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Potential using of transgenerational resistance against common bacterial blight in Phaseolus vulgaris
Crop Protection 127 (2020) 104967
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Potential using of transgenerational resistance against common bacterial blight in Phaseolus vulgaris €prü Ahmet Akko Plant Protection Department, Faculty of Agriculture, Van Yüzüncü Yıl University. Zeve Campus, 65080, Van, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Next generation priming Epigenetic Xanthomonas axonopodis pv. phaseoli Acibenzolar-S-Methyl Common bean
Direct activation of induced resistance is recommended for the prolonged and effective control of plant diseases. However, there are some potential risks to this method in field application. The priming form of resistance has less risk; nevertheless, it is necessary to increase adaptation and efficacy. Recently, it was shown that the priming as a form of resistance mechanism in plants was transferred epigenetically. In our study, the use of this parentally-inherited resistance for disease control and the possibility of increasing resistance capacity were investigated. For this aim, plant growth parameters, population dynamics of Xanthomonas axonopodis pv. phaseoli (Xap), and severity and development of the common bean bacterial blight disease were investigated in the primed generation obtained from acibenzolar-S-methyl (ASM) treated parent bean. It was revealed that the primed progeny suppressed the disease by 11%, according to the area under the disease progress curve (AUDPC). Moreover, when the primed progeny were treated with a low dose of ASM (20 μM), they showed nearly twice the resistance capacity and suppressed the disease by 60%. In primed progeny, ASM treatment significantly slowed the development of Xap populations until the 14th day, also did not adverse effects on plant development. All these results indicated that seed production with this method offers the possibility of increasing the resistance capacity of later generations, just as it ensured the protection of parents. Thus, using the appropriate dose of activator at primed progeny will ensure that the efficacy of control methods is increased, while pesticide inputs can be reduced.
1. Introduction Plants have adaptive strategies to cope with variable and unfavor able growing conditions in nature (Pikaard and Mittelsten Scheid, 2014). One of the most important of these mechanisms is induced plant resistance. This form of resistance offers significant possibilities for controlling diseases and pests by adapting plant cultivation and pro tection strategies in agriculture. Induced resistance is defined as rapid and robust activation of a basal resistance level against subsequent challenges by stimulating the plant with an appropriate stimulant (Ahmad et al., 2010; Mauch-Mani et al., 2017). Stimulants may be biotic or abiotic (environmental or chemical) and determine the type of resistance (Ton et al., 2002; Van der Ent et al., 2009). The first plant resistance system identified was systemic acquired resistance (SAR), which can be triggered by pathogens and many chemical stimulants such as salicylic acid (SA), acibenzolar-S-methyl (ASM), 2,6-dichloroiso nicotinic acid, probenazole, and cyclopropanecarboxylic acid (Walters et al., 2013). Some of these stimulants are sold as plant activators. The plant induced resistance is a complex phenomenon that is
formed by the activation of several layers and can be exhibited different forms of plant defence during different periods of the attack (Ahmad et al., 2010; Pastor et al., 2013). One of this form is priming which can be described as a physiological process by which a plant prepares itself to respond to future biotic or abiotic stress more quickly (Conrath, 2011; Mauch-Mani et al., 2017; Pastor et al., 2013). In primed plants, no sig nificant increase is observed in the activation of defence genes until being exposed to stress. Thus, the priming reduces the allocation cost of defence to the plant (van Hulten et al., 2006). In general, resistance-inducing agents establish priming. The stimu lation dose determines whether direct resistance or priming is induced. In Arabidopsis thaliana, van Hulten et al. (2006) observed that treatment with high doses of β-aminobutyric acid (BABA) triggered both direct resistance and priming simultaneously. In contrast, when the plants were treated with low doses of BABA, only priming was observed. In other words, temporary stimulation of direct resistance may ensure the long-term priming (Ahmad et al., 2010; Heil and Ton, 2008). Hence, the induced resistance phenomenon can be defined as a combination of direct defense and priming. Ahmad et al. (2010) stated that induced
E-mail address: [email protected]. https://doi.org/10.1016/j.cropro.2019.104967 Received 9 April 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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resistance is linked to the combination of stimulant dose and time period which is between stimulation and interaction of plant with the pathogen. Although the activation of direct resistance provides rapid and robust response to pathogen attack, it has a cost to the plant. Some studies revealed that direct activation of induced resistance resulted higher adverse effects on yield and development under limited growth conditions (Heil et al., 2000; van Hulten et al., 2006; Walters and Fountaine, 2009; Walters et al., 2013). On the other hand, van Hulten et al. (2006) stated that resistance activated by priming may cause minor reductions in relative growth rate and had no effect on seed production in the absence of the pathogen. They also reported that this cost could be ignored under pathogen pressure. Other researchers showed that this cost is very low; therefore, it has no significant in field conditions (Walters and Heil, 2007). The variety of epigenetic pathways in plants is notably great and they contribute significantly to plant growth, phenotypic plasticity, survival in unpredictable environments, and proliferation ability (Pikaard and Mittelsten Scheid, 2014; Springer, 2013). In recent years, some studies have reported that resistance in priming form against biotic stressors in different plants, obtained via different stimuli, can be epigenetically transferred to the offspring. Epigenetic transfer of the resistance was observed in tobacco against tobacco mosaic virus (Kathiria et al., 2010), and in Arabidopsis thaliana against Pseudomonas syringae pv. tomato (Luna et al., 2012) and Hyaloperonospora arabidopsidis (Slaughter et al., 2012). Additional observations were recorded in barley against the fungus Rhynchosporium commune (Walters and Paterson, 2012) and the common bean against P. syringae pv. phaseolicola (Martínez-Aguilar et al., 2016; Ramírez-Carrasco et al., 2017). Pieterse (2012) stated that transgenerational resistance inherited epigenetically is a form of communication between parent plants and their progeny. Epigenetic changes are reversible and do not cause any changes in nucleotide sequences. They occur through some modifications such as DNA methylation, histone modification, and through small interfering RNA activities (Conrath et al., 2015; Pikaard and Mittelsten Scheid, 2014). Luna et al. (2012) stated that transmission of priming between generations was linked to the SA and NPR1 signalling pathways. They showed that transmission occurred via hypomethylation of genes linked to SA in subsequent generations. Other detailed studies of the Arabi dopsis thaliana/P.s. pv. tomato and H. arabidopsidis pathosystem indi cated that resistance may be transmitted via RNA-directed DNA methylation and hypomethylation of the CpNpG region (Luna and Ton, 2012). It was revealed that the priming status in beans could be trans mitted with the aid of changes in the H3K4me3 and H3K36me3 marks at the promoter-exon regions of defence-associated genes. (Martínez-A guilar et al., 2016; Ramírez-Carrasco et al., 2017). The common bean (Phaseolus vulgaris L. (Fabaceae)), the model plant in the current study, is an economically important crop that is grown worldwide, because of its high protein, fiber, complex carbohydrate content, and consumption in different forms (Pathania et al., 2014). Total green and dry bean production in the world in 2017 is more than 55 million tons (FAO, 2019). However, many diseases cause severe losses (20–100%) in the yield and the quality of common bean world wide (Singh and Schwartz, 2010). One of the most important of these diseases is common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli ((Smith, 1897) Vauterin et al., 1995) (Xap). The pathogen has been observed on all continents (CABI, 2019; EPPO, 2006). Xa pv. phaseoli induces symptoms on leaves, stems, pods, and seeds. The pathogen can survive and multiply on bean and weed hosts without showing any symptom as epiphytically and endophytically (Akhavan et al., 2013; Jacques et al., 2005). One diseased plant, infected with this aggressive pathogen, in 10,000 is sufficient to cause a severe epidemic (Akhavan et al., 2013), and the disease can cause up to 40% yield loss (Boersma et al., 2015; Singh and Miklas. 2015). Such features of the pathogen make it difficult to control the disease. Some recommended methods for controlling of the disease such as
cultural methods, using resistant varieties and pesticide application may not always give the desired results (CABI, 2019). The control of the disease by chemicals is the most common method. However, Maringoni (1990) showed that spraying chemicals on the plants in the field did not give a satisfying effect. On the other hand, the pathogen has potential of the development of resistance against some chemicals which are used for controlling the disease (Griffin et al., 2017). Also, adverse effects of pesticide on the environment and human health are well understood (Bruce, 2010). Within this framework, alternative disease control methods and induced resistance in particular, have been gaining sig nificant attention for their potential. The transmission of induced resistance between generations may establish a new approach to control the diseases. This may increase the efficacy of disease control with induce systemic resistance, offering the opportunity for low-cost, reliable, and economic way. The aim of this study was to determine to potential of transgenerational priming pro vided by chemical stimulant acibenzolar-S-methyl (ASM) to control the common bacterial blight disease of bean caused by Xap. With this aim, a new generation that was produced using seeds obtained from parents with activated induced resistance, was used. In current study, we investigated: i) the effects of transmitted resistance on plant growth, Xap population density, and disease progress, and ii) the efficacy of different doses of ASM in increasing the resistance capacity of primed progeny. 2. Materials & methods 2.1. Plant material and growth conditions Common bean seeds (Phaseolus vulgaris L. cv. Gina) were planted in 300 mL plastic pots filled with sterile peat and left to develop in a climate chamber at 24 � C, with 16 h light and approximately 50% hu midity. The nutritional requirements of the seedlings were provided by regularly applying Hoagland nutritional solution. The ASM suspension (Sigma-Aldrich Chemie B.V.) was prepared at a concentration of 250 μM (Azami-Sardooei et al., 2013) and was added to 0.01% Tween solution. When the first true leaves opened, the solution was sprayed on the leaves to thoroughly wet the plant. The ASM treatment was repeated four times, once every 5 days. The same water regime was applied to control plants. Three days after the final ASM application, 40 common bean seedlings for each treatment were transplanted into a field prepared by removing weeds and adding chemical fertilizer (Nitrogen, Phosphate, Potassium; 15:15:15). The field was located within the boundaries of Gevas¸ county in Van province, Turkey (38.18� N, 43.90� E; 1684 m elevation). During the growing period, seedlings were watered regularly with a drip irrigation system, and hoed once. No pesticides were applied. The common bean pods were harvested and then completely dried. Seeds from 15 randomly selected plants were harvested from the pods and weighed. Seeds were stored in low-humidity conditions awaiting further study. 2.2. Primed generation The seedlings whose parents were treated with and without ASM were grown as primed (p) and non-primed (c) plants, respectively. These seedlings were treated at doses of 10, 20, and 80 μM ASM. The effects on disease severity and development were determined by investigating the area under the disease-progress curve (AUDPC), the Xap population dynamics, and the plant growth parameters. The constituted variants for the treatment groups and the schema of the study were given in Table 1. The primed (p) and non-primed (c) seeds were planted in pots filled with sterile peat as mentioned above and left to grow. When the first trifoliate leaves started to form, 10, 20, and 80 μM ASM was sprayed on the leaves. Three days after the ASM treatment, Xanthomonas axonopodis pv. phaseoli ((Smith, 1897) Vauterin et al., 1995) (Xap) was inoculated to plants. The pathogen isolated from common bean in Antalya, Turkey, was provided by Prof Dr Hüseyin Basım (Faculty of Agriculture, Akdeniz 2
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Crop Protection 127 (2020) 104967
Table 1 Experimental design. A, Plants were treated four times at intervals of 5 d by 250 μM ASM or water to the parent’s plant leaves. Then plants were transplanted in the field and allowed to set seed to provide. The primed and non-primed seeds (Progeny) from the parent plants constituted the main seeds material for the later experiments. In the second part of the study, in climate chamber, the progeny seedlings treated 10, 20, 80 μM doses of ASM, and then Xap inoculated. B, Experimental groups, in climate chamber studies, constituted with primed and non-primed seedlings with 10, 20, 80 μM doses of ASM. Xap population dynamics, disease progress and plant growth parameters were evaluated in the study.
Disease index ¼
AUDPC ¼
Primed plants treatments
*c (- Xap) PCc c þ ASM 10 μM c þ ASM 20 μM c þ ASM 80 μM c þ ASM 10 μM þ Xap c þ ASM 20 μM þ Xap c þ ASM 80 μM þ Xap
p (- Xap) PCp p þ ASM p þ ASM p þ ASM p þ ASM p þ ASM p þ ASM
n 1 X yi þ yiþ1 x ðtiþ1 2 i¼1
ti Þ
(2)
2.4. Determination of bacterial density in leaves With the aim of determining the population of Xap on the plants, three plants were randomly selected from each application group at 2 h, 24 h, and, 3, 7, and 14 days after pathogen inoculation. Twenty disks with a diameter of 0.5 cm were taken randomly from leaves of each seedling with a cork borer. The disks were weighed, and then crushed in polyethylene bags using a hand stomacher. Dilution steps were created from the obtained suspension and plated on modified MXP medium (Claflin et al., 1987). MXP agar is a semi-selective medium for Xap; the suspension consisted of soluble potato starch (8.0 g/L), K2HPO4 (0.8 g/L), KH2PO4 (0.6 g/L), yeast extract (0.7 g/L), potassium bromide (10.0 g/L), glucose (1.0 g/L), methyl violet 2B (30 μL/L; 1% solution in 20% ethanol), and methyl green (60 μL/L; 1% aqueous solution) and agar (15.0 g/L). This suspension was autoclaved. Antibiotic solutions containing chlorothalonil (15 mg/L), cephalexin (20 mg/L), kasugamy cin (20 mg/L), gentamycin (2 mg/L), and cycloheximide (100 mg/L) were passed through a filter and added to the autoclaved suspension after cooling to 50 � C. The petri dishes that were plated with diluted suspensions of plant extract, were incubated for 2–3 days at 27 � C. The colonies that developed were counted, and the Xap population on the plant was determined as the CFU per gram fresh plant leaf.
B Control plants treatments
Σ ðRating number x Number of leaves in the ratingÞ x100 Total number of leaves x Highest rating (1)
2.5. Plant growth parameters
10 μM 20 μM 80 μM 10 μM þ Xap 20 μM þ Xap 80 μM þ Xap
At the end of the study, 21 day after Xap inoculation, leaves number, and shoots and roots weight were determined as fresh and dry. All the leaves on the plants were counted, excluding cotyledons and bifoliate leaves to determine leaves number. The bean plants were then uprooted and cut at the root crown. Roots were cleaned of growth medium residue by washing with tap water. The water was then removed from the sur face with blotting paper before determining the fresh weights of the roots and stem. The roots and shoots were then dried in an oven at 65 � C until a stable weight was reached. The dry weights were then determined.
*c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap.
University, Antalya, Turkey). The Xap was developed for 48 h in tryptic soy agar (TSA) medium (1.7 g/L tryptone, 0.3 g/L soybean peptone, 0.25 g/L glucose, 0.5 g/L NaCl, 0.5 g/L K2HPO4, and 15 g/L agar). The concentration of Xap was set to 108 CFU/mL (A600 ¼ 0.13) and 0.01% Tween was added to the suspension. All the leaves of the common bean seedlings were then dipped in the beaker containing the pathogen sus pension for 10 s each. Immediately after pathogen application, seedlings were kept in polyethylene cabinets in the climate chamber to create high humidity for 48 h. The seedlings were kept in darkness for the first 24 h.
2.6. Data analysis Data obtained at the end of the field studies were analyzed using an unpaired two-tailed Student’s t-test (p < 0.01). The experimental vari ants for the treatments in the climate chamber were as shown in Table 1, and the studies were repeated twice. Data obtained from the climate chamber studies were analyzed using SPSS v17.0 statistical software. Significant differences between treatments were determined using Duncan’s multiple range test with a significance level of p � 0.05. The Xap population data were log-transformed prior to analysis.
2.3. Disease assessment The disease severity was determined 7, 14, and 21 days after path ogen inoculation using a 1–5 scale, where 1: no symptoms; 2: a few necrotic spots on 1–5% of leaves; 3: symptoms and necrosis on 6–25% of leaves; 4: symptoms and necrosis on 26–50% of leaves; 5: symptoms and necrosis on more than 50% of leaves or leaf death. Disease severity (1) and AUDPC (2) (Madden et al., 2007), based on disease severity values measured at certain time intervals, were calculated using the following formulas. The efficacy of the treatment was calculated as the percentage of reduction in disease severity compared to the pathogen-alone treatment.
3. Results 3.1. Obtaining priming and non-priming progeny In the field, there were no differences between seeds of common bean plants with and without ASM treatment concerning the germination rate of seeds. However, the amount of seeds obtained from the ASM-treated group (47 g/plant) was significantly (F0.383 ¼ 19.018) lower than that of the control group (24 g/plant). 3
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3.2. Disease observations
Table 2 Area Under the Disease Progress Curve (AUDPC) values calculated according to disease severity caused by Xap measured at 7, 14, and 21st days after Xap inoculation. The treatment’s efficacy was calculated according to PCc and its control groups as a percentage.
The primed (p) and non-primed (c) seedlings that were treated with different doses of ASM were tested for disease severity and progress. The primed and non-primed positive control plants (PCc and PCp) displayed greater disease severity than the other plants. However, the primed seedlings (PCp) were observed to have disease severity reduced compared to non-primed (PCc) seedlings, but this reduction was statis tically significant at 14th day observation only (Fig. 1). According to the AUDPC, which is the quantitative summary of disease severity linked to time, this was an 11% reduction (Table 2). When treated with the full dose of 80 μM, the suppression effect on disease development was calculated as 77% and 72% for primed (p80μM) and non-primed (c80μM) plants, respectively (Table 2). This effect was clearly observed during the weekly disease severity observations (Fig. 1). According to the AUDPC results, treatment of the non-primed seed lings with 10 and 20 μM ASM prevented disease development by 37% and 38%, respectively, when compared to PCc. However, application of 10 μM ASM to primed plants (p10μM) did not provide an increased reduction effect when compared to the application to non-primed plants. Contrary to this, treating primed plants with 20 μM ASM (p20μM) reduced disease severity by a significant level. This effect was identified from the first observation seven days after Xap treatment and lasted (Fig. 1). Additionally, according to the AUDPC results for the p20μM treatment, disease development was decreased to 31% compared to its control group (c20μM) and 60% compared to the PCc group (Fig. 1 and Table 2).
Treatments
AUDPC
Efficacy (%) According to its control groups
PCc* PCp c þ ASM10μM p þ ASM10μM c þ ASM20μM p þ ASM20μM c þ ASM80μM p þ ASM80μM
448 397 284 290 279 193 126 103
a
**
ab
abc abc abc bc c c
– 11 – 2 – 31 – 18
According to PCc
11 37 35 38 60 72 77
* c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap. ** Mean values followed by the same letter in a column are not significantly different according to Duncan’s Multiple Range test at P < 0.05 significance level.
effect on Xap population. While there was no significant difference in Xap population for the c10μM and c20μM groups compared to PCc, the p10μM and p20μM groups displayed significantly lower Xap population development than the PCc, c10μM, and c20μM applications. The most prominent observation was for the p20μM treatment; though the final observation point had the same degree of significance, in general, the pathogen population on the p20μM plants was significantly lower than for the c20μM plants.
3.3. Xap population dynamics Two hours after Xap inoculation, the population of the bacteria on the plants was found to be nearly 3 � 106 CFU per gram fresh weight. Generally, the growth of the pathogen population in primed plants was slower than that in non-primed plants (Table 3, line, upper case). The maximum Xap population density was reached on the 7th day for the non-primed control group, while it was reached on the 14th day for primed seedlings. Generally, excluding the last observation, all applications had lower pathogen population density (some statistically significantly lower) on primed plants than on non-primed plants (Table 3, column, lower case). Treating the control group with 80 μM ASM limited the Xap population to an extent. This effect was statistically significant on the 7th day compared to the PCc group, but was lost by the 14th day. Contrary to this, treating primed plants with 80 μM ASM had a more significant
3.4. Plant growth parameters The treatments of ASM with different doses had no effect on leaf numbers (data not shown). In the primed and non-primed (c and p) treated groups and the groups that were only inoculated with the pathogen (PCc and PCp), there were no statistically significant differ ences observed in terms of plant development parameters. Although treating primed plants with 80 μM ASM (p80μM) caused an increase in growth parameters compared to c80μM, under disease pressure, only the shoot dry weight was found to be significantly higher. The growth parameters in primed plants treated with 20 μM ASM (p20μM) generally increased (even under disease pressure) compared to non-primed plants (c20μM). This effect was statistically significant
Fig. 1. Weekly observations of disease severity caused by Xap in primed and non-primed common bean seedlings with ASM treatment (10-20-80 μM). In the first trifoliate period of progeny seedlings were treated with ASM 3 days prior to inoculation with Xap. The disease severity observed at 7, 14, and 21st days after Xap inoculation. The disease severity determined by the scale of 1–5. c: group with no application (negative control), p: plants having parents with ASM treatment, 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c plants inoculated with Xap, PCp: group p plants inoculated with Xap. The mean values followed by the same letter are not significantly different according to Duncan’s Multiple Range test at P � 0.05 significance level (N � 15). 4
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activation of defense can cause unnecessary use of resources in the plant when compared to the form of priming in the absence of the disease. Romero et al. (2001) identified this effect in peppers and found that ASM caused a reduction in the number of flower buds, open flowers, and a delay in reaching fruit maturity. Heil et al. (2000) observed that SAR induced by ASM treatment in wheat, in the absence of pathogens, led to reduced biomass and grain yield. They stated that this effect was observed more dramatically in the situations where nitrogen was limited. In the field experiment of this study, the soil was supported with chemical fertilizer to eliminate the possible phosphorus, potassium and especially nitrogen deficiency. However, significant seed losses were observed. Probably, the 250 μM dose of ASM was too high for the Gina cultivar of common bean. This overdose of ASM caused seed loses up to by 48%. Similarly, Luna et al. (2012) reported that repeated application of high doses of stimulant reduced seed quantities. This situation co incides with the findings of our study. On the other hand, van Hulten et al. (2006) stated that stimulant treatment at low doses was sufficient for priming activation. Lankinen et al. (2016) showed that 10 mM BABA (a lower dose than used by Luna et al., 2012) applied three times with 4–5 days intervals had no negative effects on the plant or the priming, which was transferred to the next generation. Similar results were found in the study conducted by Slaughter et al. (2012). They obtained the results with one stimulus application and determined the durability of the next generation. A lower level of stimulation may be sufficient to establish priming while minimizing the cost of direct induced resistance to the plant (van Hulten et al., 2006; Walters and Fountaine (2009). Direct activation of induced resistance may lead to higher costs for plants growing in conditions where resources are limited, and their ability to develop resistance may decrease (Walters et al., 2013). Therefore, it can be concluded that priming entails a lower fitness cost to the plant and carries less risk when applied in appropriate doses. For this reason, the resistance provided in the form of priming and increasing this resistance capacity are of great agricultural importance. In our study, even at doses as low as 10 μM and 20 μM, ASM appli cation on non-primed plants (c) stimulated the plant and led to the formation of resistance at rates of 37% and 38%, respectively. Furthermore, it appeared that the effect of the treatment lasted for a long duration (at least until the 21st day) when compared to PCc group. Similarly, Siegrist et al. (1997) stated that resistance was active for 4 weeks after treating bean seeds with ASM. However, priming provided by BABA treatment was found to be effective for defense in Arabidopsis for at least 4 weeks (Luna et al., 2014), and in tomato seeds for 8 weeks (Worrall et al., 2012). Pastor et al. (2013) attributed this duration of resistance to epigenetic changes in the chromatin structure via DNA methylation and post-translational modifications. They established a long-term memory and a defence mechanism in the plant in order to protect it against future attacks. Some studies have determined that induced resistance against some pathogens in different plants can be transferred to subsequent genera tions (Kathiria et al., 2010; Lankinen et al., 2016; Ramírez-Carrasco et al., 2017; Slaughter et al., 2012; Walters and Paterson, 2012). In common bean plants, Martínez-Aguilar et al. (2016) showed that the generational and transgenerational regulation of the PvWRKY29 gene, and to a lesser extent the PvPR4 gene, could be partly caused by the histone methylation status at the promoter-exon boundary region of the gene. In our study, it was revealed that priming with ASM treatment on the parents reduced disease progress by 11% in the progeny. Therefore; it could be concluded that this resistance was transferred to the progeny even if it is low (Table 2). To the best of our knowledge, no other studies have demonstrated resistance in ASM-primed progeny as we have in our study. However, Slaughter et al. (2012) found that the resistance was higher in the next generation when similar doses of BABA were applied to the primed progeny. Interestingly, the resistance to the stimulus was not observed in
Table 3 Influence of primed and non-primed common bean seedlings treated with 10, 20 and 80 μM doses of ASM on the Xap population dynamics. In the first trifoliate period of progeny, the seedlings were treated with ASM 3 days prior to inocu lation with Xap. The population of Xap was observed at 24 h, 3, 7, and 14th days after inoculation. Twenty disks were taken randomly from leaves of each seed ling. Mean Xap population sizes were estimated from this foliar tissue samples crushed. Treatment PCc* PCp c þ ASM10μM p þ ASM10μM c þ ASM 20 μM p þ ASM20μM c þ ASM80μM p þ ASM80μM
Sampling time after inoculation 24 h.
3 d.
7 d.
14 d.
7.68 a**�0.01 C 7.47 ab� 0.10 C 7.30 abc�0.20 B 6.96 bcd�0.30C 7.60 a�0.05 B 6.82 cd�0.13B 7.19 abc�0.19BC 6.54 d�0.19 C
7.10 (↓) bc�0.04D 7.18 bc�0.01C 7.68 a�0.03AB 7.28 b�0.17 BC 7.78 a�0.07 AB 6.96 bc�0.27B 6.83 c �0.06 C 6.94 bc�0.15BC
8.65 a�0.11A (→) 8.33 ab�0.22 B 7.94 bcd�0.17A 7.83 cd�0.05B 8.19 ab�0.04A 7.81 cd�0.10 A 7.88 cd�0.07AB 7.71 d�0.21AB
8.26 a� 0.11 B 8.77 a�0.07A 8.08 a�0.20 A 8.64 a�0.06A 7.94 a�0.26AB 8.07 a�0.11A 8.52 a�0.37A 8.10 a�0.46A
*c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap. ** Mean values followed by the same letter in a column (lower case) and line (upper case) are not significantly different according to Duncan’s Multiple Range test at P < 0.05 significance level (N � 3). Values obtained as CFU g fresh tissue 1 given as log transformations.
under disease pressure for root dry weight and root fresh weight (Fig. 2). For the 10 μM ASM treatment, primed plant groups without disease pressure generally displayed increased growth parameters. However, this increase was not observed under disease pressure. 4. Discussion In our study, we investigated the potential of ASM as a plant acti vator to induce resistance that can be transmitted to subsequent gen erations in the common bean. For this purpose, we studied the ability of ASM treatment to suppress common bacterial blight in the progeny of treated plants. Additionally, we examined the primed generation treated with 10, 20, and 80 μM doses of ASM to increase the resistance capacity. Previous studies have shown that ASM triggers plant resistance against pathogens in beans and also suppresses the different diseases to €fte, 2002; Siegrist et al., 1997; different extents (Bigirimana and Ho Soares et al., 2004; Vigo et al., 2012). The induced resistance by ASM treatment was observed to be very low or ineffective in some plants (Walters and Fountaine, 2009). However, it suppressed P. syringae pv. tabaci infection in tobacco plants with rates of the up to 99% (Cole, 1999). Walters and Fountaine (2009) stated that ASM displayed disease suppression at rates ranging from 4 to 99% in different pathosystems. In our study, we determined that a dose of 80 μM ASM (in non-primed plants) reduced disease development by 72% when compared to the positive control (PCc) according to the AUDPC calculation. For effective and continuous protection using induced resistance in plants under field conditions, it is necessary to repeat the ASM treatment (Huang et al., 2012) and to apply it before pathogen attack. On the other hand, direct activation of induced resistance may cause problems when €fte overdose occurs. Azami-Sardooei et al. (2013) and Bigirimana and Ho (2002) showed that high-dose ASM treatments can cause phytotoxicity, severity of which varied according to the plant species. Even in the absence of an overdose, direct activation of induced resistance is known to involve some potential risks. Walters et al. (2013) reported that direct 5
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Fig. 2. Influence of primed and non-primed common bean seedlings treated with 10, 20 and 80 μM doses of ASM on plant growth parameters. The growth pa rameters observed at 21 day after Xap inoculation. c: group with no application (negative control), p: plants having parents with ASM treatment, 10, 20, 80: doses (μM) of ASM applied to the progeny, PCc: group c plants inoculated with Xap, PCp: group p plants inoculated with Xap The mean values followed by the same letter are not significantly different according to Duncan’s Multiple Range test at P � 0.05 significance level (N � 15).
the descendants when the BABA was not applied again, but the sensi tivity to the stimulation continued. Slaughter et al. (2012) defined this situation as “progeny is primed to be primed.” Hence in our study, the increased capacity of resistance by repeated application of ASM to primed progeny may explain the higher resistance than that of non-primed plants treated with ASM. Luna et al. (2014) and Slaughter et al. (2012) stated that the epige netic mechanisms responsible for the transfer of resistance within a generation and between generations may be different. Luna et al. (2012) revealed that the resistance caused by a high level of stimulation, which disrupted the health of plants, continued in the second generation without the need for pre-stimulation. They indicated that the intensity of the stimulant or stress determined the durability of the transgenera tional resistance in the progeny. Thus, plants can adjust the stability of transgenerational defence according to the disease severity (Mauch- Mani et al., 2017). In light of these findings, it can be concluded that increasing the resistance capacity of transgenerational priming with additional stimulation provides an important advantage for plant pro tection. In this study, we observed that this protection capacity increased by up to 60%. It also caused decrease in Xap population. Moreover, in the current study, effect of ASM with different con centrations on plant growth parameters was studied on primed seed lings. According to results, primed seedlings treated with doses of 10, 20 and 80 μM ASM generally suffered no adverse effects on plant growth parameters. In fact, some of the parameters increased positively. Also, no decrease was observed in the germination rate of the primed seeds.
5. Conclusion Our results showed that the treatment of common bean plants with ASM induced a primed state that passed onto the progeny. Successful prevention of the development of the Xap population and the disease progress was observed by applying ASM at low doses. The application of 10 and 80 μM ASM did not result in a significant increase in the resis tance capacity of the priming inherited from the parents, whereas the 20 μM application increased the priming capacity by approximately two-fold. Primed generations can be sustained by strengthening their resis tance capacity with a low-dose stimulator to be used in the event of a potential pathogen attack. This approach can be applied to produce the primed seeds with appropriate stimuli that can protect against target diseases during both seed production and throughout subsequent gen erations in practice. In addition to minimizing the potential costs and risks associated with direct activation of plant resistance, a low chemical input can be achieved in this way. Moreover, this model has high inte gration potential with other plant protection and growth methods and can be an environmentally friendly, economic, and reliable one for plant diseases. Taking all these into consideration, future studies should be conducted to focus on the transgenerational priming while ensuring not losing seed efficiency and yield. Conflicts of interest The author declares no conflict of interest.
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Acknowledgements
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This work was supported by the Scientific Research Project Units of Van Yüzüncü Yıl University (grant numbers: FBA-2016-5170). References Ahmad, S., Gordon-Weeks, R., Pickett, J., Ton, J., 2010. Natural variation in priming of basal resistance: from evolutionary origin to agricultural exploitation. Mol. Plant Pathol. 11, 817–827. Akhavan, A., Bahar, M., Askarian, H., Lak, M.R., Nazemi, A., Zamani, Z., 2013. Bean common bacterial blight: pathogen epiphytic life and effect of irrigation practices. Springer Plus 2 (1), 41. https://doi.org/10.1186/2193-1801-2-41. Azami-Sardooei, Z., Seifi, H.S., De Vleesschauwer, D., H€ oft, M., 2013. Benzothiadiazole (BTH)-induced resistance against Botrytis cinerea is inversely correlated with vegetative and generative growth in bean and cucumber, but not in tomato. Australas. Plant Pathol. 42, 485. https://doi.org/10.1007/s13313-013-0207-1. Bigirimana, J., H€ ofte, M., 2002. Induction of systemic resistance to colletotrichum lindemuthianum in bean by a benzothiadiazole derivative and rhizobacteria. Phytoparasitica 30 (2), 159–168. Boersma, J.G., Hou, A., Gillard, C.L., McRae, K.B., Conner, R.L., 2015. Impact of common bacterial blight on the yield, seed weight and seed discoloration of different market classes of dry beans (Phaseolus vulgaris L.). Can. J. Plant Sci. 95, 703–710. Bruce, T.J.A., 2010. Tackling the threat to food security caused by crop pests in the new millennium. Food Secur 2, 133–141. https://doi.org/10.1007/s12571-010-0061-8. CABI., 2019. https://www.cabi.org/isc/datasheet/56962. Claflin, L.E., Vidaver, A.K., Sasser, M., 1987. MXP, a semi-selective medium Xanthomonas campestris pv. phaseoli. Phytopathology 77 (5), 730–734. Cole, D.L., 1999. The efficacy of acibenzolar-S-methyl, an inducer of systemic acquired resistance, against bacterial and fungal diseases of tobacco. Crop Protect. 18, 267–273. Conrath, U., Beckers, G.J.M., Langenbach, C.J.G., Jaskiewicz, M.R., 2015. Priming for enhanced defense. Annu. Rev. Phytopathol. 53, 97–119. Conrath, U., 2011. Molecular aspects of defence priming. Trends Plant Sci. 16 (10), 524–531. https://doi.org/10.1016/j.tplants.2011.06.004. EPPO, 2006. Data sheets on quarantine pests Xanthomonas axonopodis pv. phaseoli. CABI and EPPO for the EU under Contract 90/399003. http://www.eppo.int/QUARANT INE/bacteria/Xanthomonas_phaseoli/-XANTPH_ds.pdf. FAO, 2019. Food and agriculture organization of the united nations. http://www.fao. org/faostat/en/#data-/QC. Griffin, K., Gambley, C., Brown, P., Li, Y., 2017. Copper-tolerance in Pseudomonas syringaepv. tomato and Xanthomonas spp. and the control of diseases associated with these pathogens in tomato and pepper. A systematic literature review. Crop Protect. 96, 144–150. https://doi.org/10.1016/j.cropro.2017.02.008. Heil, M., Hilpert, A., Kaiser, W., Linsenmair, E., 2000. Reduced growth and seed set following chemical induction of pathogen defence: does systemic acquired resistance (SAR) incur allocation costs? J. Ecol. 88, 645–654. Heil, M., Ton, J., 2008. Long-distance signalling in plant defence. Trends Plant Sci. 13, 264–272. Huang, C.H., Vallad, G.E., Zhang, S., Wen, A., Balogh, B., Figueiredo, J.F.L., Behlau, F., Jones, J.B., Momol, M.T., Olson, S.M., 2012. Effect of application frequency and reduced rates of acibenzolar-S-methyl on the field efficacy of induced resistance against bacterial spot on tomato. Plant Dis. 96, 221–227. Jacques, M.A., Josi, K., Darrasse, A., Samson, R., 2005. Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans. Appl. Environ. Microbiol. 71 (4), 2008–2015. https://doi.org/10.1128/AEM.71.4.2008-2015.2005. Kathiria, P., Sidler, C., Golubov, A., Kalischuk, M., Kawchuk, L.M., Kovalchuk, I., 2010. Tobacco mosaic virus infection results in an increase in recombination frequency and resistance to viral, bacterial, and fungal pathogens in the progeny of infected tobacco plants. Plant Physiol. 153, 1859–1870. Lankinen, A., Abreha, K.B., Alexandersson, E., Andersson, S., Andreasson, E., 2016. Nongenetic inheritance of induced resistance in a wild annual plant. Phytopathology 106, 877–883. Luna, E., L� opez, A., Kooiman, J., Ton, J., 2014. Role of NPR1 and KYP in long-lasting induced resistance by β-aminobutyric acid. Front. Plant Sci. 5 (184), 1–8. Luna, E., Ton, J., 2012. The epigenetic machinery controlling transgen- gerational systemic acquired resistance. Plant Signal. Behav. 7, 615–618. https://doi.org/ 10.4161/psb.20155.
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Potential using of transgenerational resistance against common bacterial blight in Phaseolus vulgaris €prü Ahmet Akko Plant Protection Department, Faculty of Agriculture, Van Yüzüncü Yıl University. Zeve Campus, 65080, Van, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Next generation priming Epigenetic Xanthomonas axonopodis pv. phaseoli Acibenzolar-S-Methyl Common bean
Direct activation of induced resistance is recommended for the prolonged and effective control of plant diseases. However, there are some potential risks to this method in field application. The priming form of resistance has less risk; nevertheless, it is necessary to increase adaptation and efficacy. Recently, it was shown that the priming as a form of resistance mechanism in plants was transferred epigenetically. In our study, the use of this parentally-inherited resistance for disease control and the possibility of increasing resistance capacity were investigated. For this aim, plant growth parameters, population dynamics of Xanthomonas axonopodis pv. phaseoli (Xap), and severity and development of the common bean bacterial blight disease were investigated in the primed generation obtained from acibenzolar-S-methyl (ASM) treated parent bean. It was revealed that the primed progeny suppressed the disease by 11%, according to the area under the disease progress curve (AUDPC). Moreover, when the primed progeny were treated with a low dose of ASM (20 μM), they showed nearly twice the resistance capacity and suppressed the disease by 60%. In primed progeny, ASM treatment significantly slowed the development of Xap populations until the 14th day, also did not adverse effects on plant development. All these results indicated that seed production with this method offers the possibility of increasing the resistance capacity of later generations, just as it ensured the protection of parents. Thus, using the appropriate dose of activator at primed progeny will ensure that the efficacy of control methods is increased, while pesticide inputs can be reduced.
1. Introduction Plants have adaptive strategies to cope with variable and unfavor able growing conditions in nature (Pikaard and Mittelsten Scheid, 2014). One of the most important of these mechanisms is induced plant resistance. This form of resistance offers significant possibilities for controlling diseases and pests by adapting plant cultivation and pro tection strategies in agriculture. Induced resistance is defined as rapid and robust activation of a basal resistance level against subsequent challenges by stimulating the plant with an appropriate stimulant (Ahmad et al., 2010; Mauch-Mani et al., 2017). Stimulants may be biotic or abiotic (environmental or chemical) and determine the type of resistance (Ton et al., 2002; Van der Ent et al., 2009). The first plant resistance system identified was systemic acquired resistance (SAR), which can be triggered by pathogens and many chemical stimulants such as salicylic acid (SA), acibenzolar-S-methyl (ASM), 2,6-dichloroiso nicotinic acid, probenazole, and cyclopropanecarboxylic acid (Walters et al., 2013). Some of these stimulants are sold as plant activators. The plant induced resistance is a complex phenomenon that is
formed by the activation of several layers and can be exhibited different forms of plant defence during different periods of the attack (Ahmad et al., 2010; Pastor et al., 2013). One of this form is priming which can be described as a physiological process by which a plant prepares itself to respond to future biotic or abiotic stress more quickly (Conrath, 2011; Mauch-Mani et al., 2017; Pastor et al., 2013). In primed plants, no sig nificant increase is observed in the activation of defence genes until being exposed to stress. Thus, the priming reduces the allocation cost of defence to the plant (van Hulten et al., 2006). In general, resistance-inducing agents establish priming. The stimu lation dose determines whether direct resistance or priming is induced. In Arabidopsis thaliana, van Hulten et al. (2006) observed that treatment with high doses of β-aminobutyric acid (BABA) triggered both direct resistance and priming simultaneously. In contrast, when the plants were treated with low doses of BABA, only priming was observed. In other words, temporary stimulation of direct resistance may ensure the long-term priming (Ahmad et al., 2010; Heil and Ton, 2008). Hence, the induced resistance phenomenon can be defined as a combination of direct defense and priming. Ahmad et al. (2010) stated that induced
E-mail address: [email protected]. https://doi.org/10.1016/j.cropro.2019.104967 Received 9 April 2019; Received in revised form 18 September 2019; Accepted 19 September 2019 Available online 20 September 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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resistance is linked to the combination of stimulant dose and time period which is between stimulation and interaction of plant with the pathogen. Although the activation of direct resistance provides rapid and robust response to pathogen attack, it has a cost to the plant. Some studies revealed that direct activation of induced resistance resulted higher adverse effects on yield and development under limited growth conditions (Heil et al., 2000; van Hulten et al., 2006; Walters and Fountaine, 2009; Walters et al., 2013). On the other hand, van Hulten et al. (2006) stated that resistance activated by priming may cause minor reductions in relative growth rate and had no effect on seed production in the absence of the pathogen. They also reported that this cost could be ignored under pathogen pressure. Other researchers showed that this cost is very low; therefore, it has no significant in field conditions (Walters and Heil, 2007). The variety of epigenetic pathways in plants is notably great and they contribute significantly to plant growth, phenotypic plasticity, survival in unpredictable environments, and proliferation ability (Pikaard and Mittelsten Scheid, 2014; Springer, 2013). In recent years, some studies have reported that resistance in priming form against biotic stressors in different plants, obtained via different stimuli, can be epigenetically transferred to the offspring. Epigenetic transfer of the resistance was observed in tobacco against tobacco mosaic virus (Kathiria et al., 2010), and in Arabidopsis thaliana against Pseudomonas syringae pv. tomato (Luna et al., 2012) and Hyaloperonospora arabidopsidis (Slaughter et al., 2012). Additional observations were recorded in barley against the fungus Rhynchosporium commune (Walters and Paterson, 2012) and the common bean against P. syringae pv. phaseolicola (Martínez-Aguilar et al., 2016; Ramírez-Carrasco et al., 2017). Pieterse (2012) stated that transgenerational resistance inherited epigenetically is a form of communication between parent plants and their progeny. Epigenetic changes are reversible and do not cause any changes in nucleotide sequences. They occur through some modifications such as DNA methylation, histone modification, and through small interfering RNA activities (Conrath et al., 2015; Pikaard and Mittelsten Scheid, 2014). Luna et al. (2012) stated that transmission of priming between generations was linked to the SA and NPR1 signalling pathways. They showed that transmission occurred via hypomethylation of genes linked to SA in subsequent generations. Other detailed studies of the Arabi dopsis thaliana/P.s. pv. tomato and H. arabidopsidis pathosystem indi cated that resistance may be transmitted via RNA-directed DNA methylation and hypomethylation of the CpNpG region (Luna and Ton, 2012). It was revealed that the priming status in beans could be trans mitted with the aid of changes in the H3K4me3 and H3K36me3 marks at the promoter-exon regions of defence-associated genes. (Martínez-A guilar et al., 2016; Ramírez-Carrasco et al., 2017). The common bean (Phaseolus vulgaris L. (Fabaceae)), the model plant in the current study, is an economically important crop that is grown worldwide, because of its high protein, fiber, complex carbohydrate content, and consumption in different forms (Pathania et al., 2014). Total green and dry bean production in the world in 2017 is more than 55 million tons (FAO, 2019). However, many diseases cause severe losses (20–100%) in the yield and the quality of common bean world wide (Singh and Schwartz, 2010). One of the most important of these diseases is common bacterial blight caused by Xanthomonas axonopodis pv. phaseoli ((Smith, 1897) Vauterin et al., 1995) (Xap). The pathogen has been observed on all continents (CABI, 2019; EPPO, 2006). Xa pv. phaseoli induces symptoms on leaves, stems, pods, and seeds. The pathogen can survive and multiply on bean and weed hosts without showing any symptom as epiphytically and endophytically (Akhavan et al., 2013; Jacques et al., 2005). One diseased plant, infected with this aggressive pathogen, in 10,000 is sufficient to cause a severe epidemic (Akhavan et al., 2013), and the disease can cause up to 40% yield loss (Boersma et al., 2015; Singh and Miklas. 2015). Such features of the pathogen make it difficult to control the disease. Some recommended methods for controlling of the disease such as
cultural methods, using resistant varieties and pesticide application may not always give the desired results (CABI, 2019). The control of the disease by chemicals is the most common method. However, Maringoni (1990) showed that spraying chemicals on the plants in the field did not give a satisfying effect. On the other hand, the pathogen has potential of the development of resistance against some chemicals which are used for controlling the disease (Griffin et al., 2017). Also, adverse effects of pesticide on the environment and human health are well understood (Bruce, 2010). Within this framework, alternative disease control methods and induced resistance in particular, have been gaining sig nificant attention for their potential. The transmission of induced resistance between generations may establish a new approach to control the diseases. This may increase the efficacy of disease control with induce systemic resistance, offering the opportunity for low-cost, reliable, and economic way. The aim of this study was to determine to potential of transgenerational priming pro vided by chemical stimulant acibenzolar-S-methyl (ASM) to control the common bacterial blight disease of bean caused by Xap. With this aim, a new generation that was produced using seeds obtained from parents with activated induced resistance, was used. In current study, we investigated: i) the effects of transmitted resistance on plant growth, Xap population density, and disease progress, and ii) the efficacy of different doses of ASM in increasing the resistance capacity of primed progeny. 2. Materials & methods 2.1. Plant material and growth conditions Common bean seeds (Phaseolus vulgaris L. cv. Gina) were planted in 300 mL plastic pots filled with sterile peat and left to develop in a climate chamber at 24 � C, with 16 h light and approximately 50% hu midity. The nutritional requirements of the seedlings were provided by regularly applying Hoagland nutritional solution. The ASM suspension (Sigma-Aldrich Chemie B.V.) was prepared at a concentration of 250 μM (Azami-Sardooei et al., 2013) and was added to 0.01% Tween solution. When the first true leaves opened, the solution was sprayed on the leaves to thoroughly wet the plant. The ASM treatment was repeated four times, once every 5 days. The same water regime was applied to control plants. Three days after the final ASM application, 40 common bean seedlings for each treatment were transplanted into a field prepared by removing weeds and adding chemical fertilizer (Nitrogen, Phosphate, Potassium; 15:15:15). The field was located within the boundaries of Gevas¸ county in Van province, Turkey (38.18� N, 43.90� E; 1684 m elevation). During the growing period, seedlings were watered regularly with a drip irrigation system, and hoed once. No pesticides were applied. The common bean pods were harvested and then completely dried. Seeds from 15 randomly selected plants were harvested from the pods and weighed. Seeds were stored in low-humidity conditions awaiting further study. 2.2. Primed generation The seedlings whose parents were treated with and without ASM were grown as primed (p) and non-primed (c) plants, respectively. These seedlings were treated at doses of 10, 20, and 80 μM ASM. The effects on disease severity and development were determined by investigating the area under the disease-progress curve (AUDPC), the Xap population dynamics, and the plant growth parameters. The constituted variants for the treatment groups and the schema of the study were given in Table 1. The primed (p) and non-primed (c) seeds were planted in pots filled with sterile peat as mentioned above and left to grow. When the first trifoliate leaves started to form, 10, 20, and 80 μM ASM was sprayed on the leaves. Three days after the ASM treatment, Xanthomonas axonopodis pv. phaseoli ((Smith, 1897) Vauterin et al., 1995) (Xap) was inoculated to plants. The pathogen isolated from common bean in Antalya, Turkey, was provided by Prof Dr Hüseyin Basım (Faculty of Agriculture, Akdeniz 2
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Table 1 Experimental design. A, Plants were treated four times at intervals of 5 d by 250 μM ASM or water to the parent’s plant leaves. Then plants were transplanted in the field and allowed to set seed to provide. The primed and non-primed seeds (Progeny) from the parent plants constituted the main seeds material for the later experiments. In the second part of the study, in climate chamber, the progeny seedlings treated 10, 20, 80 μM doses of ASM, and then Xap inoculated. B, Experimental groups, in climate chamber studies, constituted with primed and non-primed seedlings with 10, 20, 80 μM doses of ASM. Xap population dynamics, disease progress and plant growth parameters were evaluated in the study.
Disease index ¼
AUDPC ¼
Primed plants treatments
*c (- Xap) PCc c þ ASM 10 μM c þ ASM 20 μM c þ ASM 80 μM c þ ASM 10 μM þ Xap c þ ASM 20 μM þ Xap c þ ASM 80 μM þ Xap
p (- Xap) PCp p þ ASM p þ ASM p þ ASM p þ ASM p þ ASM p þ ASM
n 1 X yi þ yiþ1 x ðtiþ1 2 i¼1
ti Þ
(2)
2.4. Determination of bacterial density in leaves With the aim of determining the population of Xap on the plants, three plants were randomly selected from each application group at 2 h, 24 h, and, 3, 7, and 14 days after pathogen inoculation. Twenty disks with a diameter of 0.5 cm were taken randomly from leaves of each seedling with a cork borer. The disks were weighed, and then crushed in polyethylene bags using a hand stomacher. Dilution steps were created from the obtained suspension and plated on modified MXP medium (Claflin et al., 1987). MXP agar is a semi-selective medium for Xap; the suspension consisted of soluble potato starch (8.0 g/L), K2HPO4 (0.8 g/L), KH2PO4 (0.6 g/L), yeast extract (0.7 g/L), potassium bromide (10.0 g/L), glucose (1.0 g/L), methyl violet 2B (30 μL/L; 1% solution in 20% ethanol), and methyl green (60 μL/L; 1% aqueous solution) and agar (15.0 g/L). This suspension was autoclaved. Antibiotic solutions containing chlorothalonil (15 mg/L), cephalexin (20 mg/L), kasugamy cin (20 mg/L), gentamycin (2 mg/L), and cycloheximide (100 mg/L) were passed through a filter and added to the autoclaved suspension after cooling to 50 � C. The petri dishes that were plated with diluted suspensions of plant extract, were incubated for 2–3 days at 27 � C. The colonies that developed were counted, and the Xap population on the plant was determined as the CFU per gram fresh plant leaf.
B Control plants treatments
Σ ðRating number x Number of leaves in the ratingÞ x100 Total number of leaves x Highest rating (1)
2.5. Plant growth parameters
10 μM 20 μM 80 μM 10 μM þ Xap 20 μM þ Xap 80 μM þ Xap
At the end of the study, 21 day after Xap inoculation, leaves number, and shoots and roots weight were determined as fresh and dry. All the leaves on the plants were counted, excluding cotyledons and bifoliate leaves to determine leaves number. The bean plants were then uprooted and cut at the root crown. Roots were cleaned of growth medium residue by washing with tap water. The water was then removed from the sur face with blotting paper before determining the fresh weights of the roots and stem. The roots and shoots were then dried in an oven at 65 � C until a stable weight was reached. The dry weights were then determined.
*c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap.
University, Antalya, Turkey). The Xap was developed for 48 h in tryptic soy agar (TSA) medium (1.7 g/L tryptone, 0.3 g/L soybean peptone, 0.25 g/L glucose, 0.5 g/L NaCl, 0.5 g/L K2HPO4, and 15 g/L agar). The concentration of Xap was set to 108 CFU/mL (A600 ¼ 0.13) and 0.01% Tween was added to the suspension. All the leaves of the common bean seedlings were then dipped in the beaker containing the pathogen sus pension for 10 s each. Immediately after pathogen application, seedlings were kept in polyethylene cabinets in the climate chamber to create high humidity for 48 h. The seedlings were kept in darkness for the first 24 h.
2.6. Data analysis Data obtained at the end of the field studies were analyzed using an unpaired two-tailed Student’s t-test (p < 0.01). The experimental vari ants for the treatments in the climate chamber were as shown in Table 1, and the studies were repeated twice. Data obtained from the climate chamber studies were analyzed using SPSS v17.0 statistical software. Significant differences between treatments were determined using Duncan’s multiple range test with a significance level of p � 0.05. The Xap population data were log-transformed prior to analysis.
2.3. Disease assessment The disease severity was determined 7, 14, and 21 days after path ogen inoculation using a 1–5 scale, where 1: no symptoms; 2: a few necrotic spots on 1–5% of leaves; 3: symptoms and necrosis on 6–25% of leaves; 4: symptoms and necrosis on 26–50% of leaves; 5: symptoms and necrosis on more than 50% of leaves or leaf death. Disease severity (1) and AUDPC (2) (Madden et al., 2007), based on disease severity values measured at certain time intervals, were calculated using the following formulas. The efficacy of the treatment was calculated as the percentage of reduction in disease severity compared to the pathogen-alone treatment.
3. Results 3.1. Obtaining priming and non-priming progeny In the field, there were no differences between seeds of common bean plants with and without ASM treatment concerning the germination rate of seeds. However, the amount of seeds obtained from the ASM-treated group (47 g/plant) was significantly (F0.383 ¼ 19.018) lower than that of the control group (24 g/plant). 3
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3.2. Disease observations
Table 2 Area Under the Disease Progress Curve (AUDPC) values calculated according to disease severity caused by Xap measured at 7, 14, and 21st days after Xap inoculation. The treatment’s efficacy was calculated according to PCc and its control groups as a percentage.
The primed (p) and non-primed (c) seedlings that were treated with different doses of ASM were tested for disease severity and progress. The primed and non-primed positive control plants (PCc and PCp) displayed greater disease severity than the other plants. However, the primed seedlings (PCp) were observed to have disease severity reduced compared to non-primed (PCc) seedlings, but this reduction was statis tically significant at 14th day observation only (Fig. 1). According to the AUDPC, which is the quantitative summary of disease severity linked to time, this was an 11% reduction (Table 2). When treated with the full dose of 80 μM, the suppression effect on disease development was calculated as 77% and 72% for primed (p80μM) and non-primed (c80μM) plants, respectively (Table 2). This effect was clearly observed during the weekly disease severity observations (Fig. 1). According to the AUDPC results, treatment of the non-primed seed lings with 10 and 20 μM ASM prevented disease development by 37% and 38%, respectively, when compared to PCc. However, application of 10 μM ASM to primed plants (p10μM) did not provide an increased reduction effect when compared to the application to non-primed plants. Contrary to this, treating primed plants with 20 μM ASM (p20μM) reduced disease severity by a significant level. This effect was identified from the first observation seven days after Xap treatment and lasted (Fig. 1). Additionally, according to the AUDPC results for the p20μM treatment, disease development was decreased to 31% compared to its control group (c20μM) and 60% compared to the PCc group (Fig. 1 and Table 2).
Treatments
AUDPC
Efficacy (%) According to its control groups
PCc* PCp c þ ASM10μM p þ ASM10μM c þ ASM20μM p þ ASM20μM c þ ASM80μM p þ ASM80μM
448 397 284 290 279 193 126 103
a
**
ab
abc abc abc bc c c
– 11 – 2 – 31 – 18
According to PCc
11 37 35 38 60 72 77
* c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap. ** Mean values followed by the same letter in a column are not significantly different according to Duncan’s Multiple Range test at P < 0.05 significance level.
effect on Xap population. While there was no significant difference in Xap population for the c10μM and c20μM groups compared to PCc, the p10μM and p20μM groups displayed significantly lower Xap population development than the PCc, c10μM, and c20μM applications. The most prominent observation was for the p20μM treatment; though the final observation point had the same degree of significance, in general, the pathogen population on the p20μM plants was significantly lower than for the c20μM plants.
3.3. Xap population dynamics Two hours after Xap inoculation, the population of the bacteria on the plants was found to be nearly 3 � 106 CFU per gram fresh weight. Generally, the growth of the pathogen population in primed plants was slower than that in non-primed plants (Table 3, line, upper case). The maximum Xap population density was reached on the 7th day for the non-primed control group, while it was reached on the 14th day for primed seedlings. Generally, excluding the last observation, all applications had lower pathogen population density (some statistically significantly lower) on primed plants than on non-primed plants (Table 3, column, lower case). Treating the control group with 80 μM ASM limited the Xap population to an extent. This effect was statistically significant on the 7th day compared to the PCc group, but was lost by the 14th day. Contrary to this, treating primed plants with 80 μM ASM had a more significant
3.4. Plant growth parameters The treatments of ASM with different doses had no effect on leaf numbers (data not shown). In the primed and non-primed (c and p) treated groups and the groups that were only inoculated with the pathogen (PCc and PCp), there were no statistically significant differ ences observed in terms of plant development parameters. Although treating primed plants with 80 μM ASM (p80μM) caused an increase in growth parameters compared to c80μM, under disease pressure, only the shoot dry weight was found to be significantly higher. The growth parameters in primed plants treated with 20 μM ASM (p20μM) generally increased (even under disease pressure) compared to non-primed plants (c20μM). This effect was statistically significant
Fig. 1. Weekly observations of disease severity caused by Xap in primed and non-primed common bean seedlings with ASM treatment (10-20-80 μM). In the first trifoliate period of progeny seedlings were treated with ASM 3 days prior to inoculation with Xap. The disease severity observed at 7, 14, and 21st days after Xap inoculation. The disease severity determined by the scale of 1–5. c: group with no application (negative control), p: plants having parents with ASM treatment, 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c plants inoculated with Xap, PCp: group p plants inoculated with Xap. The mean values followed by the same letter are not significantly different according to Duncan’s Multiple Range test at P � 0.05 significance level (N � 15). 4
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activation of defense can cause unnecessary use of resources in the plant when compared to the form of priming in the absence of the disease. Romero et al. (2001) identified this effect in peppers and found that ASM caused a reduction in the number of flower buds, open flowers, and a delay in reaching fruit maturity. Heil et al. (2000) observed that SAR induced by ASM treatment in wheat, in the absence of pathogens, led to reduced biomass and grain yield. They stated that this effect was observed more dramatically in the situations where nitrogen was limited. In the field experiment of this study, the soil was supported with chemical fertilizer to eliminate the possible phosphorus, potassium and especially nitrogen deficiency. However, significant seed losses were observed. Probably, the 250 μM dose of ASM was too high for the Gina cultivar of common bean. This overdose of ASM caused seed loses up to by 48%. Similarly, Luna et al. (2012) reported that repeated application of high doses of stimulant reduced seed quantities. This situation co incides with the findings of our study. On the other hand, van Hulten et al. (2006) stated that stimulant treatment at low doses was sufficient for priming activation. Lankinen et al. (2016) showed that 10 mM BABA (a lower dose than used by Luna et al., 2012) applied three times with 4–5 days intervals had no negative effects on the plant or the priming, which was transferred to the next generation. Similar results were found in the study conducted by Slaughter et al. (2012). They obtained the results with one stimulus application and determined the durability of the next generation. A lower level of stimulation may be sufficient to establish priming while minimizing the cost of direct induced resistance to the plant (van Hulten et al., 2006; Walters and Fountaine (2009). Direct activation of induced resistance may lead to higher costs for plants growing in conditions where resources are limited, and their ability to develop resistance may decrease (Walters et al., 2013). Therefore, it can be concluded that priming entails a lower fitness cost to the plant and carries less risk when applied in appropriate doses. For this reason, the resistance provided in the form of priming and increasing this resistance capacity are of great agricultural importance. In our study, even at doses as low as 10 μM and 20 μM, ASM appli cation on non-primed plants (c) stimulated the plant and led to the formation of resistance at rates of 37% and 38%, respectively. Furthermore, it appeared that the effect of the treatment lasted for a long duration (at least until the 21st day) when compared to PCc group. Similarly, Siegrist et al. (1997) stated that resistance was active for 4 weeks after treating bean seeds with ASM. However, priming provided by BABA treatment was found to be effective for defense in Arabidopsis for at least 4 weeks (Luna et al., 2014), and in tomato seeds for 8 weeks (Worrall et al., 2012). Pastor et al. (2013) attributed this duration of resistance to epigenetic changes in the chromatin structure via DNA methylation and post-translational modifications. They established a long-term memory and a defence mechanism in the plant in order to protect it against future attacks. Some studies have determined that induced resistance against some pathogens in different plants can be transferred to subsequent genera tions (Kathiria et al., 2010; Lankinen et al., 2016; Ramírez-Carrasco et al., 2017; Slaughter et al., 2012; Walters and Paterson, 2012). In common bean plants, Martínez-Aguilar et al. (2016) showed that the generational and transgenerational regulation of the PvWRKY29 gene, and to a lesser extent the PvPR4 gene, could be partly caused by the histone methylation status at the promoter-exon boundary region of the gene. In our study, it was revealed that priming with ASM treatment on the parents reduced disease progress by 11% in the progeny. Therefore; it could be concluded that this resistance was transferred to the progeny even if it is low (Table 2). To the best of our knowledge, no other studies have demonstrated resistance in ASM-primed progeny as we have in our study. However, Slaughter et al. (2012) found that the resistance was higher in the next generation when similar doses of BABA were applied to the primed progeny. Interestingly, the resistance to the stimulus was not observed in
Table 3 Influence of primed and non-primed common bean seedlings treated with 10, 20 and 80 μM doses of ASM on the Xap population dynamics. In the first trifoliate period of progeny, the seedlings were treated with ASM 3 days prior to inocu lation with Xap. The population of Xap was observed at 24 h, 3, 7, and 14th days after inoculation. Twenty disks were taken randomly from leaves of each seed ling. Mean Xap population sizes were estimated from this foliar tissue samples crushed. Treatment PCc* PCp c þ ASM10μM p þ ASM10μM c þ ASM 20 μM p þ ASM20μM c þ ASM80μM p þ ASM80μM
Sampling time after inoculation 24 h.
3 d.
7 d.
14 d.
7.68 a**�0.01 C 7.47 ab� 0.10 C 7.30 abc�0.20 B 6.96 bcd�0.30C 7.60 a�0.05 B 6.82 cd�0.13B 7.19 abc�0.19BC 6.54 d�0.19 C
7.10 (↓) bc�0.04D 7.18 bc�0.01C 7.68 a�0.03AB 7.28 b�0.17 BC 7.78 a�0.07 AB 6.96 bc�0.27B 6.83 c �0.06 C 6.94 bc�0.15BC
8.65 a�0.11A (→) 8.33 ab�0.22 B 7.94 bcd�0.17A 7.83 cd�0.05B 8.19 ab�0.04A 7.81 cd�0.10 A 7.88 cd�0.07AB 7.71 d�0.21AB
8.26 a� 0.11 B 8.77 a�0.07A 8.08 a�0.20 A 8.64 a�0.06A 7.94 a�0.26AB 8.07 a�0.11A 8.52 a�0.37A 8.10 a�0.46A
*c: group with no application (negative control), p: plants having parents with ASM treatment, ASM 10, 20, 80 μM: doses of ASM applied to the progeny, PCc: group c inoculated with Xap, PCp: group p inoculated with Xap. ** Mean values followed by the same letter in a column (lower case) and line (upper case) are not significantly different according to Duncan’s Multiple Range test at P < 0.05 significance level (N � 3). Values obtained as CFU g fresh tissue 1 given as log transformations.
under disease pressure for root dry weight and root fresh weight (Fig. 2). For the 10 μM ASM treatment, primed plant groups without disease pressure generally displayed increased growth parameters. However, this increase was not observed under disease pressure. 4. Discussion In our study, we investigated the potential of ASM as a plant acti vator to induce resistance that can be transmitted to subsequent gen erations in the common bean. For this purpose, we studied the ability of ASM treatment to suppress common bacterial blight in the progeny of treated plants. Additionally, we examined the primed generation treated with 10, 20, and 80 μM doses of ASM to increase the resistance capacity. Previous studies have shown that ASM triggers plant resistance against pathogens in beans and also suppresses the different diseases to €fte, 2002; Siegrist et al., 1997; different extents (Bigirimana and Ho Soares et al., 2004; Vigo et al., 2012). The induced resistance by ASM treatment was observed to be very low or ineffective in some plants (Walters and Fountaine, 2009). However, it suppressed P. syringae pv. tabaci infection in tobacco plants with rates of the up to 99% (Cole, 1999). Walters and Fountaine (2009) stated that ASM displayed disease suppression at rates ranging from 4 to 99% in different pathosystems. In our study, we determined that a dose of 80 μM ASM (in non-primed plants) reduced disease development by 72% when compared to the positive control (PCc) according to the AUDPC calculation. For effective and continuous protection using induced resistance in plants under field conditions, it is necessary to repeat the ASM treatment (Huang et al., 2012) and to apply it before pathogen attack. On the other hand, direct activation of induced resistance may cause problems when €fte overdose occurs. Azami-Sardooei et al. (2013) and Bigirimana and Ho (2002) showed that high-dose ASM treatments can cause phytotoxicity, severity of which varied according to the plant species. Even in the absence of an overdose, direct activation of induced resistance is known to involve some potential risks. Walters et al. (2013) reported that direct 5
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Fig. 2. Influence of primed and non-primed common bean seedlings treated with 10, 20 and 80 μM doses of ASM on plant growth parameters. The growth pa rameters observed at 21 day after Xap inoculation. c: group with no application (negative control), p: plants having parents with ASM treatment, 10, 20, 80: doses (μM) of ASM applied to the progeny, PCc: group c plants inoculated with Xap, PCp: group p plants inoculated with Xap The mean values followed by the same letter are not significantly different according to Duncan’s Multiple Range test at P � 0.05 significance level (N � 15).
the descendants when the BABA was not applied again, but the sensi tivity to the stimulation continued. Slaughter et al. (2012) defined this situation as “progeny is primed to be primed.” Hence in our study, the increased capacity of resistance by repeated application of ASM to primed progeny may explain the higher resistance than that of non-primed plants treated with ASM. Luna et al. (2014) and Slaughter et al. (2012) stated that the epige netic mechanisms responsible for the transfer of resistance within a generation and between generations may be different. Luna et al. (2012) revealed that the resistance caused by a high level of stimulation, which disrupted the health of plants, continued in the second generation without the need for pre-stimulation. They indicated that the intensity of the stimulant or stress determined the durability of the transgenera tional resistance in the progeny. Thus, plants can adjust the stability of transgenerational defence according to the disease severity (Mauch- Mani et al., 2017). In light of these findings, it can be concluded that increasing the resistance capacity of transgenerational priming with additional stimulation provides an important advantage for plant pro tection. In this study, we observed that this protection capacity increased by up to 60%. It also caused decrease in Xap population. Moreover, in the current study, effect of ASM with different con centrations on plant growth parameters was studied on primed seed lings. According to results, primed seedlings treated with doses of 10, 20 and 80 μM ASM generally suffered no adverse effects on plant growth parameters. In fact, some of the parameters increased positively. Also, no decrease was observed in the germination rate of the primed seeds.
5. Conclusion Our results showed that the treatment of common bean plants with ASM induced a primed state that passed onto the progeny. Successful prevention of the development of the Xap population and the disease progress was observed by applying ASM at low doses. The application of 10 and 80 μM ASM did not result in a significant increase in the resis tance capacity of the priming inherited from the parents, whereas the 20 μM application increased the priming capacity by approximately two-fold. Primed generations can be sustained by strengthening their resis tance capacity with a low-dose stimulator to be used in the event of a potential pathogen attack. This approach can be applied to produce the primed seeds with appropriate stimuli that can protect against target diseases during both seed production and throughout subsequent gen erations in practice. In addition to minimizing the potential costs and risks associated with direct activation of plant resistance, a low chemical input can be achieved in this way. Moreover, this model has high inte gration potential with other plant protection and growth methods and can be an environmentally friendly, economic, and reliable one for plant diseases. Taking all these into consideration, future studies should be conducted to focus on the transgenerational priming while ensuring not losing seed efficiency and yield. Conflicts of interest The author declares no conflict of interest.
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Acknowledgements
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