Papaya (Carica papaya L.) is originally from Central America and southern Mexico, although its area of domestication has not been entirely defined (^{Fuentes and Santamaría, 2014}). Introduction of Maradol variety (var) in Mexico displaced and put at risk of extinction those native genotypes that lack adequate properties for the market, but displaying character diversity, including “cera”, “mamey” and “coco”, sold in local markets (^{Villanueva-Jiménez et al., 2015}); in the wild is still possible to find “papaya de monte” or “pajarito” (^{Romero, 2013}). Native papaya germplasm has not been evaluated in depth, especially their susceptibility to the papaya ring spot virus (PRSV-p), Potyvirus that reduces yield between 5 and 100%, hinders plant growth and drastically affects fruit size and quality. This virus is transmitted in a non-persistent manner by aphids (Hemiptera: Aphididae), being Myzus persicae, Aphis gossypii and A. nerii some of the most important (Villanueva-Jiménez et al., 2015; ^{Hernández-Castro et al., 2015}). The Papaya Network of the National System for Plant Genetic Resources for Food and Agriculture (^{SINAREFI, 2017}) rescues native Mexican germplasm in which material tolerant to PRSV-p may exist. Therefore, the objective posed was to evaluate the response of 13 accessions (Acc) of native papaya trees and the red Maradol var regarding the incidence and severity of the PRSV-p, inoculated with the vector insect A. nerii, and its relation to plant management during its collection.

Material used. The original Acc were collected by Dr. Catarino Ávila Reséndiz (†), ex coordinator of the Papaya Network of ^{SINAREFI (2017)}. Network´s South Southeast Orthodox Seed Conservation Center provided a limited number of seeds from these collections. Being recalcitrant, these seeds face germination problems after extended periods of conservation. We obtained a low number of individuals per accession, and therefore used the Acc that produced at least three plants. Table 1 presents the characteristics of each accession and the type of management in which they were collected. In addition, the var Red Maradol by Semillas del Caribe^® was used, since it is the most widely grown in Mexico. Seeds were soaked for 48 h, replacing water every 8 h, and in gibberellic acid 0.1% for 12 h; they were placed on a folded sterilized moist cloth (15 x 20 cm) at 35 °C and 80 % relative humidity. Those that germinated were grown in trays with a mixture (1:1) of Peat Moss^® and soil rich in organic matter. Seedlings were kept under a shade screen (75 %), irrigated and transplanted into pots (20 x 15 cm); after growing 8 to 10 true leaves, they were taken to a greenhouse at the Colegio de Postgraduados, Campus Veracruz. PRSV-p was obtained from papaya leaves gathered in a commercial orchard in Jamapa, Veracruz, Mexico. The presence of the pathogen was confirmed with RT-PCR, technique for the extraction of RNA from PRSV-p, with retrotranscription to generate complementary DNA and PCR with reverse transcriptase, performed using the Zymo Research® RNA extraction kit, as indicated by the manufacturer. RT-PCR was carried out in a Multigene and Labnet® thermocycler with the Promega RT-PCR System ®: PRSV-p CP region was amplified with 3F and 11R primers described by ^{Noa-Carrazana et al. (2006}). Aphids (A. nerii) from a virus-free culture were placed on leaves with PRSV-p for an acquisition period of 45 to 55 s (^{Gonsalves et al., 2010}; ^{Osorio-Acosta et al., 2010}). Ten aphids were moved to each of 13 healthy Maradol papaya plants, kept there for 2 h, and then eliminated manually. Twenty days later, the transmission efficiency was confirmed via RT-PCR in leaves samples from each plant. Ten native plants PRSV-p positive were used as a source of inoculum.

Greenhouse test. It was established with three plants per Acc or var, which were inoculated three times with infective aphids with the methodology by ^{Osorio-Acosta el al. (2010)}. 1st inoculation: 31/10/2017; 2nd 21/11/2017; 3rd 22/01/2018. A plant with infective aphids was confined 20 to 30 min in an entomological cage. Immediately afterwards, insects were removed.

Plants were kept at 24±3 °C in a greenhouse with an anti-aphid mesh for 63 days after inoculation. Weekly evaluations of PRSV-p incidence and severity of symptoms were carried out on leaves, stems and petioles (Table 2).

Incidence. The final incidence evaluated by symptoms was confirmed using the PRSV-p AGDIA^® kit. Tissue was taken from 42 plants of the 13 Acc and the Red Maradol var; it was placed in bags and taken cold to the Virology Laboratory (Plant Health Program, Colegio de Postgraduados). Each sample was processed in a grinding mill (Dayton Electric^®, Mod: 4Z522); the extract was gathered with 100 µl of the extraction buffer. Two replications were added per sample in a 96-well plate along with positive and negative controls. For the incubations, the enzyme-substrate mixture and the buffer washings were carried out following indications according to the manufacturer. Wells were read at 405 nm (Thermo Scientific^®, Multiskan FC). A≥2.0 nm value was considered positive for PRSV-p. The percentage of incidence (I%) of the disease was determined using the formula: 1% = (n ₁/N _j )*100, where: n ₁ = number of diseased plants per experimental plot during observation, and N _j = total number of plants evaluated per experimental plot. Visual symptoms of the PRSV-p were displayed in 64.29% of inoculated plants from the Acc and Maradol var, confirmed by DAS-ELISA. Number of diseased plants in the 13 Acc and Red Maradol var increased with time, with final incidences of the PRSV-p inoculated with A. nerii between 33.3 and 100%. In other words, all Acc were susceptible to PRSV-p under greenhouse conditions, with clear differences in the proportion of infected plants (Table 3). Acc 65, 94a, 205 were the first to show symptoms of PRSV-p in a plant, on the 3rd week after inoculation (WAI), while red Maradol began with symptoms in two plants; Acc 80a, 210b, 147a, 60 and 203 displayed a plant with symptoms on the 4th WAI, and Acc 188a and 169a displayed two plants with symptoms; Acc 64, 150 and 197a were the last to show the first symptoms of PRSV-p, both in two plants up to the 5th WAI.

The proportion of plants affected in red Maradol and in Acc 188a and 80 increased gradually until they reached a final incidence of 100 %, while in Acc 169, 65, 210b, 94a, 64a, 150 and 197a, incidence increased slowly until it reached 67 %. Acc 147a, 60, 203 and 205 maintained the same proportion of affected plants during the entire period (33%), and they were less affected by the virus. This might be due to a lower susceptibility to transmission by A. nerii on these accessions. Selecting Acc with transmission indices of less than 100% might guarantee the production of fruit in the orchard; this factor, although it is not the strongest, might provide tolerance to the virus. ^{Rodríguez et al. (2013}) also mentioned that all Acc of papaya plants evaluated in the field were sensitive to PRSV-p, with differences in time of infection between them. It is therefore likely that the differences in tolerance of the Acc to the PRSV-p have a genetic origin. ^{Gonsalves et al. (2010}) indicate that no natural resistance to this virus has been found in C. papaya, although tolerant lines have been found in the Philippines, Taiwan, Thailand, and Florida, USA, which, once are diseased, do not produce adequate fruits for the market, either. However, delaying the beginning of the infection allows for a production of quality fruits (^{Hernández-Castro et al., 2015}), which is why it is crucial to recognize the Acc with a long incubation period to help obtain the first healthy fruits. In the fields of Taiwan, the incidence of the disease in non-transgenic papaya lines appeared 29 days after transplanting, while transgenic lines acquired the disease five months later (^{Bau et al., 2004}).

Severity. The visual estimation of the severity value per plant was transformed into the mean percentage rank indicated in the description of symptoms (Table 2); this constituted the level of damage during the evaluation (X _ki ), which was used to calculate the mean severity index (IS) per accession, using the formula , where, in addition, N _ki = number of plants with the level of damage at the moment of evaluation; and N _j = total number of plants evaluated. All Acc and the var red Maradol displayed an increase in the proportion of symptoms evaluated in the nine weeks after inoculation (WAI), although there were differences between them. The highest PRSV-p severity was observed in the Red Maradol var (with a mean severity index of 4.8), followed by Acc 88, 169 and 188a (4.3), Acc 210b and 65 (3.3), Acc 150 (2.8) and 64a (2.5), and were therefore considered the most susceptible to the disease (Figure 1, Right). The high susceptibility of red Maradol to the PRSV-p is probably caused by not been selected for tolerance to the virus. From those Acc that presented the latest incidence, 205 and 203 displayed the lowest mean severity index (1.0) on week nine. Acc 60 and 147a displayed a mean severity index of 1.7, followed by 94a with a value of 2.0, and by 197a with 2.2 (Figure 1, Left). These genotypes have the potential of being used as a source of tolerance in genetic breeding programs for papaya. In Cuba, ^{Rodríguez et al. (2013}) evaluated four promising accessions for seven months under field conditions; in three of them they obtained a low mean final severity of the symptoms reported for stem, petiole, foliage and fruit: “Sapote de Pilón en Cuba” (2.3), “Amarilla de Duaba” (2.4) and “Amarilla de Nava” (2.5); the Acc “Tallo Morado de Nava” was the most effective in the trial (3.1), yet in none of the four was the final severity in fruit above 2.5; the authors considered these materials as tolerant and with potential to be used as base material in genetic breeding programs for papaya in that country. The level of tolerance by severity in this investigation is comparable with that of 10 transgenic lines that ^{Bau et al. (2003}) classified as resistant, since they kept symptoms moderate five WAI. Because the present test was carried out during nine WAI, it is possible for the severity to increase during the lifetime of the plants. However, a delay in the infection of at least 5 weeks at the beginning of the development of the plant may produce differences in yield of over 50%, even in susceptible materials such as Red Maradol (^{Hernández-Castro et al., 2010}). On the other hand, advanced intergenetic crosses (F6) of C. papaya (var Arka Surya) with Vasconcellea cauliflora carried out by ^{Yanthan et al. (2017}) produced seven progenies tolerant to PRSV-p under field conditions. In previous years, the group of ^{Gonsalves et al. (2010}) introduced the capside of the PRSV-p strain HA 5-1 to transgenic varieties of the Solo group and developed PRSV-p resistance. However, Mexico has still not allowed the production of transgenic varieties of papaya.

Figure 1 Dynamics of the mean severity index PRSV-p inoculated with A. nerii for nine weeks after inoculation. Left: six accessions with the lowest severity index. Right: Red Maradol variety and seven accessions with the highest severity index. 

Management of accessions during collections. IS data were analyzed by Kruskal and Wallis to separate the groups of Acc regarding the management observed during the collection of the material. The analysis showed differences (P=0.032) between the percentage of symptoms of the Acc related to management. Acc found in the wild with no management presented the lowest severity, in comparison with the commercial var Red Maradol, while Acc with backyard management and planted in orchards did not present different percentage of severity between them (Figure 2). This confirms that it is in wild genotypes where the largest gene bank may lie for tolerance to the infection of the virus, since these are genotypes that have not yet been selected by man. In this regard, ^{d’Eeckenbrugge et al. (2014}) mention that within Caricaceae there are 21 wild species of the genus Vasconcellea, which have a potential to resist the virus PRSV-p, and could be used to generate resistance in commercial populations. In the Philippines, ^{Alviar et al. (2012}) indicate that Sinta is a papaya hybrid with a parent line of the genus Cariflora that inherits to the hybrid a high PRSV-p tolerance, therefore infected plants do not reduce their yields. This highlights the importance of evaluating a var or accession regarding the infection of the virus as a strategy for the development of resistant varieties.

Despite its preliminary connotation, we suggest to replicate this investigation with more accessions, evaluating them until fruit set. Due to the limitations for the use of transgenics, Mexican efforts to maintain accession banks of wild papaya material should take advantage of the papaya complete genome availability to identify genetic markers and identify the tolerance or resistance genes in outstanding C. papaya materials (^{Porter et al. 2013}), so the advancement of development programs for resistance genotypes to this and other viruses can be systematized in a quick and efficient manner.

Figure 2 Distribution of severity in groups of accessions of papaya (Carica papaya L.), according to their management.