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Morphological and physiological characteristics of endornavirus-infected and endornavirus-free near-isogenic lines of bell pepper (Capsicum annuum)
Scientia Horticulturae 250 (2019) 104–112
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Morphological and physiological characteristics of endornavirus-infected and endornavirus-free near-isogenic lines of bell pepper (Capsicum annuum)
T
C. Escalante, R.A. Valverde
⁎
Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA
ARTICLE INFO
ABSTRACT
Keywords: Bell pepper Bell pepper endornavirus Endornaviridae Plant virus Seed germination
Plant endornaviruses are persistent viruses that infect plants without causing symptoms. Although endornaviruses have been reported in many economically important plant species, little is known about the effect they have on their hosts. Bell pepper endornavirus (BPEV) has been reported in cultivated peppers (Capsicum spp.). In commercial cultivars of bell pepper (C. annuum) grown in the United States, this virus is found at prevalence rates of 100%. Two bell pepper near-isogenic lines (NILs), one BPEV-infected and the other BPEVfree, were generated and used to investigate the type of symbiotic relationship between BPEV and bell pepper. Selected morphological and physiological characteristics of the NILs were evaluated. The overall appearance of the two NILs was similar. When compared with the BPEV-infected line, the BPEV-free line had a significantly higher percentage of seed germination. The plant height, number of fruits, and total fruit weight was higher in plants of the BPEV-free line than in plants of the BPEV-infected line; however, in most experiments, the differences were not statistically significant (p < 0.05). Other characteristics between the two lines, such as, stem diameter, percentage of dry weight, fruit volume, chlorophyll, carotenoid, and anthocyanin content, were similar. The results obtained in this investigation suggest that for the evaluated characters, BPEV appears to have a weak parasitic relationship with the host.
1. Introduction Peppers (Capsicum spp.) are perennial plants native to the tropical Americas and were one of the first crops domesticated in the Western Hemisphere and are an economically important food crop grown worldwide (Lin et al., 2013; Pernezny et al., 2003). The genus Capsicum includes five domesticated species: C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens; however, C. annuum is the most commonly cultivated (Pickersgill, 1997; Bosland et al., 1996; DeWitt and Bosland, 1996). There are several C. annuum horticultural types, which include bell, cayenne, cherry, jalapeño, serrano, and poblano, among others. In the United States, the bell type is the most commonly grown and consumed (Correl and Thornsbury, 2013). Based on the type of symbiotic relationship with the host, plant viruses can be grouped as acute or persistent (Roossinck, 2010). With a few exceptions, acute viruses are in a parasitic relationship with the host and cause negative effects such as leaf distortions and discolorations, fruit malformations, plant stunting, and low fruit yields (Hull, 2014). In contrast, persistent viruses do not appear to affect the phenotype of the host (Boccardo et al., 1987; Fukuhara, 1999; Khankhum
and Valverde, 2018; Okada et al., 2011; Valverde and Gutierrez, 2008; Zabalgogeazcoa et al., 1993). There have been reports of associations of persistent viruses with changes in some plant morphological and physiological characters (Chen et al., 2016; Grill and Garger, 1981; Khankhum and Valverde, 2018; Sabanadzovic et al., 2009); however, the type of symbiotic relationship between persistent viruses and the host is poorly understood. Endornaviruses are viruses with linear RNA genomes that range from 9.7 to 17.6 kb; have been reported infecting plants, fungi, and oomycetes; and are transmitted through the gametes at rates of nearly 100% (Fukuhara and Gibbs, 2012; Moriyama et al., 1996; Valverde and Gutierrez, 2007; Stielow et al., 2011). Plant endornaviruses are persistent viruses which do not appear to cause symptoms, and indirect evidence suggests that they lack cell-to-cell movement and are transmitted only vertically (Fukuhara and Gibbs, 2012; Moriyama et al., 1996; Valverde and Gutierrez, 2007). The type of symbiotic relationship between endornaviruses and the host is poorly understood. Although endornaviruses have been reported in many economically important plant species, there have been only two reports of associations between endornaviruses and changes in some plant morphological and
Abbreviations: BPEV, bell pepper endornavirus; NIL, near-isogenic line; PvEV1, Phaseolus vulgaris endornavirus 1; PvEV2, Phaseolus vulgaris endornavirus 2 ⁎ Corresponding author. E-mail address: [email protected] (R.A. Valverde). https://doi.org/10.1016/j.scienta.2019.02.043 Received 19 November 2018; Received in revised form 12 February 2019; Accepted 14 February 2019 Available online 20 February 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 250 (2019) 104–112
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physiological characteristics (Grill and Garger, 1981; Khankhum and Valverde, 2018). Vicia faba endornavirus has been associated with cytoplasmic male sterility (Grill and Garger, 1981). Furthermore, Khankhum and Valverde (2018) reported that two endornaviruses infecting common bean (Phaseolus vulgaris) were associated with faster seed germination, longer radicle, lower chlorophyll content, higher carotenoid content, longer pods, higher weight of 100 seeds. In general, endornaviruses have been reported only in specific crop cultivars (Fukuhara, 1999; Okada et al., 2011, 2013; Valverde et al., 2011; Zabalgogeazcoa et al., 1993). This suggests that crop domestication and modern plant breeding may have affected their occurrence and distribution within infected crop species. Bell pepper endornavirus (BPEV) has been reported infecting cultivated peppers throughout the world (Okada, et al., 2011; Chen et al., 2015; Sela et al., 2012; Muñoz-Baena et al., 2017), and it has been in silico identified from C. annuum transcriptomes (Jo et al., 2016). In commercial bell pepper cultivars grown in the United States, this virus is found at prevalence rates of 100% (Okada et al., 2011). Nevertheless, an endornavirus-free bell pepper plant of the cultivar Marengo has been identified (Okada et al., 2011). Two near-isogenic lines (NILs) were generated, one BPEV-infected and the other BPEV-free. With the aim of determining the type of symbiotic relationship between BPEV and bell pepper, we evaluated some morphological and physiological characters of these two NILs.
0.15EC (Syngenta Crop Protection, Inc., Greensboro, NC) following the manufacturer’s recommendation. For seed germination experiments, four plants of each NIL were transplanted into eight 11-L plastic pots containing 10-L of soil mix and kept in the greenhouse until fruits matured. For pigment content experiments, eight seedlings of each NIL were transplanted into 0.4-L clay pots containing 0.3 L of soil mix and kept in a growth room under artificial light (54 W/120 V 60 Hz/4.0 A Lamps) with an average temperature of 23 °C and a 15 h photoperiod. Four plantings (pots with plants placed on the gravel plot) were conducted during March-April and October-November 2015 and March-April and October-November 2016. An additional planting was conducted on 6 March 2017. Plants for seed germination experiments were planted in the greenhouse on March 2015. Plants for pigment content experiments were planted on May 2018. To confirm the presence or absence of BPEV, all experimental plants were tested using gel electrophoretic analyses of viral dsRNA and RT-PCR as described above. 2.3. Fruit evaluations The fruits of each NIL were harvested two times during each of the five plantings. The first harvest was conducted when the fruits reached 7.0 cm long (only fruit that were 7.0 cm or longer were collected) and the second harvest was conducted three weeks after, following the described minimum length. The total number of fruit per plant was recorded at the end of each harvest. After harvesting, fruits were placed into Ziploc™ bags, transferred to the laboratory, and weighed using a digital balance. The total fruit weight per plant was recorded at the end of the second harvest. To determine the fruit volume, five fruits per plant were randomly selected and fruit dimensions (diameter, length, and weight) were individually measured using an electric digital caliper (784EC 6″ SE, Guangdong, China). The fruit volume was determined using the equation Yi = 19.226859 + 0.139562Xi – 0.256142Zi + 1.429122Ti; were, Yi = volume of the fruit (cm3), Xi = diameter of the fruit (mm), Zi = length of the fruit (mm) and Ti = weight of the fruit (g) (Kadri and Kilic, 2010).
2. Materials and methods 2.1. Generation of near-isogenic lines Plants of the two previously identified bell pepper cv Marengo lines (Okada et al., 2011) were planted in steam sterilized soil mix which consisted of two parts of soil, one part of sand, and one part of MiracleGro® potting mix (Scotts Miracle-Gro, Marysville, OH, USA). Plants were kept in a greenhouse with an average temperature of 28 °C located on the Baton Rouge campus of Louisiana State University (30°24′38.5″N 91°10′22.5″W). Using the backcross breeding method (Harlan and Pope, 1922), we conducted five backcrosses using the BPEV-infected line as the donor parent and the BPEV-free line as the recurrent parent. To confirm the presence or absence of BPEV in plants of the backcross progenies, viral double-stranded RNA (dsRNA) was extracted using the method reported by Khankhum et al. (2017), analyzed in agarose gel electrophoresis, and used in reverse-transcription polymerase chain reactions (RT-PCR) as described by Okada et al. (2011). From each backcross, we tested an average of 10 plants of the F1 generation and selected one BPEV-infected plant for the backcross with the recurrent parent. During all stages of the development of the NIL, plants of the two lines were kept physically separated in screen cages to avoid pollen contaminations.
2.4. Plant phenotype The plant morphological characters, including leaf shape, size, and color, of the NILs grown in the gravel plot and growth room were visually evaluated by weekly-inspections conducted during all plant growth stages. The plant height, stem diameter, and dry weight of plants placed on the gravel plot were evaluated after the second fruit harvest. Plant height was determined by measuring the length of the main stem from the soil level to the highest apical bud. The diameter of the stem was measured at 1.0 cm above the soil level using the electric digital caliper. The dry weight was determined by collecting plants cut at the soil level, cutting them into approximately 25 cm pieces and placing them in paper bags. The fresh plant material was weighed and then bags were placed in an oven at 60 °C until the plants reached a constant weight (Abu-Zahra, 2012). The weight of the dry tissue over the fresh weight tissue ratio was determined and expressed as percentage.
2.2. Planting For the evaluation of plant height, stem thickness, fruit number, fruit weight, fruit volume, dry weight, and seed germination, seeds of the two NILs were planted in the soil mix as described above and kept in the greenhouse. One month after planting, 20 seedlings, 10 of each NIL, were transplanted into twenty 11-L plastic pots containing approximately 10-L of soil mix and kept in the greenhouse. Two weeks after transplanting, the pots were placed outside the greenhouse on a gravel plot in a completely randomized design (Fig. A1). A week after placing the pots on the gravel plot, 35.0 g of Osmocote® (19-6-3) (Scotts Miracle-Gro) was added to the soil surface of each pot. Plants were inspected daily and watered as needed. To control arthropod pests, plants placed in the gravel plot were sprayed at least once with Avid
2.5. Seed germination For the seed germination tests, seeds were collected from four fruits (one per plant) of each of the two NILs that matured (at least half of the fruit turning yellow) at the same time. Fruits were dissected in the laboratory, seeds collected, dried at room temperature for two days, and kept at 4 °C for 4 to 6 months. Before the germination test, seeds were immersed in 10% sodium hypochlorite solution for 10 min and rinsed with sterile distilled water. Plastic petri dishes (100 mm x 15 mm)
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containing two layers of brown paper towel were prepared, and 10 seeds were placed on the paper towel. Deionized water (8 ml) was added to moisten the paper, and a folded kimwipe was placed over the seeds (Fig. A2). Three plates were used for each NIL. Plates were incubated (Ambi-Hi-Lo® Chamber, Lab-Line Instruments, Inc., Ill, USA) at 23 °C for 12 days. Three days after placing the plates in the incubator, seeds were inspected and germination recorded daily. Seed germination tests were replicated three times using the same seed source. The viability of the non-germinated seed was not tested.
3. Results 3.1. Development of NILs The development of the NILs is illustrated in Figure A3. Testing an average of 10 plants of the F1 generation from each backcross resulted in BPEV-infected and BPEV-free plants. The results were similar for each F1 of the corresponding backcross. A typical result of the gel electrophoresis analysis of viral dsRNA is shown in Fig. 1. A BPEV dsRNA of approximately 16.0 kb was readily obtained from BPEV-infected plants (Fig. 1). After five backcrosses, seeds of the BPEV-infected and the BPEV-free NILs were increased in a greenhouse by self-pollination for at least two generations. Seeds were dried at room temperature (23 °C) for 4 days, stored at 4 °C and used in all comparative experiments.
2.6. Pigment content To determine the pigment content, four 2-month-old plants of each NIL were randomly selected from a group of eight plants. All leaves of each plant were ground in liquid nitrogen with a mortar and pestle and kept at −70 °C. For chlorophyll and carotenoid analyses, 0.3 g of ground tissue was used. Chlorophyll a and b content was determined following the method described by Arnon (1949), and carotenoid content following the method described by Kirk and Allen (1965). Total anthocyanin content was determined using 0.6 g of ground tissue following the method described by Mancinelli et al. (1975). Chlorophyll content was measured at 663 and 645 nm for chlorophyll a and b respectively, anthocyanin content at 530 and 657 nm, and carotenoid content at 480 nm in addition to 663 and 645 nm using a Unico® 1000 spectrophotometer (Unico, Dayton, NJ, USA). This experiment was repeated two times.
3.2. Plant morphology and fruit evaluations The overall appearance of plants of the two NILs was similar throughout all growth stages (Fig. 2). There were no visible differences between the two NILs in the plant architecture or leaf phenotype. In all five experiments, evaluating the fruit weight between the two NILs, the BPEV-free line yielded higher fruit weights than the BPEVinfected line (Fig. 3A, Table A1). However, differences were statistically significant only in one experiment. In three experiments, the BPEV-free line yielded higher number of fruits per plant than the BPEVinfected line; however, the data was statistically significant in only two experiments (Fig. 3B, Table A1). In two experiments the BPEV-free line yielded fruits with larger volume than the BPEV-infected line; however, in two other experiments, opposite results were obtained (Table A1). Nevertheless, the differences in volume obtained in all four experiments were not statistically significant. In four experiments evaluating the plant height, the BPEV-free line consistently yielded higher values than the BPEVinfected line, although the differences were not statistically significant (Fig. 4A, Table A2). Similarly, in three experiments, the stem diameter values were higher for the BPEV-free plants, but the differences were not statistically significant (Fig. 4B, Table A2). The percentage of dried weight between the two lines did not show significant variations among the four experiments (Table A2).
2.7. Experimental design and statistical analysis A completely randomized design was used in all the experiments. The averaged data from each NIL was analyzed by one-way ANOVA using SPSS (IBM© SPSS© Statistics Version 24) the analysis was performed through the General Desktop Virtual Lab of the Louisiana State University, Baton Rouge. Statistical analysis for morphological characteristics and fruit yield was carried out between NILs and between plantings (replicates). In the case of analysis between replicates, the data was pooled when there were no statistical differences. The comparisons were considered statistically significant at p < 0.05.
3.3. Seed germination In the three seed germination experiments, seeds from the BPEVfree line showed significantly higher percentage of germination (Fig.5, Table A3). The average of seed germination did not differ among replicates. At day 12 the average percentages of seed germination from the three experiments were 92.20% and 66.66% for the BPEV- line and the BPEV + line respectively. 3.4. Pigment content The total chlorophyll, carotenoid, and anthocyanin content between the two NILs were similar (Fig. A4). The total chlorophyll content was 2.74 ± 0.11 and 2.74 ± 0.17 mg/g for the BPEV- and the BBPEV + line respectively. The carotenoid content was 0.42 ± 0.02 and 0.41 ± 0.04 mg for the BPEV- and the BPEV + line respectively. The anthocyanin content (absorbance at 530 nm/g F.W.) was higher in the BPEV- line (1.23 ± 0.12) than in the BPEV + line (1.08 ± 0.06), but the differences were not statistically significant.
Fig. 1. Agarose (1.2%) gel electrophoresis of dsRNAs extracted from seven F1 plants that resulted from crossing a BPEV-infected plant (donor parent) with a BPEV-free plant (recurrent parent). Lanes 1, 2, 3, 5, and 7 BPEV-free plants; lanes 4 and 6 BPEV-infected plants; lane 8, 1 kb Molecular Ruler (Bio-Rad, Hercules, CA, USA).
4. Discussion Many plant species exist in symbiotic relationships with fungi,
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Fig. 2. Near-isogenic lines of bell pepper cv. Marengo, one infected with bell pepper endornavirus (BPEV +) and the other free of bell pepper endornavirus (BPEV-).
than the one used in this investigation. Using two lines selected from the common bean cv. Black Turtle Soup, one infected with Phaseolus vulgaris endornavirus 1 (PvEV1) and Phaseolus vulgaris endornavirus 2 (PvEV2) and the other endornavirusfree, Khankhum and Valverde (2018) conducted a comparative study on their physiological and morphological characters. They reported that the endornavirus-infected line yielded seed with similar percentage of seed germination but germinated faster than the endornavirus-free line. Moreover, the virus-infected line contained higher carotenoid content. Nevertheless, the infected line yielded lower total chlorophyll content. In our investigations with bell pepper NILs, we obtained different and, in some cases, contrasting results than those reported by Khankhum and Valverde (2018) for common bean. Under the conditions of seed harvest, storage, and germination experiments used in this investigation, the BPEV-free line yielded significantly higher percentage of seed germination than the BPEV-infected line, although the speed of germination was similar. It has been reported that infections of Arabidopsis thaliana by the acute virus cucumber mosaic virus cause a significant reduction of seed germination (Bueso et al., 2017). Similarly, Mandhare and Gawade, (2010) reported a significant reduction of soybean seed germination in soybean infected by soybean mosaic virus, another acute virus. Hormone contents, signaling, and interactions play important roles in determining the physiological state of the seed and in regulating the germination process (Kucera et al., 2005). It is possible that BPEV, acting as a biotic stress, interferes with transcript regulation of genes involved in these processes. In our experiments, the percentage of seed germination of both NILs did not reach 100%, suggesting expression of seed dormancy, which could have been caused by early fruit harvest or the low temperature conditions in which the seed germination experiments were conducted (Randle and Honma, 1981; Hartz et al., 2008). Although many commercial bell pepper cultivars are 100% infected with BPEV, it is likely that their seed germination percentage has not been compared with BPEV-free cultivars. It has been reported that chlorophyll and carotenoids content are negatively affected by viral infections; nevertheless, we did not obtain significant differences in these characters between the two NILs (DeBlasio et al., 2018; Kapinga et al., 2009; Rahoutei et al., 2000; Reinero and Beachy, 1986). In contrast to our results, Khankhum and Valverde (2018) reported that the endornavirus-infected common bean line yielded significantly lower chlorophyll and higher carotenoid
bacteria, and viruses and the nature of the symbiotic relationship can range from mutualistic to parasitic (Agrios, 2005; Hayat et al., 2010; Ikeda et al., 2012; Marquez et al., 2007; van Molken et al., 2012; Roossinck, 2010, 2013; Xu et al., 2008). Parasitic relationships have been well documented; however, limited information is available about mutualistic relationships (Perez-Brocal et al., 2011; Roossinck, 2011, 2015). One approach taken to determine the type of symbiotic relationship between plants and microorganisms is inoculation experiments. In the case of plant endornaviruses, inoculation poses a challenge because these viruses lack cell-to-cell movement; therefore, the use of conventional virus-inoculation methods, such as mechanical or graft inoculations, do not result in systemic viral infections (Valverde and Gutierrez, 2007). Because of these BPEV properties, we could not conduct inoculations of the virus to the endornavirus-free line. Nevertheless, the approach we used to generate the NILs could be considered as a novel “virus-inoculation” method. Therefore, the development of NILs provided us with a practical approach to evaluate possible effects of BPEV in some of the host characteristics. Results from this and the previous investigations by Khankhum and Valverde (2018) support that endornaviruses do not appear to change the host phenotype. It appears that the plant is able to suppress the expected ability of the virus to cause symptoms. Alternatively, it is also possible that the virus may lack that ability. In any case, the plant does not appear to be able to stop the virus replication. Under the experimental conditions of this investigation, we found that when compared with the BPEV-positive line, the BPEV-negative line showed higher values in most of the evaluated characters. Although, most differences were not statistically significant, the results of this investigation suggest that BPEV negatively affects bell pepper fruit number and fruit weight which translates in a negative effect on fruit yield. These results are not surprising because it has been shown that acute plant viral infections often leads to lower fruit yield and quality (Anderson et al., 2004; Boualem et al., 2016; Padgett et al., 1990; Spence et al., 2006). In some cases even though there is an absence of symptoms, there is a reduction of plant fitness (Christov et al., 2007). As is the case in many other crops, fruit weight is an important parameter because commercially, bell pepper fruits are sold by weight (Hartz et al., 2008; Fonsah, 2009). Nevertheless, to evaluate the possible negative effect of BPEV on fruit yield, from the commercial standpoint, it will be necessary to conduct experiments on a larger scale
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Fig. 3. Fruit weight (A) and fruit number (B) of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
values than the endornavirus-free line. It is possible that different endornavirus species vary in the effect they may have to their hosts. Furthermore, mixed infections of endornaviruses, as in the case of Black Turtle Soup common bean, could result in host effects that vary from those of single infections such as bell pepper infected with BPEV. During the early stages of NILs development (backcross 2), we conducted a limited number of experiments on the transcriptome of the BPEV-free and the BPEV-infected lines (Escalante et al., unpublished). Data analyses showed over 1000 differentially expressed genes. The preliminary transcriptome results and reports of de novo genome assembling of endornaviruses from small RNAs, suggests an active interaction between BPEV and the host (Chen et al., 2015; Sela et al., 2012). Transcriptome data, combined with the biological characteristics of the two NILs presented in this investigation should provide information that will elucidate the potential effect(s) of BPEV in bell pepper. The overall results obtained in this investigation suggest that BPEV has a weak parasitic relationship with the host. However, because we
only evaluated a limited number of characteristics, we cannot conclude that the symbiotic relationship between BPEV and bell pepper is only parasitic. Because plant breeders in the United States have bred only BPEV-infected cultivars, it is possible that BPEV provides an unknown beneficial effect to the plant. The results of this investigation, together with those of Khankhum and Valverde (2018), suggest that the type of symbiosis between endornaviruses and their plant host may depend on the characters being evaluated, the host, and the virus species involved. To gain more information that will help to determine the type of symbiotic relationships between plant endornaviruses and their hosts, evaluations of other host characteristics such as tolerance to biotic and abiotic stresses should be conducted using the NILs. 5. Conclusions In this comparative study using two NILs of bell pepper, one BPEV-infected and the other BPEV-free; we determined that BPEV is not associated with significant changes in the plant phenotype.
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Fig. 4. Plant height (A) and stem thickness (B) of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
Fig. 5. Progress of seed germination of two near-isogenic lines of bell pepper Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
However, we found an association of BPEV with lower percentage of seed germination. Other evaluated characters were not significantly different between the two NILs. This investigation
constitutes the first report investigating associations of a plant endornavirus with physiological and morphological characteristics of the host using NILs. 109
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Declaration of interest
USA for providing the original endornavirus-free line of Marengo bell pepper. The National Institute of Food and Agriculture, USDA and a Feasibility Study Grant (US-4725-14F) from the US-Israel Binational Agricultural Research and Development Fund provided partial funding for this investigation.
The authors have no potential conflict of interests. Acknowledments The authors wish to thank MJ Roossinck, Penn State University, Appendix A
Table A1 Fruit weight, fruit number and fruit volume of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Experiment No.
Mean†
NIL
1
BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+
2 3 4 5
Fruit weight (g)
Fruit number
Fruit volume (cm3)
484.2 358.6 407.1 339.0 332.8 291.8 221.4 134.2 491.3 400.4
21.0 ± 2.7a 16.0 ± 1.9a 9.0 ± 0.7a 6.0 ± 0.5b 8.0 ± 0.7a 8.0 ± 0.2a 6.0 ± 0.5a 7.0 ± 1.2a 17.0 ± 0.8a 13.0 ± 0.9b
79.2 85.5 72.0 69.8 69.1 52.2 68.4 72.2 ND ND
± ± ± ± ± ± ± ± ± ±
58.7a 48.6a 25.5a 26.0a 13.4a 31.0a 17.8a 20.2b 43.7a 31.9a
± ± ± ± ± ± ± ±
3.1a 5.8a 2.9a 6.1a 7.0a 4.1a 4.7a 4.6a
ND = Not determined. † = Each value represents the mean ± standard error of each treatment. Table A2 Plant height, stem thickness and percentage of dry weight of two near-isogenic lines. (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Means followed by different lowercase letters are significantly different between BPEV- and BPEV + in each experiment. Experiment No.
Mean†
NIL
1
BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+
2 3 4 5
Plant height (cm)
Stem thickness (mm)
Dry weight (%)
63.8 58.6 35.9 36.6 46.8 45.6 46.2 38.4 41.4 41.0
13.8 ± 0.7a 13.5 ± 0.2a 9.6 ± 0.3a 10.1 ± 0.2a 12.3 ± 0.5a 11.2 ± 0.6a 10.7 ± 0.2a 9.6 ± 0.4b 13.9 ± 0.4a 14.5 ± 0.4a
18.3 17.6 17.1 17.2 16.0 15.3 16.8 18.3 ND ND
± ± ± ± ± ± ± ± ± ±
2.8a 2.4a 1.1a 1.4a 1.8a 2.6a 1.9a 2.4b 0.7a 1.5b
± ± ± ± ± ± ± ±
0.4a 0.7a 0.3a 1.2a 0.5a 0.6a 0.8a 1.6a
ND = Not determined. † = Each value represents the mean ± standard error of each treatment. Table A3 Seed germination (three experiments) of two near-isogenic lines of bell pepper Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Values represent the percent seed germination throughout a period of 12 days. Day
Percent germination Experiment 1
3 4 5 6 7 8 9 10 11 12
Experiment 2
Experiment 3
BPEV-
BPEV+
BPEV-
BPEV+
BPEV-
BPEV+
10.0 80.0 86.7 86.7 86.7 90.0 90.0 90.0 90.0 90.0
6.7 30.0 40.0 46.7 46.7 56.7 66.7 70.0 70.0 70.0
0.0 73.3 86.7 93.3 93.3 93.3 93.3 93.3 93.3 93.3
23.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3
0.0 3.3 43.3 60.0 66.7 70.0 80.0 83.3 86.7 93.0
0.0 3.3 23.3 36.7 53.3 60.0 70.0 73.3 73.3 76.7
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Appendix B. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.02.043.
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Morphological and physiological characteristics of endornavirus-infected and endornavirus-free near-isogenic lines of bell pepper (Capsicum annuum)
T
C. Escalante, R.A. Valverde
⁎
Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA
ARTICLE INFO
ABSTRACT
Keywords: Bell pepper Bell pepper endornavirus Endornaviridae Plant virus Seed germination
Plant endornaviruses are persistent viruses that infect plants without causing symptoms. Although endornaviruses have been reported in many economically important plant species, little is known about the effect they have on their hosts. Bell pepper endornavirus (BPEV) has been reported in cultivated peppers (Capsicum spp.). In commercial cultivars of bell pepper (C. annuum) grown in the United States, this virus is found at prevalence rates of 100%. Two bell pepper near-isogenic lines (NILs), one BPEV-infected and the other BPEVfree, were generated and used to investigate the type of symbiotic relationship between BPEV and bell pepper. Selected morphological and physiological characteristics of the NILs were evaluated. The overall appearance of the two NILs was similar. When compared with the BPEV-infected line, the BPEV-free line had a significantly higher percentage of seed germination. The plant height, number of fruits, and total fruit weight was higher in plants of the BPEV-free line than in plants of the BPEV-infected line; however, in most experiments, the differences were not statistically significant (p < 0.05). Other characteristics between the two lines, such as, stem diameter, percentage of dry weight, fruit volume, chlorophyll, carotenoid, and anthocyanin content, were similar. The results obtained in this investigation suggest that for the evaluated characters, BPEV appears to have a weak parasitic relationship with the host.
1. Introduction Peppers (Capsicum spp.) are perennial plants native to the tropical Americas and were one of the first crops domesticated in the Western Hemisphere and are an economically important food crop grown worldwide (Lin et al., 2013; Pernezny et al., 2003). The genus Capsicum includes five domesticated species: C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens; however, C. annuum is the most commonly cultivated (Pickersgill, 1997; Bosland et al., 1996; DeWitt and Bosland, 1996). There are several C. annuum horticultural types, which include bell, cayenne, cherry, jalapeño, serrano, and poblano, among others. In the United States, the bell type is the most commonly grown and consumed (Correl and Thornsbury, 2013). Based on the type of symbiotic relationship with the host, plant viruses can be grouped as acute or persistent (Roossinck, 2010). With a few exceptions, acute viruses are in a parasitic relationship with the host and cause negative effects such as leaf distortions and discolorations, fruit malformations, plant stunting, and low fruit yields (Hull, 2014). In contrast, persistent viruses do not appear to affect the phenotype of the host (Boccardo et al., 1987; Fukuhara, 1999; Khankhum
and Valverde, 2018; Okada et al., 2011; Valverde and Gutierrez, 2008; Zabalgogeazcoa et al., 1993). There have been reports of associations of persistent viruses with changes in some plant morphological and physiological characters (Chen et al., 2016; Grill and Garger, 1981; Khankhum and Valverde, 2018; Sabanadzovic et al., 2009); however, the type of symbiotic relationship between persistent viruses and the host is poorly understood. Endornaviruses are viruses with linear RNA genomes that range from 9.7 to 17.6 kb; have been reported infecting plants, fungi, and oomycetes; and are transmitted through the gametes at rates of nearly 100% (Fukuhara and Gibbs, 2012; Moriyama et al., 1996; Valverde and Gutierrez, 2007; Stielow et al., 2011). Plant endornaviruses are persistent viruses which do not appear to cause symptoms, and indirect evidence suggests that they lack cell-to-cell movement and are transmitted only vertically (Fukuhara and Gibbs, 2012; Moriyama et al., 1996; Valverde and Gutierrez, 2007). The type of symbiotic relationship between endornaviruses and the host is poorly understood. Although endornaviruses have been reported in many economically important plant species, there have been only two reports of associations between endornaviruses and changes in some plant morphological and
Abbreviations: BPEV, bell pepper endornavirus; NIL, near-isogenic line; PvEV1, Phaseolus vulgaris endornavirus 1; PvEV2, Phaseolus vulgaris endornavirus 2 ⁎ Corresponding author. E-mail address: [email protected] (R.A. Valverde). https://doi.org/10.1016/j.scienta.2019.02.043 Received 19 November 2018; Received in revised form 12 February 2019; Accepted 14 February 2019 Available online 20 February 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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physiological characteristics (Grill and Garger, 1981; Khankhum and Valverde, 2018). Vicia faba endornavirus has been associated with cytoplasmic male sterility (Grill and Garger, 1981). Furthermore, Khankhum and Valverde (2018) reported that two endornaviruses infecting common bean (Phaseolus vulgaris) were associated with faster seed germination, longer radicle, lower chlorophyll content, higher carotenoid content, longer pods, higher weight of 100 seeds. In general, endornaviruses have been reported only in specific crop cultivars (Fukuhara, 1999; Okada et al., 2011, 2013; Valverde et al., 2011; Zabalgogeazcoa et al., 1993). This suggests that crop domestication and modern plant breeding may have affected their occurrence and distribution within infected crop species. Bell pepper endornavirus (BPEV) has been reported infecting cultivated peppers throughout the world (Okada, et al., 2011; Chen et al., 2015; Sela et al., 2012; Muñoz-Baena et al., 2017), and it has been in silico identified from C. annuum transcriptomes (Jo et al., 2016). In commercial bell pepper cultivars grown in the United States, this virus is found at prevalence rates of 100% (Okada et al., 2011). Nevertheless, an endornavirus-free bell pepper plant of the cultivar Marengo has been identified (Okada et al., 2011). Two near-isogenic lines (NILs) were generated, one BPEV-infected and the other BPEV-free. With the aim of determining the type of symbiotic relationship between BPEV and bell pepper, we evaluated some morphological and physiological characters of these two NILs.
0.15EC (Syngenta Crop Protection, Inc., Greensboro, NC) following the manufacturer’s recommendation. For seed germination experiments, four plants of each NIL were transplanted into eight 11-L plastic pots containing 10-L of soil mix and kept in the greenhouse until fruits matured. For pigment content experiments, eight seedlings of each NIL were transplanted into 0.4-L clay pots containing 0.3 L of soil mix and kept in a growth room under artificial light (54 W/120 V 60 Hz/4.0 A Lamps) with an average temperature of 23 °C and a 15 h photoperiod. Four plantings (pots with plants placed on the gravel plot) were conducted during March-April and October-November 2015 and March-April and October-November 2016. An additional planting was conducted on 6 March 2017. Plants for seed germination experiments were planted in the greenhouse on March 2015. Plants for pigment content experiments were planted on May 2018. To confirm the presence or absence of BPEV, all experimental plants were tested using gel electrophoretic analyses of viral dsRNA and RT-PCR as described above. 2.3. Fruit evaluations The fruits of each NIL were harvested two times during each of the five plantings. The first harvest was conducted when the fruits reached 7.0 cm long (only fruit that were 7.0 cm or longer were collected) and the second harvest was conducted three weeks after, following the described minimum length. The total number of fruit per plant was recorded at the end of each harvest. After harvesting, fruits were placed into Ziploc™ bags, transferred to the laboratory, and weighed using a digital balance. The total fruit weight per plant was recorded at the end of the second harvest. To determine the fruit volume, five fruits per plant were randomly selected and fruit dimensions (diameter, length, and weight) were individually measured using an electric digital caliper (784EC 6″ SE, Guangdong, China). The fruit volume was determined using the equation Yi = 19.226859 + 0.139562Xi – 0.256142Zi + 1.429122Ti; were, Yi = volume of the fruit (cm3), Xi = diameter of the fruit (mm), Zi = length of the fruit (mm) and Ti = weight of the fruit (g) (Kadri and Kilic, 2010).
2. Materials and methods 2.1. Generation of near-isogenic lines Plants of the two previously identified bell pepper cv Marengo lines (Okada et al., 2011) were planted in steam sterilized soil mix which consisted of two parts of soil, one part of sand, and one part of MiracleGro® potting mix (Scotts Miracle-Gro, Marysville, OH, USA). Plants were kept in a greenhouse with an average temperature of 28 °C located on the Baton Rouge campus of Louisiana State University (30°24′38.5″N 91°10′22.5″W). Using the backcross breeding method (Harlan and Pope, 1922), we conducted five backcrosses using the BPEV-infected line as the donor parent and the BPEV-free line as the recurrent parent. To confirm the presence or absence of BPEV in plants of the backcross progenies, viral double-stranded RNA (dsRNA) was extracted using the method reported by Khankhum et al. (2017), analyzed in agarose gel electrophoresis, and used in reverse-transcription polymerase chain reactions (RT-PCR) as described by Okada et al. (2011). From each backcross, we tested an average of 10 plants of the F1 generation and selected one BPEV-infected plant for the backcross with the recurrent parent. During all stages of the development of the NIL, plants of the two lines were kept physically separated in screen cages to avoid pollen contaminations.
2.4. Plant phenotype The plant morphological characters, including leaf shape, size, and color, of the NILs grown in the gravel plot and growth room were visually evaluated by weekly-inspections conducted during all plant growth stages. The plant height, stem diameter, and dry weight of plants placed on the gravel plot were evaluated after the second fruit harvest. Plant height was determined by measuring the length of the main stem from the soil level to the highest apical bud. The diameter of the stem was measured at 1.0 cm above the soil level using the electric digital caliper. The dry weight was determined by collecting plants cut at the soil level, cutting them into approximately 25 cm pieces and placing them in paper bags. The fresh plant material was weighed and then bags were placed in an oven at 60 °C until the plants reached a constant weight (Abu-Zahra, 2012). The weight of the dry tissue over the fresh weight tissue ratio was determined and expressed as percentage.
2.2. Planting For the evaluation of plant height, stem thickness, fruit number, fruit weight, fruit volume, dry weight, and seed germination, seeds of the two NILs were planted in the soil mix as described above and kept in the greenhouse. One month after planting, 20 seedlings, 10 of each NIL, were transplanted into twenty 11-L plastic pots containing approximately 10-L of soil mix and kept in the greenhouse. Two weeks after transplanting, the pots were placed outside the greenhouse on a gravel plot in a completely randomized design (Fig. A1). A week after placing the pots on the gravel plot, 35.0 g of Osmocote® (19-6-3) (Scotts Miracle-Gro) was added to the soil surface of each pot. Plants were inspected daily and watered as needed. To control arthropod pests, plants placed in the gravel plot were sprayed at least once with Avid
2.5. Seed germination For the seed germination tests, seeds were collected from four fruits (one per plant) of each of the two NILs that matured (at least half of the fruit turning yellow) at the same time. Fruits were dissected in the laboratory, seeds collected, dried at room temperature for two days, and kept at 4 °C for 4 to 6 months. Before the germination test, seeds were immersed in 10% sodium hypochlorite solution for 10 min and rinsed with sterile distilled water. Plastic petri dishes (100 mm x 15 mm)
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containing two layers of brown paper towel were prepared, and 10 seeds were placed on the paper towel. Deionized water (8 ml) was added to moisten the paper, and a folded kimwipe was placed over the seeds (Fig. A2). Three plates were used for each NIL. Plates were incubated (Ambi-Hi-Lo® Chamber, Lab-Line Instruments, Inc., Ill, USA) at 23 °C for 12 days. Three days after placing the plates in the incubator, seeds were inspected and germination recorded daily. Seed germination tests were replicated three times using the same seed source. The viability of the non-germinated seed was not tested.
3. Results 3.1. Development of NILs The development of the NILs is illustrated in Figure A3. Testing an average of 10 plants of the F1 generation from each backcross resulted in BPEV-infected and BPEV-free plants. The results were similar for each F1 of the corresponding backcross. A typical result of the gel electrophoresis analysis of viral dsRNA is shown in Fig. 1. A BPEV dsRNA of approximately 16.0 kb was readily obtained from BPEV-infected plants (Fig. 1). After five backcrosses, seeds of the BPEV-infected and the BPEV-free NILs were increased in a greenhouse by self-pollination for at least two generations. Seeds were dried at room temperature (23 °C) for 4 days, stored at 4 °C and used in all comparative experiments.
2.6. Pigment content To determine the pigment content, four 2-month-old plants of each NIL were randomly selected from a group of eight plants. All leaves of each plant were ground in liquid nitrogen with a mortar and pestle and kept at −70 °C. For chlorophyll and carotenoid analyses, 0.3 g of ground tissue was used. Chlorophyll a and b content was determined following the method described by Arnon (1949), and carotenoid content following the method described by Kirk and Allen (1965). Total anthocyanin content was determined using 0.6 g of ground tissue following the method described by Mancinelli et al. (1975). Chlorophyll content was measured at 663 and 645 nm for chlorophyll a and b respectively, anthocyanin content at 530 and 657 nm, and carotenoid content at 480 nm in addition to 663 and 645 nm using a Unico® 1000 spectrophotometer (Unico, Dayton, NJ, USA). This experiment was repeated two times.
3.2. Plant morphology and fruit evaluations The overall appearance of plants of the two NILs was similar throughout all growth stages (Fig. 2). There were no visible differences between the two NILs in the plant architecture or leaf phenotype. In all five experiments, evaluating the fruit weight between the two NILs, the BPEV-free line yielded higher fruit weights than the BPEVinfected line (Fig. 3A, Table A1). However, differences were statistically significant only in one experiment. In three experiments, the BPEV-free line yielded higher number of fruits per plant than the BPEVinfected line; however, the data was statistically significant in only two experiments (Fig. 3B, Table A1). In two experiments the BPEV-free line yielded fruits with larger volume than the BPEV-infected line; however, in two other experiments, opposite results were obtained (Table A1). Nevertheless, the differences in volume obtained in all four experiments were not statistically significant. In four experiments evaluating the plant height, the BPEV-free line consistently yielded higher values than the BPEVinfected line, although the differences were not statistically significant (Fig. 4A, Table A2). Similarly, in three experiments, the stem diameter values were higher for the BPEV-free plants, but the differences were not statistically significant (Fig. 4B, Table A2). The percentage of dried weight between the two lines did not show significant variations among the four experiments (Table A2).
2.7. Experimental design and statistical analysis A completely randomized design was used in all the experiments. The averaged data from each NIL was analyzed by one-way ANOVA using SPSS (IBM© SPSS© Statistics Version 24) the analysis was performed through the General Desktop Virtual Lab of the Louisiana State University, Baton Rouge. Statistical analysis for morphological characteristics and fruit yield was carried out between NILs and between plantings (replicates). In the case of analysis between replicates, the data was pooled when there were no statistical differences. The comparisons were considered statistically significant at p < 0.05.
3.3. Seed germination In the three seed germination experiments, seeds from the BPEVfree line showed significantly higher percentage of germination (Fig.5, Table A3). The average of seed germination did not differ among replicates. At day 12 the average percentages of seed germination from the three experiments were 92.20% and 66.66% for the BPEV- line and the BPEV + line respectively. 3.4. Pigment content The total chlorophyll, carotenoid, and anthocyanin content between the two NILs were similar (Fig. A4). The total chlorophyll content was 2.74 ± 0.11 and 2.74 ± 0.17 mg/g for the BPEV- and the BBPEV + line respectively. The carotenoid content was 0.42 ± 0.02 and 0.41 ± 0.04 mg for the BPEV- and the BPEV + line respectively. The anthocyanin content (absorbance at 530 nm/g F.W.) was higher in the BPEV- line (1.23 ± 0.12) than in the BPEV + line (1.08 ± 0.06), but the differences were not statistically significant.
Fig. 1. Agarose (1.2%) gel electrophoresis of dsRNAs extracted from seven F1 plants that resulted from crossing a BPEV-infected plant (donor parent) with a BPEV-free plant (recurrent parent). Lanes 1, 2, 3, 5, and 7 BPEV-free plants; lanes 4 and 6 BPEV-infected plants; lane 8, 1 kb Molecular Ruler (Bio-Rad, Hercules, CA, USA).
4. Discussion Many plant species exist in symbiotic relationships with fungi,
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Fig. 2. Near-isogenic lines of bell pepper cv. Marengo, one infected with bell pepper endornavirus (BPEV +) and the other free of bell pepper endornavirus (BPEV-).
than the one used in this investigation. Using two lines selected from the common bean cv. Black Turtle Soup, one infected with Phaseolus vulgaris endornavirus 1 (PvEV1) and Phaseolus vulgaris endornavirus 2 (PvEV2) and the other endornavirusfree, Khankhum and Valverde (2018) conducted a comparative study on their physiological and morphological characters. They reported that the endornavirus-infected line yielded seed with similar percentage of seed germination but germinated faster than the endornavirus-free line. Moreover, the virus-infected line contained higher carotenoid content. Nevertheless, the infected line yielded lower total chlorophyll content. In our investigations with bell pepper NILs, we obtained different and, in some cases, contrasting results than those reported by Khankhum and Valverde (2018) for common bean. Under the conditions of seed harvest, storage, and germination experiments used in this investigation, the BPEV-free line yielded significantly higher percentage of seed germination than the BPEV-infected line, although the speed of germination was similar. It has been reported that infections of Arabidopsis thaliana by the acute virus cucumber mosaic virus cause a significant reduction of seed germination (Bueso et al., 2017). Similarly, Mandhare and Gawade, (2010) reported a significant reduction of soybean seed germination in soybean infected by soybean mosaic virus, another acute virus. Hormone contents, signaling, and interactions play important roles in determining the physiological state of the seed and in regulating the germination process (Kucera et al., 2005). It is possible that BPEV, acting as a biotic stress, interferes with transcript regulation of genes involved in these processes. In our experiments, the percentage of seed germination of both NILs did not reach 100%, suggesting expression of seed dormancy, which could have been caused by early fruit harvest or the low temperature conditions in which the seed germination experiments were conducted (Randle and Honma, 1981; Hartz et al., 2008). Although many commercial bell pepper cultivars are 100% infected with BPEV, it is likely that their seed germination percentage has not been compared with BPEV-free cultivars. It has been reported that chlorophyll and carotenoids content are negatively affected by viral infections; nevertheless, we did not obtain significant differences in these characters between the two NILs (DeBlasio et al., 2018; Kapinga et al., 2009; Rahoutei et al., 2000; Reinero and Beachy, 1986). In contrast to our results, Khankhum and Valverde (2018) reported that the endornavirus-infected common bean line yielded significantly lower chlorophyll and higher carotenoid
bacteria, and viruses and the nature of the symbiotic relationship can range from mutualistic to parasitic (Agrios, 2005; Hayat et al., 2010; Ikeda et al., 2012; Marquez et al., 2007; van Molken et al., 2012; Roossinck, 2010, 2013; Xu et al., 2008). Parasitic relationships have been well documented; however, limited information is available about mutualistic relationships (Perez-Brocal et al., 2011; Roossinck, 2011, 2015). One approach taken to determine the type of symbiotic relationship between plants and microorganisms is inoculation experiments. In the case of plant endornaviruses, inoculation poses a challenge because these viruses lack cell-to-cell movement; therefore, the use of conventional virus-inoculation methods, such as mechanical or graft inoculations, do not result in systemic viral infections (Valverde and Gutierrez, 2007). Because of these BPEV properties, we could not conduct inoculations of the virus to the endornavirus-free line. Nevertheless, the approach we used to generate the NILs could be considered as a novel “virus-inoculation” method. Therefore, the development of NILs provided us with a practical approach to evaluate possible effects of BPEV in some of the host characteristics. Results from this and the previous investigations by Khankhum and Valverde (2018) support that endornaviruses do not appear to change the host phenotype. It appears that the plant is able to suppress the expected ability of the virus to cause symptoms. Alternatively, it is also possible that the virus may lack that ability. In any case, the plant does not appear to be able to stop the virus replication. Under the experimental conditions of this investigation, we found that when compared with the BPEV-positive line, the BPEV-negative line showed higher values in most of the evaluated characters. Although, most differences were not statistically significant, the results of this investigation suggest that BPEV negatively affects bell pepper fruit number and fruit weight which translates in a negative effect on fruit yield. These results are not surprising because it has been shown that acute plant viral infections often leads to lower fruit yield and quality (Anderson et al., 2004; Boualem et al., 2016; Padgett et al., 1990; Spence et al., 2006). In some cases even though there is an absence of symptoms, there is a reduction of plant fitness (Christov et al., 2007). As is the case in many other crops, fruit weight is an important parameter because commercially, bell pepper fruits are sold by weight (Hartz et al., 2008; Fonsah, 2009). Nevertheless, to evaluate the possible negative effect of BPEV on fruit yield, from the commercial standpoint, it will be necessary to conduct experiments on a larger scale
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Fig. 3. Fruit weight (A) and fruit number (B) of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
values than the endornavirus-free line. It is possible that different endornavirus species vary in the effect they may have to their hosts. Furthermore, mixed infections of endornaviruses, as in the case of Black Turtle Soup common bean, could result in host effects that vary from those of single infections such as bell pepper infected with BPEV. During the early stages of NILs development (backcross 2), we conducted a limited number of experiments on the transcriptome of the BPEV-free and the BPEV-infected lines (Escalante et al., unpublished). Data analyses showed over 1000 differentially expressed genes. The preliminary transcriptome results and reports of de novo genome assembling of endornaviruses from small RNAs, suggests an active interaction between BPEV and the host (Chen et al., 2015; Sela et al., 2012). Transcriptome data, combined with the biological characteristics of the two NILs presented in this investigation should provide information that will elucidate the potential effect(s) of BPEV in bell pepper. The overall results obtained in this investigation suggest that BPEV has a weak parasitic relationship with the host. However, because we
only evaluated a limited number of characteristics, we cannot conclude that the symbiotic relationship between BPEV and bell pepper is only parasitic. Because plant breeders in the United States have bred only BPEV-infected cultivars, it is possible that BPEV provides an unknown beneficial effect to the plant. The results of this investigation, together with those of Khankhum and Valverde (2018), suggest that the type of symbiosis between endornaviruses and their plant host may depend on the characters being evaluated, the host, and the virus species involved. To gain more information that will help to determine the type of symbiotic relationships between plant endornaviruses and their hosts, evaluations of other host characteristics such as tolerance to biotic and abiotic stresses should be conducted using the NILs. 5. Conclusions In this comparative study using two NILs of bell pepper, one BPEV-infected and the other BPEV-free; we determined that BPEV is not associated with significant changes in the plant phenotype.
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Fig. 4. Plant height (A) and stem thickness (B) of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
Fig. 5. Progress of seed germination of two near-isogenic lines of bell pepper Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Bars indicate standard error.
However, we found an association of BPEV with lower percentage of seed germination. Other evaluated characters were not significantly different between the two NILs. This investigation
constitutes the first report investigating associations of a plant endornavirus with physiological and morphological characteristics of the host using NILs. 109
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Declaration of interest
USA for providing the original endornavirus-free line of Marengo bell pepper. The National Institute of Food and Agriculture, USDA and a Feasibility Study Grant (US-4725-14F) from the US-Israel Binational Agricultural Research and Development Fund provided partial funding for this investigation.
The authors have no potential conflict of interests. Acknowledments The authors wish to thank MJ Roossinck, Penn State University, Appendix A
Table A1 Fruit weight, fruit number and fruit volume of two near-isogenic lines (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other one free of BPEV (BPEV-). Mean values followed by different lowercase letters are significantly different (p < 0.05) between BPEV- and BPEV + in each experiment. Experiment No.
Mean†
NIL
1
BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+
2 3 4 5
Fruit weight (g)
Fruit number
Fruit volume (cm3)
484.2 358.6 407.1 339.0 332.8 291.8 221.4 134.2 491.3 400.4
21.0 ± 2.7a 16.0 ± 1.9a 9.0 ± 0.7a 6.0 ± 0.5b 8.0 ± 0.7a 8.0 ± 0.2a 6.0 ± 0.5a 7.0 ± 1.2a 17.0 ± 0.8a 13.0 ± 0.9b
79.2 85.5 72.0 69.8 69.1 52.2 68.4 72.2 ND ND
± ± ± ± ± ± ± ± ± ±
58.7a 48.6a 25.5a 26.0a 13.4a 31.0a 17.8a 20.2b 43.7a 31.9a
± ± ± ± ± ± ± ±
3.1a 5.8a 2.9a 6.1a 7.0a 4.1a 4.7a 4.6a
ND = Not determined. † = Each value represents the mean ± standard error of each treatment. Table A2 Plant height, stem thickness and percentage of dry weight of two near-isogenic lines. (NILs) of bell pepper cv. Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Means followed by different lowercase letters are significantly different between BPEV- and BPEV + in each experiment. Experiment No.
Mean†
NIL
1
BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+ BPEVBPEV+
2 3 4 5
Plant height (cm)
Stem thickness (mm)
Dry weight (%)
63.8 58.6 35.9 36.6 46.8 45.6 46.2 38.4 41.4 41.0
13.8 ± 0.7a 13.5 ± 0.2a 9.6 ± 0.3a 10.1 ± 0.2a 12.3 ± 0.5a 11.2 ± 0.6a 10.7 ± 0.2a 9.6 ± 0.4b 13.9 ± 0.4a 14.5 ± 0.4a
18.3 17.6 17.1 17.2 16.0 15.3 16.8 18.3 ND ND
± ± ± ± ± ± ± ± ± ±
2.8a 2.4a 1.1a 1.4a 1.8a 2.6a 1.9a 2.4b 0.7a 1.5b
± ± ± ± ± ± ± ±
0.4a 0.7a 0.3a 1.2a 0.5a 0.6a 0.8a 1.6a
ND = Not determined. † = Each value represents the mean ± standard error of each treatment. Table A3 Seed germination (three experiments) of two near-isogenic lines of bell pepper Marengo; one infected with BPEV (BPEV+) and the other free of BPEV (BPEV-). Values represent the percent seed germination throughout a period of 12 days. Day
Percent germination Experiment 1
3 4 5 6 7 8 9 10 11 12
Experiment 2
Experiment 3
BPEV-
BPEV+
BPEV-
BPEV+
BPEV-
BPEV+
10.0 80.0 86.7 86.7 86.7 90.0 90.0 90.0 90.0 90.0
6.7 30.0 40.0 46.7 46.7 56.7 66.7 70.0 70.0 70.0
0.0 73.3 86.7 93.3 93.3 93.3 93.3 93.3 93.3 93.3
23.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3 53.3
0.0 3.3 43.3 60.0 66.7 70.0 80.0 83.3 86.7 93.0
0.0 3.3 23.3 36.7 53.3 60.0 70.0 73.3 73.3 76.7
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Appendix B. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.02.043.
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