantly different according to LSD test (P < 0.05)

The means comparison revealed that NaCl salinity and P. ultimum mainly had a reducing effect on seedling dry weight of the cultivars (Table 3). In pathogen-free environments, enhancing the salinity decreased seedlings dry weight, but in pathogen-inoculated conditions, dry weight first increased from 0 to -10 bar and then decreased in the higher salinities (Table 3). In the absence of the pathogen, the highest seedling dry weight belonged to cultivars LRV5151 and Arak2811 (Fig. 5). In P. ultimum infected environ-ments, the highest seedling dry weight in 0 bar belonged to LRV5151 and in salinities -10, -14 and -18 bar, Arak2811 (Fig. 6). In a non-saline condi-tion (0 bar), infecting with the pathogen increased CV% for seedling dry weight; however, in saline environments, the CV% was reduced (Table 4).

Fig. 5. Effect of salinity level without Pythium ultimum inoculation on seedling dry weight of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity

level, means followed by the same letter are not significantly different according to LSD test (P < 0.05)

Fig. 6. Effect of salinity level with Pythium ultimum inoculation on seedling dry weight of four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity

level, means followed by the same letter are not significantly different according to LSD test (P < 0.05)

The results showed that the effect of pathogen on percent and rate of seed germination and seedling dry weight in safflower was quite different, depending

on the presence or absence of salinity stress (Table 3). For example, the pres-ence of the pathogen led to an 8.2% reduction of seed germination in non-saline environments, whereas over all the saline environments, it created an almost 5.5% increment (Table 3). Reduction of dry and fresh weight of seedlings in safflower due to salinity has been reported before by Siddiqi et al., (2007). Soil salinity is also an important factor in the establishment of rice seedlings in the presence of Pythium torulosum, as shown by Eberle et al (2008), so that in the electrical conductivity above 2022 μS × cm-1, salinity reduced root and seedling weight and increased leaf necrosis. This study also suggested that interaction between seedlings' pathogens, generally common in soil, and salinity may cause more damage to the plant establishment.

With the addition of salinity, the average rate of damping-off was re-duced from 0.086 (in non-saline environment) to 0.013 (in -18 bar salinity; Table 3 and Fig. 7). In asparagus, sodium choleric salinity also reduced damage of Fusarium rot (Reid et al., 2001). The rate of damping-off indi-cates how fast the seedlings of a genotype die. The highest rate of seedling damping-off over all salinities belonged to Arak2811 and the genotype with the lowest rate varied, depending on the salinity level (LRV5151in non-saline and Dinger in non-saline environments; Fig. 7). The salinity enhanced the CV% for rate of damping-off caused by P. ultimum; however, the superior-ity of Arak2811 over the other cultivars was more obvious in -18 bar (Table 4).

Fig. 7. Effect of salinity level with Pythium ultimum inoculation on rate of damping off in four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811). In each salinity

level means followed by the same letter are not significantly different according to LSD test (P < 0.05)

Increasing salinity from 0 to -10 bar lowered the ratio of damping-off to germination; however, further increases from -14 to -18 bar dropped the ratio (Table 3 and Fig. 8). The highest ratio of damping-off to germination in all salinities belonged to Arak2811, and the lowest one depended on the salinity level (Fig. 8). In the non-saline environment, the lowest ratio of damping-off to germination belonged to LRV5151, and in saline environ-ments (-10, -14 and -18 bar) to Dinger and Aceteria (Fig. 8). Salinity also increased the difference between the cultivars for the ratio of damping-off to germination, so that CV% was 33.18% in salinity 0 bar and 56.27% in -18 bar (Table 4). Although salinity increased CV% for the rate and ratio of damping-off, it reduced CV% for percent and rate of germination and also seedling dry weight (Table 4). This finding suggests that decisions about recommendations regarding the best salt-tolerant cultivar for cultivation in soil infected with Pythium ultimum can be made mainly on the basis of the potential of genotypes, determined through rate and ratio of damping-off.

Fig. 8. Effect of salinity level with Pythium ultimum inoculation on ratio of damping off to seed germination in four safflower cultivars (Acteria, Dinger, LRV5151 and Arak2811).

In each salinity level, means followed by the same letter are not significantly different according to LSD test (P < 0.05)

According to definition, interaction between two or more factors is cre-ated when the main effect of one of the factors changes over the levels of the other factor (Snedecor and Cochran, 1989). Although researchers be-lieve that interaction makes interpretation of results very difficult and delu-sive, the effect of other factors should be considered in explaining the ef-fects of one particular factor on the variable(s) being investigated. The oc-currence of significant two or three-way interactions between factors in the

present study indicates that seed germination, damping-off and seedling dry weight of safflower in Pythium-free and -inoculated environments depend on the level of salinity and the cultivated cultivar. From a practical point of view, depending on the NaCl and Pythium-infected conditions of the field, different varieties are recommended to achieve an acceptable establishment and production. For example, while the highest rate of germination in con-trol conditions (0 bar and pathogen-free condition) was observed in LRV5151, in the presence of pathogen and salinity -18 bar, Arak 2811 had the highest rate of germination (Fig. 3 and 4). Since the salinity affects growth and pathogenicity of Pythium ultimum in addition to germination and growth of safflower, the occurrence of significant two or three-way in-teractions in this experiment is not surprising (Pankhurst et al., 2001). Changes in the consequences of salinity in the presence or absence of a pathogen have been reported in other studies. The results of Howell et al. (1994) also showed that in the absence of Verticillium albo-atrum, salinity 5.7 Dsz/m2 significantly reduced the performance of two varieties of alfalfa in relation to control. In a non-saline environment, the percent of stems in-fected with V. albo-atrum was significantly higher and the hardiness of ne-crotic roots greater in variety Moapo-69 than in NK-89786. Also, in the salt -free soils, V. albo-atrum had no significant influence on the performance of the varieties; however, the simultaneous presence of the pathogen and salinity significantly reduced the performance. A study on Verticillium wilt disease of pistachio in a hydroponic environment showed that the interac-tion of sodium chloride and the pathogen Verticillium dahliae had more effect on reduction of dry weight and concentration of ions, Na, K, Cl in shoot and root organs than a separate treatment of the salt and pathogen (Mohammadi and Banihashemi, 2007). Salinity has been reported to in-crease the incidence of Phytophthora root rot in chrysanthemum (MacDonald, 1982) and tomato (Snapp et al., 1991).

In the salinity -10 bar, the pathogen slightly increased germination and dry weight,and this was contrary to what was observed in salinities 0, -14 and -18 bar (Table 3). This shows that in the lower salinities, the presence of the pathogen increased the germination and growth. Increased growth of seedlings in reaction to biotic and abiotic stresses has been reported before in halophytes plants such as Atriplex (Saikachout et al., 2009). Another in-teresting point is that the highest germination and rate of pathogen-caused damping-off was observed in Arak-2811 (Fig. 1 and 7). This makes the de-cision about the introduction of the most tolerant genotype a little difficult, because the genotype showing the highest seed germination in pathogen-infected environments also has the highest rate of damping-off. So, instead of using the rate of damping-off, the ratio of damping-off to seed germina-tion was calculated (Fig. 8). The ratio of damping-off to seed germinagermina-tion actually indicates what proportion of the seedlings is infected by the

patho-gen. Since some of the seeds rot because of the pathogen before germina-tion begins, this ratio offers real criteria of resistance to or tolerance of seedlings to the pathogen. Percent of germinated seed in Pythium-infected environment represents the number of seeds not infected, as more germina-tion shows more resistance to the seed rot. So it can be deduced that geno-types having a lower ratio of damping-off to seed germination are more re-sistant to seedling damping-off. The results showed that the difference be-tween genotypes in terms of damping-off was more than their differences for seed rot (Table 4). Percent of non-germinated seeds for Arak2811, LRV5151, Dinger and Aceteria was 12.0, 17.0, 30.5 and 36.5 %, respec-tively; however, their ratio of damping-off to seed germination was 90.5, 39.6, 58.0 and 46.7 %, respectively (data not shown). Therefore, the most tolerant genotype in terms of seed rot (or pre-emergence death) was Arak2811 and for damping-off (or post-emergence death) was LRV515. As a finding, the resistance to seed rot in Arak2811 was not related to resis-tance of the seedlings to P. ultimum. The existence of both pre and post-emergence damping-off caused by P. ultimum has been reported before in safflower (Mundel et al., 1995).

The simultaneous presence of NaCl salinity and P. ultimum reduced ad-verse effects of both factors when applied separately (Table 3). Aceteria and Dinger can be used for cultivation in saline soils infected with the pathogen P. ultimum, since both of them had lower rate and ratio of damp-ing-off in these conditions.

In the present study, the combined effects of pathogen Pythium ultimum and NaCl salinity were examined on germination and seedling growth of safflower genotypes, but in real conditions of the field, other important fac-tors such as moisture, temperature and soil structure affect these interac-tions which makes the prediction difficult. Therefore, here we recommend studying the interaction of all these factors on the pathogenesis of P.

ulti-mum in safflower cultivation. Our results also suggest that it may be useful

to include salt tolerant cultivars of safflower in breeding programs for Py-thium damping-off resistance to minimize further yield decline in areas where both factors occur together.

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