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IDENTIFICATION OF RESISTANCE TO SINGLE AND MIXED INFECTIONS OF PEPPER GOLDEN MOSAIC VIRUS (PepGMV) AND THE HUASTECO PEPPER VIRUS IN CHILI PEPPERS (Capsicum chinense Jacq.). J. L. Anaya-López1,2; Y. Godínez-Hernández1,2; C. I. Muñoz-Sánchez2; L. Guevara-Olvera2; R. G. Guevara-González2; R. F. Rivera-Bustamante3; M. M. González-Chavira1; I. Torres-Pacheco1¶ 1
Unidad de Biotecnología del Bajío, Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias, Campo Experimental Bajío. Carretera Celaya-San Miguel de Allende km 6.5, Apartado Postal 112, Celaya, Guanajuato, México. ( ¶Corresponding author). E-mail:
[email protected] 2 Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Avenida Tecnológico y Antonio García Cubas s/n, Col. FOVISSSTE, Apartado postal 57, Celaya, Guanajuato., México. 3 Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del IPN Unidad Irapuato, Carretera Irapuato-León km 9.6, Libramiento Norte, Apartado Postal 629, Irapuato, Guanajuato, México.
SUMMARY In this study, resistance to single and mixed infections to pepper golden mosaic virus (PepGMV) in Capsicum chinense Jacq. individuals were evaluated. Plant inoculation was carried out by biolistics and grafting. To 140 plants from seven accessions of C. chinense Jacq., inoculations were preformed with PepGMV. Of these plants, 99 were asymptomatic, although the virus was detected by ELISA and PCR techniques. Additionally, resistance to geminivirus (PepGMV and Pepper huasteco virus or PHV) mixed infections, was evaluated in these accessions. Thirty-one plants from the accessions with the best characteristics of resistance to single infections by PepGMV were evaluated, and 15 individuals displayed resistance in different degrees. Resistance to geminivirus mixed infections was grouped as: delayed symptoms, symptom remission and asymptomatic plants. DNA levels of both geminiviruses correlated positively with symptom severity. ADDITIONAL KEY WORDS: geminivirus, resistance, mixed infection, ELISA, PCR.
INTRODUCTION Losses in the chili pepper crop of Mexico between 20 and 100 % are due principally to diseases of viral etiology, among those of major importance are the geminiviruses (Godinez-Hernandez et al., 2001; Torres-Pacheco et al., 1996). In nature a single virus or mixes of these can be found affecting crops. Thus, mixed infections that involve geminiviruses in Mexico have been found in crops such as chili pepper and tomato, and even in weeds (Vera-Aguado et al., 1999). Geminiviruses are plant viruses with a “geminate” morphology under an electron microscope, monopartite or bipartite genomic structure, and are transmitted by insects of the order Homoptera (Hanley-Bowdoin et al., 1999; Lazarowitz, 1992). Today in Mexico, two geminiviruses are considered the main pathogens in chili pepper: Pepper huasteco virus (PHV) and Pepper golden mosaic virus (PepGMV), formerly known as Texan chili virus, Torres-
Received: August 18, 2001 Accepted: January 16, 2003
Pacheco et al., 1996). These two geminiviruses have been found in association, affecting chili crops in Mexico (VeraAguado et al., 1999). For this reason, it is necessary to identify sources of resistance to these viruses in chili pepper. Much effort has been made to identify natural sources of resistance to PHV in chili with good results (GodinezHernandez et al., 2001; Hernandez-Verdugo et al., 1998, 2001). Nevertheless, it is also necessary to identify sources of resistance to PepGMV alone or in mixed infections together with PHV. The objective if this study was to identify natural resistance to PepGMV, alone or mixed with PHV, in collections of Capsicum chinense, which exhibited good resistance to simple PHV geminivirus infections (GodinezHernandez et al., 2001). Our results demonstrate identification of several sources of resistance to simple PHV and mixed with PepGMV infections. Also, a positive correlation was found between the level of viral DNA and the severity of the symptoms in plants infected by mixed PepGMV-PHV.
Revista Chapingo Serie Horticultura 9(2): 225-234, 2003.
232 MATERIALS AND METHODS Plant material C. chinense Jacq. plants from seven collections conducted in the state of Yucatan, Mexico, were used in this study. The collections were previously denominated (TrujilloAguirre and Díaz-Plaza, 1995): UX-SMH-1, UX-SMH-5, UXSMH-18, US-SMH-22, UX-SMH-24, UX-SMH-26, AND UXSMH-55. As the experimental unit for this study, individual plants were considered and not the collections. In all of the experiments conducted, Capsicum annuum cv. Sonora Anaheim, which is highly susceptible to infection of PepGMV and PHV, was used as the control (Torres-Pacheco, 1997). The collections that were evaluated in this work are available in the National Institute of Research in Forestry, Agriculture and Livestock (INIFAP), Experimental Station Bajío, located on the Celaya-San Miguel de Allende Highway, Celaya, Guanajuato, Mexico. Inoculation of the viruses The plants were inoculated firstly by the biobalistic method, using a device for conduction of the microparticles (Model PDS 1000, Dupont). For biobalistic inoculation, 2.5 mg of DNA of each component (A and B) of the viruses PepGMV or PHV was used for every six plants, depending on whether the infection was simple or mixed, and 3 mg of tungsten microparticles (Sylvania No. 10) was deposited on the surface. Firing was done in a vacuum at a distance of 1.2 cm and a pressure of 56.4 kg⋅cm-2 (Garzon-Tiznado et al., 1993). Inoculation was done on an area of the apex of plants with four to six true leaves. Inoculated plants were kept in a greenhouse at a temperature between 24 and 32 °C (Gondinez-Hernandez et al., 2001). The plants that did not exhibit typical symptoms 30 days after inoculation (DPI) were inoculated by grafting with a slip of cv. Sonora Anaheim plants infected with PepGMV alone or PepGMV and PHV together. Each slip was previously verified with PCR (see below) for the presence of the viruses before carrying out the grafting. Evaluation of resistance and detection of geminivirus Plants inoculated by biobalistics and/or grafting were incubated for three months in the greenhouse. Samples of apical and basal leaves were collected 11, 21, 31, 45, 59, and 90 days after inoculation. Severity of the disease was evaluated qualitatively using a scale reported by TorresPacheco (1997). In some cases PepGMV was detected with ELISA, with a geminivirus detection set (Agdia Inc., Elkhart, IN). Both PepGMV and PHV were detected with PCR, which was performed with DNA extracted from leaf samples with the method described by Dellaporta et al. (1983). Detection of each virus by PCR was done with the following specific oligonucleotides for PepGMV: (260) 5’ TGG TGT AGG ACT CCA GCA GAG TC 3’, y (261) 5’ TAG GCC CAC ACC TTG GTG ACC AAG 3’. For the detection of PHV, the oligonucleotides were (240) 5’ GGC TTA TTT GTA ATA AGA GAG Identification of resistance...
GTG T 3’ Y (241) 5’ GAA TTA AAG GTA CAT GGA CCA CTT 3’. For PepGMV, an amplified band of the common region of the virus of 200 pb was expected, while for PHV the size of the band of the common region of the virus was 350 pb. The reactions of PCR were performed in a thermocycler (Parker Elmer) and contained the following components: 0.75 µl of each disoxynucleotide (2.5 mM), 2 ml of oligonucleotide (50 ng⋅µl-1), 0.5 ml of Taq polymerase (6 U⋅µl-1), 1 ml of plant DNA (100 ng⋅µl-1) in a reaction volume of 50 µl. The conditions for amplification by PCR were: for PepGMV, 1 min at 94 °C, 2 min at 55 °C, and 2 min at 72 °C. For PHV, 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C. Statistical analysis Analyses of variance and correlations between levels of viral DNA in different interactions of resistance and symptomatic severity were performed with the SAS Proc GLM software (SAS Institute, 1990). The data were transformed to the square root of x+1 (x=each value of densitometry) for optimization of the analyses.
RESULTS Phenotypic response of the collections of habanero chili peppers to simple infection by PepGMV The infectivity of PepGMV varied from 15 % up to 50 %. Also, severity was high in the plants that were susceptible to infection by PepGMV alone (Table 1). The plants that were susceptible to infection by PepGMV exhibited symptoms in the first 5 DPI; these symptoms appeared mainly in the apical zone, although after a lapse of approximately 25 DPI they could also be perfectly observed in the lower and middle zones of the plants. Moreover, the presence of the virus was confirmed with PCR from DNA samples from apical and basal leaves of the susceptible plants (Figure 1). In addition to the presence of plants susceptible to PepGMV, 99 individuals showing no symptoms were detected; these were shown to have the virus (data not shown) with an ELISA test. These plants were considered tolerant to infection by PepGMV. Moreover, in some of these tolerant individuals the symptoms appeared nearly 70 DPI.
TABLE 1. Percentage of plants infected by the Pepper golden mosaic virus and severity of symptoms in each of the collections of Capsicum chinense Jacq. evaluated. Accession
Infected Plants (%)
Severity (range1-10)z
UX-SMH-1
20
UX- SMH-5
50
9 8
UX- SMH-18
20
7
UX- SMH-22
38
8
UX- SMH-24
38
7
UX- SMH-26
15
8
233 Relationship between levels of viral DNA and symptom severity.
UX- SMH-55
20
8
z
Values based on the scale reported by Torres-Pacheco (1997).
Figure 1.Detection by PCR of the presence of the Pepper golden mosaic virus (PepGMV) on apical and basal leaves of habanero chili peppers. Lanes 1-6 and 7-12 correspond to samples taken 25 and 44 days after infection, respectively. Lanes 1 to 3 and 7 to 9 are apical leaves, and lanes 4 to 6 and 10 to 12 are basal leaves. The amplification was carried out with the oligonucleotides 260 and 261. The arrow indicates the position at 350 pb.
Phenotypic response in habanero chili peppers to mixed infection by PepGMV and PHV Thirty-one individuals from different collections were inoculated with a 1:1 mixture of the two geminiviruses. Of the 31 individuals inoculated, 16 were susceptible, 12 had remission of symptoms, two were asyntomatic and one had a delay in the appearance of symptoms (Table 2). Also, during 90 DPI severity of the symptoms was evaluated in one representative individual of each interaction observed, whose kinetics of severity was high in the susceptible plant and nil in the asyntomatic plant during practically the entire period of incubation (Table 2). In the plant with delayed symptoms, symptoms did not appear until 45 DPI, while in the plant with symptom remission, the symptoms reached maximum 21 DPI and later diminished during the experiment (Table 2).
TABLE 2. Phenotypic response of collections of Capsicum chinense Jacq. to Pepper golden mosaic virus (PepGMV) and pepper huasteco virus (PHV), as well as severity v kinetics in one representative individual of each interaction found. Days After Inoculation 11
21
31
45
59
90
Susceptiblez
2
5
5
5
6
5
Delayedy symtoms
0
0
0
3
3
2
Remission of symtoms 0
4
2
2
2
1
w
0
0
0
0
0
Phenotype
x
Asyntomatic z
0
6 susceptible plants. y One plant with delayed symptoms. x 12 plants with symptom remission. w Two asymtomatic plants. v Severity, according to Torres-Pacheco (1997).
To determine whether there was correlation between symptom severity and the presence of viral DNA in plants inoculated with the mixture of PepGMV and PHV, these were analyzed in terms of their DNA levels for PepGMV and PHV in apical and basal leaves. For PepGMV, two statistical groups were found in both upper and lower leaves; group a= susceptible plants and group b= asymptomatic plants, with symptom remission and delay. The analysis of correlation indicated a positive relationship between symptom severity and levels of PepGMV in apical and basal leaves (r= 0.68 and 0.66, respectively; P≤0.01) (Table 3). For PHV, three statistical groups were identified for apical leaves: group a= susceptible plants, ab= symptom remission and delay in appearance of symptoms, and b= asymptomatic plants. For basal leaves, two statistical groups were identified: a= susceptible plants and b= symptom remission and delay and asymptomatic. As in the case of PepGMV, for PHV there was a positive correlation between symptom severity and levels of DNA in apical and basal leaves (r= 0.70 and 0.78, respectively; P≤0.01).
TABLE 3. Average of viral DNA levels and symptom severity in habanero chili plants, expressing four phenotypic responses to mixed infections by pepper golden mosaic virus (PepGMV) and pepper huasteco virus (PHV). Averagez of DNA Vira Levell Apical Leaves
Basal Leaves Totaly Severityx
Fenotype
PHV
PepGMV
PHV
PepGMV
28.02 aw
13.31 a
7.79 a
15.44 a
26.04 a
2.36 a
Delayed Syntoms
5.69 ab
2.93 b
2.17 b
4.70 b
7.38 b
1.62 b
Remission of Syntoms
5.91 ab
3.47 b
3.77 b
3.58 b
9.57 b
1.46 b
Asyntomatic Plants
1.90 b
1.33 b
2.31 b
2.64 b
3.69 b
1.0 c
Susceptible
z
Average of six observations. y Total DNA of PepGMV and PHV (sum of PepGMV+PHV). x Average of six observations. Values according to Torres-Pacheco (1997). w Values in each column followed by different letters indicate that they are different, according to the Tukey’s test at a P≤0.01.
Finally, considering the sum of DNA from PepGMV and PHV in both apical and basal leaves, two statistical groups were identified: a=susceptible plants and b=asymptomatic plants, as well as those with remission and symptom delay. The analysis of correlation for this latter case indicated a greater relationship between the levels of DNA and symptom severity (r=0.84; P≤0.01).
DISCUSSION The results obtained in this work demonstrated the presence of C. chinense Jacq. individuals with traits of reRevista Chapingo Serie Horticultura 9(2): 225-234, 2003.
234 sistance to simple infections by PepGMV or mixed infections by PepGMV and PHV (Tables 1 and 3). The number of phenotypic responses found in the analyzed plants was greater for mixed infections by PepGMV and PHV than for simple infections by PepGMV. These results could suggest that the more viral particles there are in the infection in plants, such as those analyzed in this study, the larger the number of phenotypic responses. Also, it was evident in all of the cases evaluated in mixed geminivirus infections that the severity of the symptoms was closely associated with the levels of viral DNA. These results coincide with the data found by other authors in the identification of sources of natural resistance in chili to PHV (Hernández-Verdugo et al., 1998, 2001). The individuals found to be resistant are excellent genetic resources with which studies on the genetics of resistance could be conducted and which could be included in future chili improvement programs.
F. 1993. Inoculation of peppers with infectious clones of a new geminivirus by a biolistic procedure. Phytopathology 53: 514-521. GODÍNEZ-HERNÁNDEZ, Y.; ANAYA-LÓPEZ, J. L.; DÍAZ-PLAZA, R.; GONZÁLEZ-CHAVIRA, M.; RIVERA-BUSTAMANTE, R. F.; TORRES-PACHECO, I.; GUEVARA-GONZÁLEZ, R. G. 2001. Characterization of resistance to pepper huasteco geminivirus in chili peppers (Capsicum chinense) from Yucatán, México. HortScience 36(1): 139-142. HANLEY-BOWDOIN, L.; SETTLAGE, S.B.; OROZCO, B.M.; NAGAR, S.; ROBERTSON, D. 1999. Geminiviruses: Models for plant replication, transcription, and cell regulation. Critical Review Plant Sciences 18: 71-106. HERNÁNDEZ-VERDUGO, S.; GUEVARA-GONZÁLEZ, R. G.; RIVERABUS-TAMANTE, R. F.; OYAMA, K. 1998. Los parientes silvestres del chile (Capsicum spp.) como recursos genéticos. Boletín Sociedad Botánica de México. 62: 171181. HERNÁNDEZ-VERDUGO, S.; GUEVARA-GONZÁLEZ, R. G.; RIVERABUSTAMANTE, R. F.; OYAMA, K. 2001. Screening wild plants of Capsicum annuum for resistance to pepper huasteco virus (PHV): Presence of viral DNA and differentiation among populations. Euphytica 122: 31-36.
CONCLUSIONS
LAZAROWITZ, S. G. 1992. Geminiviruses: Genome structure and gene function. Critical Review of Plant Sciences 11: 327-349.
Individual habanero chili plants were identified with traits of resistance to simple infections by PepGMV and mixed infection by PepGMV and PHV.
LOAIZA-FIGUEROA, F.; RITLAND, K.; LABORDE-CANSINO, J. A.; TANKSLEY, S.D. 1989. Patterns of genetic variation of the genus Capsicum (Solanaceae) in México. Plant Systematics and Evolution 165:159-188.
ACKNOWLEDGEMENTS
PICKERSGILL, B. 1984. Migration of chili peppers, Capsicum spp. in the Americas, pp. 105-123. In: Papers of the Peabody Museum of Archeology and Ethnology. D. Stone (ed.). Harvard University Press. Cambridge, Massachusetts, USA.
This study was funded by the Miguel Hidalgo Research System (SIHGO) (1998-0201018), CONCYTEG (98-09-01049), International Foundation for Science (AS/11325), and CONACYT (K325-P9702 and J31638-B). We are grateful to I. Torres-Pacheco and R.G. Guevara-Gonzalez, J.L. AnayaLópez, and Y. Godinez-Hernandez and, for the scholarships granted, to COSNET, SIHGO, and International Foundation for Science (AS/11325).
LITERATURE CITED ANÓNIMO.1990. SAS/STAT user´s guide. Version 6. 4 th ed. SAS Institute, Cary, NC, USA. DELLAPORTA, S. L; WOOD, J.; HICKS, J. B. 1983. A plant DNA minipreparation: version II. Plant Molecular Biology Reports 1: 19-21. GARZÓN-TIZNADO, J. A.; TORRES-PACHECO, I.; ASCENCIO-IBAÑEZ, J. T.; HERRERA-ESTRELLA, L.; RIVERA-BUSTAMANTE, R.
Identification of resistance...
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