The wild type of Momordica charantia is not infected by potyviruses that cause disease in papaya and cucurbit crops

The wild type of Momordica charantia is not infected by potyviruses that cause disease in papaya and cucurbit crops

David Marques de Almeida Spadotti^I; José Edivaldo Buriolla^I; Jorge Alberto Marques Rezende^I; Vinicius Castro Souza^II

^IDepartamento de Fitopatologia e Nematologia, ESALQ, Universidade de São Paulo, 13418-900, Piracicaba, SP, Brazil;

^IIDepartamento de Ciências Biológicas, ESALQ, Universidade de São Paulo, 13418-900, Piracicaba, SP, Brazil

^{Correspondence}

ABSTRACT

In the present work, the susceptibility of wild and domesticated plants of Momordica charantia to viruses from the genus Potyvirus that cause diseases in papaya (Carica papaya) and cucurbit crops was evaluated. The plants were subjected to experimental and natural infection with Papaya ringspot virus types P and W (PRSV-P and PRSV-W) and Zucchini yellow mosaic virus (ZYMV). None of the potyviruses infected the wild type of M. charantia through mechanical or aphid inoculation or under field exposition, whereas the domesticated type was only infected by isolates of ZYMV. In addition, both wild and domesticated types of M. charantia were not infected in natural conditions by an isolate of Zucchini lethal chlorosis virus, genus Tospovirus, transmitted by Frankliniella zucchini. These data clearly indicated that this wild type of M. charantia does not seem to have a role in the epidemiology of the diseases caused by these three potyviruses in Brazil.

Key words:Carica papaya, Cucurbita pepo, aphid transmission, disease management, potyvirus

Papaya ringspot virus, types P and W (PRSV-P; PRSV-W) and Zucchini yellow mosaic virus (ZYMV), family Potyviridae, genus Potyvirus, cause significant diseases in economically important crops around the world. PRSV-P causes the ringspot disease of papaya (Carica papaya L.), which is considered a limiting factor for papaya production everywhere this tropical species is grown (Gonsalves, 1998). In addition to papaya, PRSV-P can also experimentally infect wild species of Vasconcellea, which were traditionally included in the genus Carica (Conover, 1964; Torres & Giacometti, 1966; Cook & Milbrath, 1971), and some cucurbit species/varieties show variable susceptibility according to the virus isolate (Capoor & Varma, 1958; Sánchez de Luque & Martínez, 1977; Sureka et al., 1977; Yeh et al., 1984). Under natural conditions PRSV-P is not important for cucurbit crops. Isolates of PRSV-W and ZYMV cause the common and yellow mosaic diseases, respectively, in many cucurbit species, dramatically reducing the yield, particularly for the most susceptible species/varieties, including zucchini squash (Cucurbita pepo L) (Rezende & Pacheco, 1998; Lecoq et al., 2009). All the three potyviruses are transmitted by several species of aphids in a non-persistent manner (Purcifull et al., 1984; Desbiez & Lecoq, 1997).

The control of these diseases is very difficult throughout the world. Except for the resistant transgenic papaya hybrids commercially grown in Hawaii (Tripathi et al., 2008) and the interspecific hybrid developed by Siar et al. (2011), all varieties of papaya are susceptible to infection by PRSV-P. For more than 25 years, management of papaya ringspot disease in Brazil has been performed through the adoption of several cultural practices that minimise the problems associated with PRSV-P in the commercial orchards. These practices include the following: the use of virus-free seedlings for new plantings; the eradication of old and abandoned orchards before starting new crops; the avoidance of cucurbit plants, which may harbour the virus within the proximity of the orchard; and, in particular, the roguing of diseased plants by means of systematic inspections of the crops by well-trained personnel (Ventura et al., 2004). Although growers are recommended to avoid the planting of cucurbit plants within or near the orchard, recent studies conducted in Brazil by Mansilla et al. (2013) revealed that these species do not have a significant importance on the epidemiology of papaya ringspot disease. The control of PRSV-W and ZYMV relies mainly on the use of resistant/tolerant cucurbit varieties whenever available. In addition, growers are also recommended to eradicate old and abandoned crops before starting new ones and avoid the planting of any other cucurbit plants (wild and/or cultivated) near the crops to reduce the sources of virus inocula (Zitter et al., 1996). Among the wild cucurbit plants that should not be allowed within the vicinity of papaya and cucurbit crops is Momordica charantia L. (bitter gourd, balsam pear), a plant that has shown contradictory results of susceptibility to these potyviruses (Adsuar, 1950, Capoor & Varma, 1958; Conover, 1964; Sánchez de Luque & Martínez, 1977; Pearson & Liyanage, 1997; Chin et al., 2007; Silveira et al., 2009). Therefore, the purpose of this work was to investigate the susceptibility of wild and domesticated types of M. charantia to experimental and natural infections with PRSV-P, PRSV-W, and ZYMV. In addition, natural infection with the tospovirus Zucchini lethal chlorosis virus (ZLCV) was also evaluated.

Wild and domesticated types of M. charantia, also known as 'nigauri' or 'goya', C. pepo cv. Caserta and C. papaya cv. Golden were used for the experiments. The wild and domesticated types of M. charantia are mainly differentiated by the size of their fruits and seeds. The fruits are spindle-shaped, measuring approximately 5 cm long in the wild type population and up to 25 cm long in the domesticated type. The seed size is proportional to the fruit size. The leaves of domesticated type are also larger than those of the wild type population. Seeds of both types of M. charantia were obtained from plants naturally grown in the campus Luiz de Queiroz, University of São Paulo, Piracicaba, São Paulo state, Brazil.

ZYMV isolates were obtained in the states of São Paulo (ZYMV-RI), Rio Grande do Sul (ZYMV-RS), Mato Grosso (ZYMV-MT), and Brasilia, DF (ZYMV-Fe, ZYMV-DF). The PRSV-P isolates were from the states of São Paulo (PRSV-P CF), Pernambuco (PRSV-P PE), and Santa Catarina (PRSV-P SC). The PRSV-W isolates were from São Paulo (PRSV-W C) and Brasilia, DF (PRSV-W DF). The experimental transmission tests were performed through mechanical and aphid inoculations. For the mechanical inoculation, infected leaves were ground in 0.02 M phosphate buffer, pH 7.0, containing 0.02 M sodium sulfite, diluted 1:10 (w/v). The inoculum was rubbed on leaves dusted with carborundum. The vector transmission tests were performed using virus-free Myzus persicae Sulzer reared on sesame (Sesamum indicum L.) and wild radish (Raphanus raphanistrum L). Viruliferous aphids were starved for 60 min and then transferred to the source of inoculum for an acquisition access period of 30 min. Afterward, the aphids were transferred in groups of 15 to each test plant. The inoculation access period was 2 h after which the aphids were eliminated with insecticide. For each transmission test, two plants of C. pepo were used as control for the infectivity of PRSV-W and ZYMV isolates, whereas two plants of C. papaya were used as control for PRSV-P isolates.

Virus infections in the inoculated plants were confirmed by symptomatology, RT-PCR, and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). Back inoculation to C. pepo for detection of PRSV-W and ZYMV and to C. papaya for detection of PRSV-P was done whenever the other assays did not yield conclusive results. Total RNA was extracted from the plant tissues according to Bertheau et al. (1998) and one-step RT-PCR was performed using the PCR Master kit reagents (Fermentas) according to the manufacturer's instructions. The sense ZY2 and antisense ZY3 (Thomson et al., 1995) specific primers, whose amplicon has 1186 base pairs, were used to detect isolates of ZYMV. The PV1 antisense (Gibbs & Mackenzie, 1997) and sense primer designed for the WCIEN motif of the CP gene of potyviruses (Mota et al., 2004), which amplify a fragment of 800 base pairs, were used to detect the PRSV strains. PTA-ELISA (Mowat & Dawson, 1987) was performed using polyclonal antisera raised against ZYMV, PRSV-P, and PRSV-W. Antisera against these potyviruses were from the collection of the Laboratory of Plant Virology, ESALQ/USP, Piracicaba, SP. Primary antisera were diluted 1:1000, whereas secondary antiserum (SIGMA A-8025) was diluted 1:32000. The appropriate positive and negative controls were included in duplicate wells for each assay. The absorbance values (405 nm) were measured at 30 and 60 min after the substrate was added. The samples were considered positive when the average absorbance value was at least three times higher than the average absorbance value of the negative control. Detection of ZLCV was done only by PTA-ELISA using polyclonal antiserum provided by Dr. Alice K. Inoue-Nagata, Embrapa, CNPH, Brasília, DF, following the same procedure described above.

From November 2010 to March 2012, four experiments were conducted in a greenhouse to test the susceptibility of wild and domesticated types of M. charantia to different isolates of ZYMV, PRSV-W and PRSV-P by means of mechanical and aphid inoculations. The number of inoculated wild and domesticated M. charantia plants differed according to the virus isolate (Table 1). Zucchini squash plants were included in all of the tests as a control for the infectivity of ZYMV and PRSV-W, whereas papaya plants were used as the control for the infectivity of PRSV-P. The plants were maintained in the greenhouse, and 15 days after inoculation, samples from the inoculated leaves were tested by PTA-ELISA, RT-PCR and biological assays for virus detection. The biological recovery test consisted of the mechanical inoculation of zucchini squash or papaya plants with extracts from the M. charantia plants inoculated with the different potyviruses. Thirty days later, a systemic infection on the upper leaves of wild and domesticated types was evaluated based on the expression of symptoms, and virus detection was performed as described above.

During March 2011 and April 2012, nine plants of each type of M. charantia were evaluated for natural infection with ZYMV, PRSV-W, and PRSV-P, and ZLCV. Control plants of C. pepo cv. Caserta and C. papaya were planted near M. charantia types to serve as the controls for the infection with these viruses. The C. pepo plants were renewed three times during that period, with a total of 30 plants. Evaluations were also based on the expression of symptoms, PTA-ELISA, and RT-PCR as described before. Samples from symptomatic C. pepo were tested in groups of five plants.

Mechanical and aphid transmission tests performed with all three potyviruses under greenhouse conditions revealed that only the ZYMV isolates were able to infect the domesticated type of M. charantia. The ZYMV-MT isolate was only detected on the inoculated leaf of one plant, but did not move systemically. The ZYMV-RS isolate was able to systemically infect two out of four plants when inoculated mechanically and all four plants when the inoculation was performed by aphids. ZYMV-RI and ZYMV-DF, when inoculated by aphids were detected by PTA-ELISA and RT-PCR on the upper leaves of three and one out of four plants, respectively. None of the wild type of M. charantia plants inoculated (mechanically or by aphids) with the three potyviruses was infected (Table 1). Although some plants inoculated with isolates ZYMV-RI, PRSV-W-C, and PRSV-W-DF tested positive by PTA-ELISA, RT-PCR and biological recovery assays did not confirm systemic infections. All C. pepo and C. papaya plants used for the control of the infectivity of the virus isolates were infected (data not shown). When exposed for 14 months to natural infection in the field, only one domesticated type plant was infected with ZYMV. The zucchini squash plants used as control were infected with PRSV-W and/or, ZYMV and/or, ZLCV, whereas the papaya plants were infected with PRSV-P. None of the M. charantia plants exposed in the field were infected with ZLCV, which was detected in plants used as the control (Table 2).

As initially mentioned, reports on the susceptibility of M. charantia to PRSV-P, PRSV-W and ZYMV are inconsistent. In Jamaica, Chin et al. (2007) were able to recover PRSV-P from symptomatic M. charantia plants present in papaya orchards. Chin et al. (2007) also found that high rates of virus transmission by A. gossypii were obtained in tests from M. charantia to papaya (77-83%), papaya to M. charantia (90-93%), and M. charantia to M. charantia (60-70%). In Florida, USA, Adlerz (1972) identified M. charantia as source of Watermelon mosaic virus 1 (WMV-1), presently identified as PRSV-W, but Adlerz et al. (1983) were not able to experimentally infect this species with ZYMV and WMV-2, presently identified as WMV. On Reunion Island, Africa, however, ZYMV was reported as infecting systemically M. charantia (Desbiez & Lecoq, 1997). A survey of cucurbit viruses in Western Samoa found M. charantia infected with ZYMV but not with PRSV-W (Pearson & Liyanage, 1997). In contrast, M. charantia was not infected by mechanical inoculation with PRSV-P, as reported by Adsuar (1950) in Puerto Rico, Conover (1964) in Florida, USA, Pinto (1972) in Venezuela, and Sánchez de Luque & Martínez (1977) in Colombia.

In the present work, the wild-type of M. charantia was not infected with any of the tested potyviruses through mechanical and aphid inoculations or under field exposure for 14 months, whereas the domesticated type was only infected with isolates of ZYMV under experimental and natural conditions. These results are in accordance with previous reports on the reaction of wild type of M. charantia to the same potyviruses in Brazil. The absence of natural infection of this cucurbit species with PRSV-P was first reported by Barbosa & Paguio (1982) in a survey in the state of Pernambuco. In an assessment in the states of Pernambuco and Bahia for the natural infection of M. charantia with PRSV-W, ZYMV, WMV, and Cucumber mosaic virus (CMV), Silveira et al. (2009) did not find any infected plants. Yuki et al. (2000) were not able to infect M. charantia experimentally with PRSV-W, ZYMV, WMV, and CMV through mechanical and M. persicae inoculations. Altogether, these data clearly indicate that wild type of M. charantia, widely distributed in Brazil, does not seem to have importance in the epidemiology of the cucurbit diseases caused by ZYMV and PRSV-W and papaya ringspot disease associated with PRSV-P in Brazil, as based on the high resistance to infection with all three potyviruses. The same is true for the domesticated type of M. charantia with regard to the diseases caused by strains of PRSV-P and PRSV-W. Recently, Mansilla et al. (2013) showed the relative importance of some cucurbit species in the epidemiology of papaya ringspot disease in Brazil. These authors recommended that the control of the disease through roguing should focus mainly on diseased papaya trees, a method that has been practiced successfully in the country for many years, and on those cucurbits known to be particularly susceptible to natural infection with PRSV-P.

Received 15 November 2012

Accepted 5 May 2013

TPP 2012-0128 -

Section Editor: Alice K. Inoue-Nagata

• Adlerz WC (1972) Momordica charantia as a source of watermelon mosaic virus 1 for cucurbit crops in Palm Beach County, Florida. Plant Disease Reporter 56:563-564.
• Adlerz WC, Purcifull DE, Simone GW, Hiebert E (1983) Zucchini yellow mosaic virus: A pathogen of squash and other cucurbits in Florida. Proceedings of the Florida State for Horticultural Society 96:72-74.
• Adsuar J (1950) Studies on virus diseases of papaya (Carica papaya) in Puerto Rico: IV - Preliminary studies on the host range of papaya mosaic. University of Puerto Rico, Agricultural Experiment Station, Río Piedras. Tech. Paper 5:1-5.
• Barbosa FR, Paguio OR (1982) Papaya ringspot virus: Incidence and yield loss in papaya. Fitopatologia Brasileira 7:365-373.
• Bertheau Y, Fréchon N, Toth IK, Hyman, LJ (1998) DNA amplification by polymerase chain reaction (PCR). In: Pérombelon MCM, Van Der Wolff JM (Eds.) Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica on Potatoes: A Laboratory Manual. Dundee Scotland. Scottish Crop Research Institute. pp. 38-48.
• Capoor SP, Varma PM (1958) A mosaic disease of papaya in Bombay. Indian Journal of Agricultural Science 27:225-234.
• Chin M, Ahmad MH, Tennant P (2007) Momordica charantia is a weed host reservoir for Papaya ringspot virus type P in Jamaica. Plant Disease 91:1518.
• Conover RA (1964) Distortion ringspot, a severe virus disease of papaya in Florida. Proceedings of the Florida State for Horticultural Society 77:440-444.
• Cook AA, Milbrath GM (1971) Virus diseases of papaya on Oahu (Hawaii) and identification of additional diagnostic host plants. Plant Disease Reporter 55:785-788.
• Desbiez C, Lecoq H (1997) Zucchini yellow mosaic virus. Plant Pathology 46:809-829.
• Gibbs A, Mackenzie A (1997) A primer pair for amplifying part of the genome of all potyvirids by RT-PCR. Journal of Virological Methods 63:9-16.
• Gonsalves D (1998) Control of Papaya ringspot virus: a case of study. Annual Review of Phytopathology 36:415-437.
• Lecoq H, Wipf-Scheibel C, Chandeysson C, Le Van A, Fabre F, Desbiez C (2009) Molecular epidemiology of Zucchini yellow mosaic virus in France: An historical overview. Virus Research 141:190-200.
• Mansilla PJ, Moreira AG, Mello APOA, Rezende JAM, Ventura JA, Yuki VA, Levatti FJ (2013) Importance of cucurbits in the epidemiology of Papaya ringspot virus - type P. Plant Pathology 62:571-577.
• Mowat WP, Dawson S (1987) Detection of plant viruses by ELISA using crude sap extracts and unfractionated antisera. Journal of Virological Methods 15:233-247.
• Mota LDC, Della Vechia MGS, Gioria R, Kitajima EW, Rezende JAM, Camargo LEA, Amorim L (2004) Pfaffia mosaic virus: A new potyvirus found infecting Pfaffia glomerata in Brazil. Plant Pathology 53:368-373.
• Pearson MN, Liyanage AD (1997) Records of cucurbit viruses infecting vegetable crops in Western Samoa. Australasian Plant Pathology 26:188-191.
• Pinto OL (1972) Identificacion de las virosis de la lechosa (Carica papaya L.) em Venezuela. Revista de la Faculdad de Agronomia 6:5-36.
• Purcifull DE, Edwardson JR, Hebert E, Gonsalves D (1984) Papaya ringspot virus. CMI/AAB Descriptions of Plant Viruses no. 292. Kew UK. Association of Applied Biologists.
• Rezende JAM, Pacheco DA (1998) Control of Papaya ringspot virus - type W in zucchini squash by cross-protection in Brazil. Plant Disease 82:171-175.
• Sánchez de Luque C, Martínez G (1977) Identificación de plantas hospedantes del virus de la mancha anular de la papaya. Fitopatología Colombiana 6:112-121.
• Siar SV, Beligan GA, Sajise, AJC, Villegas VN, Drew RA (2011) Papaya ringspot virus resistance in Carica papaya via introgression from Vasconcellea quercifolia Euphytica 181:159-168.
• Silveira LM, Queiroz MA, Lima JAA, Nascimento AKQ, Neto ISL (2009) Serological survey of virus in cucurbit species in the lower middle São Francisco river basin, Brazil. Tropical Plant Pathology 34:123-126.
• Sureka SK, Mathur K, Shukla DD (1977) Virus diseases of papaya (Carica papaya) in Udaipur. Indian Journal of Mycology & Plant Pathology 7:115-121.
• Thomson KG, Dietzgen RG, Gibbs AJ, Tang YC, Liesack W, Teakle DS, Stackebrandt E (1995) Identification of Zucchini yellow mosaic potyvirus by RT-PCR and analysis of sequence variability. Journal of Virological Methods 55:83-96.
• Torres MR, Giacometti DC (1966) Virosis de la papaya (Carica papaya L.) en el Valle del Cauca. Agricultura Tropical 22:27-38.
• Tripathi S, Suzuki JY, Ferreira SA, Gonsalves D (2008) Papaya ringspot virus - P: characteristics, pathogenicity, sequence variability and control. Molecular Plant Pathology 9:269-280.
• Ventura JA, Costa H, Tatagiba JS (2004) Papaya diseases and integrated control. In: Naqvi SAMH (Ed.) Diseases of Fruits and Vegetables: Diagnosis and Management. London UK. Kluver Academic Publishers. pp. 201-268.
• Yeh SD, Gonsalves D, Provvidenti R (1984) Comparative studies on host range and serology of papaya ringspot virus and watermelon mosaic virus I. Phytopathology 74:1081-1085.
• Yuki VA, Kuniyuki H, Rezende JAM, Pavan MA, Groppo GA, Kitajima EW (2000) O melão de São Caetano não deve ser hospedeiro de alguns vírus de importância para as cucurbitáceas. Fitopatologia Brasileira 25:451.
• Zitter TA, Hopkins DL, Thomas CE (1996) Compendium of Cucurbit Diseases. St. Paul MN, USA. APS Press.

Publication Dates

• Publication in this collection
  27 Aug 2013
• Date of issue
  Oct 2013

History

• Received
  15 Nov 2012
• Accepted
  05 May 2013