Plant material

A sampling of papaya plants and weeds was conducted in the state of Colima, Mexico, from March to September 2014, in three plantations at La Cumbre and Los Asmoles, localities from the municipality of Colima, and Camino to Chanchopa from the municipality of Tecomán. Leaves of various weed species showing overall viral symptoms (leaf malformation, chlorosis, mosaic and corrugated leaves) were sampled. Leaves of different papaya plants with PRSV characteristic symptoms were also collected at the same plantations. To identify the gender and species of the collected weeds, dichotomous keys were used according to ^{Fryxell, (1988)}.

Serological detection of PRSV

The serological detection of PRSV in all leaf samples was performed using DAS-ELISA and the PhatoScreen^® (Agdia) kit. The samples were macerated in an extraction shock-absorber and then 100 µL of raw extract were taken and placed in ELISA plates along with the positive and negative controls provided in the kit. The incubations, the enzyme-substrate conjugate, as well as the corresponding washings were conducted following the manufacturer’s instructions, this is, measuring wells at 405 nm. The absorbance value three times higher than that of the absorbance of the negative control was taken as a positive value. Each analysis was carried out in triplicate. Based on those results, a descriptive analysis was performed to determine the percentage of the samples positive to PRSV.

Extraction and purification of RNA

To extract PRSV viral RNA, 0.5 g of leaves were weighed and powdered with liquid nitrogen. Fifty mg of powder were immediately dissolved in 500 μL de TRIZOL^® (LifeTechnologies™). The mixture was homogenized and incubated for 5 min at room temperature. It was centrifuged at 12,000 rpm for 10 min at 4 °C, and the aqueous phase was transferred to a new tube adding 100 µL of chloroform; the mixture was vigorously agitated and incubated at room temperature for 3 min. The mixture was centrifuged at 12,000 rpm for 15 min at 4 °C. To the aqueous phase recovered, 250 µL of cold isopropyl alcohol were added, and then centrifuged at 12,000 rpm for 10 min at 4 °C. The tablet obtained in the previous process was washed in 500 µL of 75% ethanol, dried at room temperature and re-suspended in RNA-free water treated with diethyl dicarbonate. The RNA concentration and purity were verified in a NanoDrop spectrophotometer with an absorbance ratio of 260 and 280 nm, while the RNA integrity was verified in 1% agarose gel electrophoresis with 10 mM sodium borate buffer stained with ethidium bromide.

Detection of Potyvirus through reverse transcription polymerase chain reaction (RT-PCR)

The RNA reverse transcription (RT) was performed with the kit “GoScript™ Reverse Transcription System” (Promega A5001. Madison, WI. USA). The RNA was denatured at 70 °C for 10 min while the RT reaction mixture was prepared; the mixture contained 2 µL of ribonuclease inhibitor (Recombinant RNasin), 8 U of AMV reverse transcriptase (0.32 µL), 0.25 µg of random oligonucleotides (0.5 µL) and 5 µL of total RNA (5 ng). The mixture was incubated at room temperature for 10 min and then at 48 °C for 45 min. Immediately afterward it was incubated at 94 °C for 2 min, and finally at 5 °C. The DNAc was amplified using PCR and universal oligonucleotides for Potyvirus as described by ^{Zheng et al. (2010)}. The last reaction mixture for PCR was prepared in a 25 µL volume containing 17.5 µL of MQ water, 2.5 µL of 10X polymerase buffer, 1 µL of MgCl₂ 50 mM, 0.5 µL of dNTPs 10 mM, 1 µL of each of the oligonucleotides NIb2F (GTI TGY GTI GAY GAY TTY AAY AA) and NIb3R (TCI ACI ACI GTI GAI GGY TGN CC), 0.5 µL of Taq DNA polymerase (5U/µL) (Invitrogen) and 1 µL of DNAc. The reaction mixture was incubated in a thermocycler under the following conditions: denaturation at 95 °C for 3 min, followed by 35 denaturation cycles at 95 °C for 45 sec, alignment at 45 °C for 45 sec, extension at 72 °C for 45 sec, and a final extension at 72 °C for 5 min. When the experiment ended, the mixture was kept at 4 °C. 12.5 µL of the PCR product was tested by 1% agarose gel electrophoresis using SB (sodium borate, 10 mM) at 100 V. Finally, the amplification products were visualized using a transilluminator with UV light.

Cloning of PCR products

The PCR products were purified using the “PCR Clean-up” (Lamda Biotech) kit, but those showing more than one amplified band were purified using agarose gel and the “Rapid Gel Extraction System” kit (Marligen). Later on, the purified fragments were cloned in the commercial vector “pGEM-T Easy” (Promega) and inserted into competent E. coli cells JM109 (Promega), according to the methodology of ^{Riley et al. (2008)}.

Secuencing and secuencing analysis

The extraction of plasmidic DNA was performed using the “Pure Link^® Quick Plasmid Miniprep” (Invitrogen) kit; the plasmid was then sequenced in a ABI PRISM 310 Genetic Analyzer by the termination method using Big Dye from Applied Biosystems. The bioinformatics analysis was performed using CLC Genomics Workbench version 4. The similarity of the obtained sequences was compared with the sequences reported for PRSV in the NCBI database using the “Basic Local Alignment Search Tool” (BLAST) to determine the origin of the isolates.

Identifying weeds and alternative hosts for PRSV

The genus of the grass-type weed was identified as Eragrostis spp. Another group of weeds were identified within the Herissantia genus. In this research, all the weed species that gave positive for PRSV were identified according to their morphology and the use of descriptors, according to ^{Fryxell (1988)}. Papaya plantations showed different phenology and cultural labor to control weeds (Figure 1). The papaya crops at La Cumbre and Chanchopa were being harvested at 13 and 12 months of age, respectively. At La Cumbre there were several papaya plantations that were different ages; the weed samples were collected from a one-month old crop that was at the vegetative development stage. Weeds were managed adequately at the plantations at La Cumbre and Chanchopa, while at the Los Asmoles site there was no weed management; as a result, a great diversity and quantity of weed species sprang up there. This condition represents a source of infection for phytopathogens, because when crops are abandoned, or there are no weed management practices, mixed viral infections become increasingly frequent (^{Bermúdez-Guzmán et al., 2016}). In some cases, weeds are interspersed with plants in the form of tramp crops. However, it is very important to ensure that such weeds are not virus hosts that attract possible vectors like aphids that contribute to the rapid spread of the virus (^{Kalleshwaraswamy and Kumar, 2008}). Overall, the sampled papaya plants showed mosaic, leaf deformation, oily stains in the stem and ring-like spots in fruit, all of them characteristic symptoms of PRSV (Figure 1). The symptoms observed in the field were similar to those reported in papaya plants in several states of Mexico, as well as in Hawaii, Florida, Taiwan and Thailand, to name a few (^{Noa-Carranza et al., 2006}; ^{Tripathi et al., 2008}).

Figure 1 A) Symptoms of PRSV in a papaya plantation where there are abundant interspersed weeds; B) Plantation with full control of weeds; C) Distorted leaves of papaya with chlorosis and mosaic symptoms; D) Fruit with charac-teristic rings caused by PRSV. 

^{Martins et al. (2016)} recently collected in Brazil aphid vectors present in 22 weed species in papaya plantations, most of them belonging to the Euphorbiaceae, Commelinaceae, Brassicaceae and Malvaceae families. They also identified one Solanaceae species: Solanum americanum. ^{Chin et al. (2007)} reported that Momordica charantia is a PRSV-P carrier; however, ^{Spadotti et al. (2013)}, after evaluating it through mechanical inoculation and aphid vectors, stated that Momordica charantia is not susceptible to any of the PRSV strains (P and W). In this research, we found a M. charantia specimen from Los Asmoles that appeared healthy and gave negative DAS-ELISA and RT-PCR test results. However, in the future it would be useful to analyze a greater number of samples of this species to determine if M. charantia can act as a reservoir of the virus.

The expression of the multiple viral symptoms in weeds associated with papaya crop in three localities in Colima is shown in Figure 2. The weedsamples that developed symptoms account for 72% of the total sampling, except for Eragrosis spp. and Herissantia spp. Mosaics were observed in plants identified as abutilon (Abutilon abutilastrum), spurred anoda (Anoda cristata) and buffalobur nightshade (Solanum rostratum). The latter species also developed leaf distortion and chlorosis, the same as summer squash (Cucurbita pepo), whose leaf deformation was quite severe. On the other hand, an asymptomatic specimen of Momordica charantia was found in Los Asmoles, which has been reported as host for several viruses; C. pepo, S. rostratum and A. cristata are susceptible to 62, 8 and 1 viruses, respectively; for the Abutilon genus, five viral phytopathogens have been reported (^{Brunt et al., 1996}. When weeds serve as a reservoir of a large number of viruses, this often means that the symptoms observed are the result of the synergic expression of a mixture of viral infections (^{Zhang et al., 2001}; ^{Hii et al., 2002}; ^{Gastélum et al., 2007}; ^{Bermúdez-Guzmán et al., 2016}).

Figure 2 Diverse weeds in three papaya plantations with viral symptoms. A) A. abutilastrum with mosaics in leaves. B) C. pepo with severe deformation of leaves. C) S. rostratum with mosaics and chlorosis. D) A. cristata with mosaic in leaves. E) Momordica charantia asymptomatic. 

Detection of PRSV

Data on the detection of PRSV through DAS-ELISA and RT-PCR in plants of C. papaya and weeds associated with the crop from three Colima sites are shown in Table 1. From the 130 collected samples, 57 collected at La Cumbre were analyzed using DAS-ELISA and gave positive to PRSV in C. papaya and C. pepo. This result may be explained by the fact that the analyzed samples did not reach the minimum viral concentration since, according to reports, the DAS-ELISA technique does not detect virus in plants if its concentration level is less than 0.1 ng of virus per mL (^{Salazar, 1996}). In this study, up to 50% of positive samples was reported by DAS-ELISA, a percentage highly significant and different from the percentage obtained by ^{Hernández de la Cruz et al. (2007)}, who detected 2.43% of PVYNTN in potato tubers using the DAS-ELISA technique.

The BLASTn analysis of the 13 sequences obtained in the present research that correspond to the NIb region of PRSV allowed for the first time the identification of A. abutilastrum, A. cristata and S. rostratum as new natural host species for PRSV. Isolated sequences of C. papaya and C. pepo were also included, but they have been extensively reported as hosts for PRSV (^{Noa-Carranza et al., 2006}; ^{Sreenivasulu and Sai-Gopal, 2010}; ^{Goyal et al., 2012}; ^{Mansilla et al., 2013}). Phylogenetics (Figure 3) was useful to identify three similar sequence groups. A clear grouping is seen according to the plantation zone; for example, it was observed that sample 11 and 12 corresponding to C. pepo and A. abutilastrum, respectively, was the same as the one found in C. papaya corresponding to sequences 9 and 8 from Los Asmoles. This finding clearly confirms that the weed transmits the same virus to papaya plants. Another major finding was the diverse sequences identified for the Colima zone which are markedly different from other isolates, even from the one reported for Veracruz (AY231130) with 92% homology. Such diversity of PRSV isolates has been observed in regions of China due to the high mutation rate of the virus (^{Zhao et al., 2016}), which makes difficult to implement control strategies. As expected, other Potyvirus such as: Japanese yam mosaic potyvirus (JYMV), Potato virus Y (PVY), Bean yellow mosaic virus (BYMV) and Tobacco vein banding mosaic virus (TVBMV) were located farther away in the dendrogram with 70 to 73% identity in the sequence.

Figure 3 Phylogeny based on the similarity of the sequences of the NIb region of the Papaya ringspot virus (PRSV) genome from isolates collected in Colima, Mexico (1-13) and compared with isolates from Brazil, Hawaii, India, Taiwan, Thailand, and other Potivirus: Japanese yam mosaic potyvirus (JYMV), Potato virus Y (PVY), Bean yellow mosaic virus (BYMV), Tobacco vein banding mosaic virus (TVBMV). 

28.15% of papaya plants analyzed in this research developed viral symptoms but they gave negative to PRSV when tested using RT-PCR. This may be due to the fact that C. papaya is susceptible to more than eight phytopathogen viruses such as Papaya apical necrosis virus (PANV), Papaya lethal yellowing virus (PLYV), Papaya Mosaic Virus (PMV), Papaya leaf distortion mosaic virus (PLDMV), among others (^{Lastra and Quintero, 1981}; ^{Brunt et al., 1996}), so it may be that the viral symptom may be caused by some of those viruses.