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Potyviridae as a major challenge to growing cucurbits in Puerto Rico

J.C.V. Rodrigues1, I. Quintero-Lopez1 and L. Wessel-Beaver2

1University of Puerto Rico, Agricultural Exp. Station, San Juan, PR 00926, USA 2Department of Crops & Agroenvironmental Sciences, University of Puerto Rico, PO Box

9000, Mayagüez, PR 00681-9000, USA

Keywords: Cucurbita, plant breeding, molecular variability, disease, potyvirus, ZYMV, PRSV.

AbstractPotyviridae causes substantial yield losses in watermelon, pumpkin,

zucchini, melon and squash, and are the most frequent family of viruses reported in cucurbits in Puerto Rico. Sampling conducted between 2006 and 2011 showed that PRSV (Papaya ringspot virus) and ZYMV (Zucchini yellow mosaic virus), both transmitted by aphids, are the most frequent virus species infecting various cucurbit species and the invasive species SqVYV (Squash Vein Yellow Virus), transmitted by whitefly, is severely affecting watermelons. The presence of wild cucurbit species provides a constant source of inoculum to crops and vice-versa. The most common wild cucurbit species in Puerto Rico, balsam pear (Momordica charantia L.) can be found everywhere on the island. West Indian gherkin (Cucumis anguria L.) and hedgehog gourd (Cucumis dipsaseus Ehrenb. ex Spach), are more frequently found at lower elevations. ELISA, immunostrip tests, RT-PCR and sequencing of a coat protein gene fragment were conducted to identify and characterize viruses affecting cucurbits. Mechanical transmission to Cucurbita moschata ‘Waltham’ was conducted and symptoms were evaluated in order to select virus isolates to challenge resistant lines of C. moschata. The different virus isolates induced a broad range of symptoms to inoculated C. moschata, indicating their high biological variability, which was confirmed by the genetic diversity of their sequences. More than one virus or strain usually infects cucurbits and their potential interaction is an additional challenge to cucurbit breeding programs.

INTRODUCTIONCucurbits provide important basic ingredients for the Caribbean diet and

pumpkins are the second most important vegetable crop in terms of revenue generated in Puerto Rico. Virus and severe virus vector outbreaks are a frequent and major cause of low yields and phytosanitary limitations to growing cucurbits Cucurbitaceae 2012, Proceedings of the Xth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (eds. Sari, Solmaz and Aras) Antalya (Turkey), October 15-18th, 2012

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in Puerto Rico. Continuous growing throughout the year and overlapping of susceptible crops makes Puerto Rico, a highly diverse island, an excellent and dynamic environment for plant viruses to evolve. Those aspects are also a major challenge for the development of control strategies. A survey was conducted to assess the types and prevalence of viruses infecting cucurbits. The virus isolates were mechanically transmitted, serological assays were conducted to identify the various viruses and RT-PCR followed by sequencing was done for the potyviruses, which were the most frequently encountered viruses in this survey.

MATERIALS AND METHODS A total of ninety-nine cucurbit plants (mainly pumpkin and watermelon) showing virus-like symptoms were sampled in 14 municipalities of Puerto Rico (Adjuntas, Barranquitas, Caguas, Coamo, Corozal, Isabela, Juana Diaz, Mayagüez, Morovis, Orocovis, Santa Isabel, Vega Alta, Villalba, Yauco). Assays were conducted at the Rio Piedras Experimental Station under greenhouse and laboratory conditions. ELISA and ‘immunostrips’ assays (Agdia Inc.) were conducted to identify the occurrence and predominance of the following virus tests: potyvirus, PRSV, ZYMV, CMV, and SqMV. Viral extracts from field samples were inoculated to Cucurbita moschata ‘Walthan’ using phosphate buffer and carborundum. Inoculated plants were observed for 21 days for development of symptoms. Infected tissues were freeze-dried and added to the UPR/AES viral collection for preservation.

A core portion of the coat protein subunit gene was amplified in a PCR reaction using the degenerate primers MJ1 (5’-ATGGTHTGGTGYATHGARAAYGG-3’) and MJ2 (5’-TGCTGCKGCYTTCATYTG-3’) (Grisoni et al. 2006). Bands were cut from the gel, cleaned, and sequenced with ABI Dye Terminator kit (2 times per sample) (Sambrook and Russell 2001). Sequences were edited and aligned using Codon Aligner Software. Sequences of known PRSV and ZYMV isolates deposited at GenBank (http://www.ncbi.nlm.nih.gov/genbank/) were used as control for taxonomical information. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Ney 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 5). The analysis involved 32 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 209 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011).

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RESULTS AND DISCUSSION One hundred percent of the samples were positive for at least one of the tested viruses. Serological tests showed more than 90 percent of the samples to be positive for potyvirus, with 45% positive for PRSV and 40% positive for ZYMV. Ten percent of the samples were from an unknown potyvirus. WMV, SqMV and CMV were reported in less than 2 percent of the samples. Occurrence of co-infection with 2 or more viruses was common.

Following mechanical transmission to Cucurbita moschata ‘Waltham’ a wide range of symptoms was observed 2 weeks after inoculation with the various isolates, indicating a high degree of variability. Some isolates produced noticeably stronger symptoms than others.

RNA was extracted from some of the field samples and inoculated plants. RT-PCR was conducted with potyvirus degenerate primers and the amplicons were sequenced to identify more specifically the potyviruses. Sample sequences were highly diverse (Table 1), confirming the biological variability observed in the greenhouse assays. A phylogenetic tree produced by Neighboring Join (NJ) Method with Bootstrap values showed a diversity of virus isolates (Fig. 1).

The results of this research have been used to select virus strains to challenge resistant pumpkin and watermelon lines in our breeding program at the University of Puerto Rico Virus diversity, frequent co-infection events of related viruses, and abundance of natural reservoirs (weed species) are major forces challenging the successful breeding of tropical cucurbit species for virus resistance.

ACKNOWLEDGMENTSTo USDA-NIFA (T-STAR, Tropical-Subtropical Research Program) grant

numbers 2006-34135-17545, 2008-34135-19505 and 2010-34135-21022) for financing and UPR Experimental extension personnel for advise during collecting trips. Thanks to the Sequencing and Genotyping Facility at UPR-RP, S.C.O.R.E Grant S06GM08102 & NIH I.N.B.R.E. grant P20RR16470.

Literature citedGrisoni M. Moles M, Farreyrol K, Rassaby L, Davis,R, Pearson M (2006) Identification of

potyviruses infecting vanilla by direct sequencing of a short RT-PCR amplicon. Plant Pathology 55(4): 523-529

Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425

Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791

Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using

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the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101: 11030-11035

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution (In Press)

Table 1. Summary of the BLAST results from potyvirus coat protein (CP) gene fragments amplified from cucurbit plant samples collected in Puerto Rico.

Closest match at GenBank

ID Host plantVirus

species AccessionMax score

Query coverage E value

Max. identity within species (%) pb

809 Melothia sp. PRSV AB127935.1 280 82% 4.00E-72 81-77 376

570 Cucumis sp. PRSV AB127935.1 248 94% 2.00E-62 81-77 282

804 C. pepo PRSV AB127935.1 315 99% 2.00E-82 83-81 305

862 C. pepo PRSV AB127935.1 257 78% 3.00E-65 86-78 284

866 Melothia sp. PRSV AB127935.1 320 96% 4.00E-84 83-77 315

829 C. pepo PRSV AB127935.1 352 99% 6.00E-94 86-78 308

836 Melothia sp. PRSV AB127935.1 307 99% 2.00E-80 87-82 254

841 Luffa sp. PRSV AB127935.1 289 99% 5.00E-75 85-83 262

1031 C. moschata PRSV AB127935.1 205 88% 2.00E-49 82-74 245

1F C. lanatus ZYMV JN561294.1 471 95% 9.00E-130 98-94 291

2F C. lanatus ZYMV JN561294.1 479 96% 6.00E-132 98-95 287

3i C. lanatus PRSV AB127935.1 385 99% 1.00E-103 87-80 314

3ii C. lanatus ZYMV JF317296.1 547 97% 2.00E-152 97-94 336

4i C. lanatus ZYMV JF317296.1 527 99% 2.33E-156 98-94 308

4 C. lanatus PRSV AB127935.1 284 96% 2.00E-73 83-78 285

4ii C. lanatus PRSV AB127935.1 320 98% 3.00E-84 87-80 269

3 C. lanatus ZYMV JF792444.1 489 91% 4.00E-135 96-92 331

6 C. lanatus ZYMV JF792444.1 547 97% 2.00E-152 97-94 333

7 C. lanatus PRSV FJ467933.1 547 99% 2.00E-152 97-95 328

8 C. lanatus PRSV FJ467933.1 520 100% 2.00E-144 98-95 306

13 C. lanatus PRSV JN132457.1 535 95% 1.00E-148 97-95 333

18 C. lanatus PRSV JN132457.1 545 97% 6.00E-152 97-95 335

19 C. lanatus PRSV JN132457.1 542 98% 7.00E-151 97-95 330

20 C. lanatus PRSV FJ467933.1 549 97% 5.00E-153 97-95 337

21 C. lanatus PRSV FJ467933.1 551 99% 1.00E-153 98-95 328

22 C. lanatus PRSV FJ467933.1 468 95% 1.00E-128 97-95 294

23 C. lanatus PRSV JN132428.1 416 90% 6.00E-113 95-93 290

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Fig. 1. A phylogenetic tree based on coat protein (CP) gene fragments sequences was produced by using the Neighbor-Joining method with Bootstrap (1000 replicates) on nodes.

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6

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