M Pitrat'

C Wipf-Scheibef

D Besombes'

C Desbiez

H Lecoq

Resistance of melon to Cucumber Vein Yellowing Virus (CVYV)

Keywords: Germplasm evaluation, Cucumis mela, inheritance, genetic control, . . necrosis, mOSaIC

Cucumber vein yellowing virus (CVYV) belongs to the Ipomovirus genus, family Potyviridae. It is transmitted by the whitefly Bemisia tabaci, but can be transmitted mechanically in artificial inoculation. A collection of 1188 melon accessions has been inoculated with a CVYV strain isolated from melon in Spain. Five phenotypes have been observed: 46 % of the accessions are susceptible with mosaic and vein-banding symptoms like the control V édrantais; 50 % are highly susceptible with a severe mosaic, yellowing and stunting. Ouzbeque 2 is one of these accessions; few accessions exhibited necrotic symptoms with a rapid death of the plants. HSD 93-20-A from Sudan is a representative ofthis phenotype; only one accession (HSD 2458 from Sudan) was tolerant with very mild mottle but the virus can be detected by DAS-ELISA or RT-PCR in the plant apex; only an inbred line derived from the accession PI 164323 from India was resistant, exhibiting no symptom and the virus cannot be detected in non-inoculated leaves either by DAS-ELISA or RT-PCR. Inheritance of these behaviours was studied in FI' F 2 and BC progenies between the above mentioned accessions. Three loci seem ta be involved. At a fust locus tentatively named Cucumber vein yellowing resistance (symbol Cvy-l), three alleles have been identified: Cvy-r for susceptibility (present in Védrantais), Cvy-J1 controlling resistancè in PI 164323 and Cvy-P controlling necrosis'in HSD 93-20-A. At an independent second locus, the recessive allele cvy-2 presentin HSD 2458 controls the tolerance. And at a third locus the allele Cvy-3, present in Ouzbèque 2, controls the highly susceptible type of symptoms.

INTRODUCTION

Cucumber vein yellowing virus (CVYV) has been first described in Israel (Cohen and Nitzany 1960), then in ether countries of the Middle-East and more recently in the western part of the Mediterranean area (Cuadrado et al. 2001; Cucurbitaceae 2012, Proceedings of the X" EUCARPIA meeting on genetics and breeding of Cucurbitaceae (eds. Sari, Solmaz and Aras) Antalya (Turkey), October 15-18 1 11, 2012

MATERIALS AND METHODS Plant material

A total number of 1188 accessions of a 'melon collection maintained in France by a network associating INRA and private seed companies have been sown in trays with 10 seeds per accession. Sorne accessions with a typical phenotype were then used to study the inheritance ofthese traits, namely: 'Védrantais', 'Ouzbèque 2', PI 164323, HSD 2458 and HSD 93-20-A, The FI' F 2 and back-cross progenies were produced. A recombinant inbred lines population (126 RILs) was developed between Védrantais and PI 164323.

- Lecoq et al. 2007;Lecoq and Desbiez 2012). It belongs to the Ipomovirus genus, family Potyviridae and is transmitted in the semi-persistent manner by the whitefly Bemisia tabaci, but can be transmitted mechanically in artificial inoculation. It induces mosaic and yellowing symptoms on the leaves and can severely reduce the yield and the fruit quality.

Resistance has been described in cucumber as controlled by a dominant gene (Pico et al. 2008). Wild Cucumis species (C prophetarum, C africanus, C dipsaceus) have been described as resistant (Marco et al. 2003) but up to date no accession of C melo has been observed to be resistant (Montoro et al. 2004).

ln this paper, we describe the screening of melon germplasm and the inheritance of observed different phenotypes.

Virus material and inoculation method

A CVYV strain isolated from melon in Spain in 2003 was used for mechanical inoculation on the cotyledons at the pointing first leaf-stage. Symptoms were visually rated. DAS-ELISA and RT-PCR tests were performed on plants with no symptoms or very weak symptoms to assess the presence or absence of the virus. DAS-ELISA was done using an antiserum produced against the CVYV coat protein expressed in bacteria and RT-PCR using primers previously described (Lecoq et al. 2007).

RESULTS

Germplasm evaluation

Among the 1188 accessions, a first group (46.0% of the accessions) exhibited typical mosaic symptoms like the control 'Védrantais' and a second group (50.3%) exhibited very severe mosaic symptoms with yellowing and stunting. The accession 'Ouzbèque 2' was selected as a representative ofthis second group for inheritance studies. A majority of accessions [ielonging to the botanical varieties cantalupensis, reticulatus andflexuosus were in the first group with mosaic symptoms. A majority of accessions of the botanical varieties agrestis, acidulus, ameri, chate, chinensis, dudaim, makuwa, momordica, and tibish were in the second group with severe symptoms. About 50% of the accessions of the botanical varieties chito, conomon and inodorus had mosaic symptoms and 50% severe symptoms. Twenty-three accessions exhibited a systemic necrosis a few days after inoculation; most of them originated from Sudan. HSD 93-20-A was' used in the inheritance studies as a representative of this group. One accession from Sudan, HSD 2458, exhibited very mild symptoms of mottling but the virus can be detected by DAS-ELISA or by RT-PCR. Finally, an inbred line in an accession from India, PI 164323, was the only resistant accession withno symptoms and no detection of the virus in noninoculated leaves, although very rarely few plants presented the necrotic phenotype. Interestingly, all plants from other inbred lines of PI 164323 developed a systemic necrosis few days after inoculation.

Genetic control

Inheritance of resistance ln FI' F 2 and BC progenies from the cross 'Védrantais' x PI 164323, four phenotypes were observed: no symptom (like PI 164323), necrosis (like HSD 93-20-A), mosaic (like 'Védrantais') and severe mosaic, yellowing and stunting (like Ouzbèque 2) (Table la). The necrotic symptom, of the FI and a majority ofplants in the F2 and BC progenies, correspond to the heterozygous status. ln the segregating progenies, the pooled numbers of plants with no symptom or necrosis versus mosaic or severe mosaic can be explained by one dominant gene. ln the F2' 267 vs 100 (X 2 for 3:1 = 0.989, Prob = 33%); in the BC s 85 vs 87 (X2 for 1:1 = 0.002, Prob = 88%) and no susceptible plant in the BC r but one; in the RILs population, 44 RILs vs 38 RILs (X2 for 1:1 = 0.439, Prob = 51%). ln the BCr, no susceptible plant (but one with severe mosaic) was observed. These segregations fitted a "one dominant gene for resistance" hypothesis with necrosis at the heterozygous status. We propose to name this gene Cucumber vein yellowing virus resistance (symbol Cvy-l).

_ This genetic control was confinned in crosses between PI 164323 and HSD 2458 (Table 1c), when pooling the numbers of plants with no symptom or necrosis versus mild mottle, mosaic or severe mosaic. ln the F2' 393 vs 134 (X2 for 3: 1 = 0.051, Prob = 82%); in the BC s 45 vs 50 (X2 for 1:1 = 0.263, Prob = 61%) and no susceptible plant in the BC r but one.

Inheritance of necrosis

The necrotic symptom observed in the accession HSD 93-20-A (Table 1b and1e) was also observed occasionally in PI 164323 and frequently in the FI' F2 and BC progenies with PI 164323 as a parent (Table la and1c). The segregation observed in the F 2 between Ouzbèque 2 and HSD 93-20-A (Table 1b) fitted a 3 (resistant or necrotic) vs 1 (mosaic) segregation corresponding to a monogenic dominant control

(x 2 = 2.881, Prob = 9%).
ln crosses between PI 164323 and HSD 93-20-A, the FI was resistant and in the F 2 no susceptible plant was observed (157 resistant and 38 necrotic). It can be concluded that the same locus Cvy-l is involved in the genetic control of necrosis in HSD 93-20-A but that another allele is present in HSD 90-20-A. We propose the symbol Cvy-I' for the allele in PI 164323 and Cvy-J2 for the allele in HSD 93-20-A with the following symptoms: resistance for the homozygous Cvy-l' / Cvy-l' or the heterozygous Cvy-JI / Cvy-J2 and necrosis for the homozygous Cvy-J2 / Cvy-J2 or the heterozygous (Cvy-Jf / Cvy-L: or Cvy-J2 / Cvy-F],

Inheritance of tolerance

The accession HSD 2458 exhibited very weak symptoms (mild mottle) but the virus can be detected in DAS-ELISA or RT-PCR. ln the F 2 between HSD 2458 and 'Y édrantais' (Table 1d), a 1 tolerant: 3 mosaic segregation was observed (X 2 = 0.114, Prob = 74%) and in the F 2 with Ouzbèque 2, a 1 tolerant: 3 mosaic or severe mosaic segregation was observed (X2 = 0.346, Prob = 56%). We propose the symbol cvy-2 for this recessive gene controlling tolerance in HSD 2458. This genetic control was confirmed in the F 2 between HSD 2458 and PI 164323 (Table le) with a 1 tolerant: 15 resistant or necrotic or mosaic or severe mosaic segregation (X2 = 0.790, Prob = 37%). The same 1:15 segregation was also observed in the F 2 between HSD 2458 and HSD 93-20-A ( Id) by the absence of plants with severe mosaic in the F 2 between "Védrantais' and HSD 2458 and the presence of plants with severe mosaic or mosaic in the F 2 between Ouzbèque 2 and HSD 2458.

Linkage and epistasis between the three l~ci

The locus Cvy-l is independent from Cvy-3 as shown for instance by the 1:1 segregation for mosaic vs severe mosaic among the susceptible RILs (Table la).

The alleles Cvy-1' and Cvy-F are epistatic and dominant on Cvy-3 according to the higher number of resistant or necrotic plants over the susceptible (mosaic or severe mosaic) or tolerant plants. Moreover the two Iines PI 164323 and HSD 93-20-A have respectively the genotypes [Cvy-1' Cvy-3] and [Cvy-P Cvy-3] confinning the epistatic effect over Cvy-3. However thé allele Cvy-3 is not necessary for the resistance as it can be conc1uded from the observation of F2 progenies between 'Védrantais' and RILs which were resistant: sorne ofthese F 2 were segregating with plants with no symptom and plants with mosaic but no plants with severe mosaic indicating that these RILs had the genotype [Cvy-l' The FI between HSD 2458 and Ouzbèque 2, heterozygous for both cvy-2 and Cvy-3, exhibited mosaic symptoms (Table Id). ln the F 2 progeny, we observed about 25% oftolerant plants corresponding to the homozygous [cvy-2 cvy-2] (see above inheritance of tolerance). Plants heterozygous for cvy-2 or without the cvy-2 allele [cvy-2 cvy-2+] or [cvy-2+ cvy-2+] exhibited mosaic or severe mosaic symptoms. The observed 7:5 segregation (mosaic vs severe mosaic) (X 2 = 2.489, Prob = Il %), could be explained by the following interactions: plants heterozygous for both genes [cvy-2 cvy-2+ Cvy-3 Cvy-3+] like the Fior homozygous for Cvy-3+ exhibited mosaic symptoms and plants homozygous cvy-2+ and homo-or heterozygous Cvy-3 [cvy-]+ cvy-2+ Cvy-3 -] exhibited severe mosaic symptoms.

From the segregation observed in the F2 and BC between PI 164323 and HSD 2458 (Table 1c), we can conc1ude that Cvy-l is epistatic dominant over cvy-2: the plants homozygous Cvy-l' Cvy-l' were resistant whatever the allele at the cvy-2 locus and the plants heterozygous at the locus Cvy-l were necrotic whatever the allele at the cvy-2 locus.

DISCUSSION AND CONCLUSIONS

After the evaluation of the melon genetic resources with one isolate of CVYV, five phenotypes have been observed. Resistance has been found in only one accession as was the case for other viruses, Zucchini yellow mosaic virus (ZYMV) or Papaya ringspot virus-watermelon strain (PRSV-W), where a low nurnber of resistant accessions have been identified. This resistance is controlled by one dominant allele but the heterozygous plants exhibited a necrotic reaction. ln commercial FI cultivars resulting from breeding prograrns, both parents must be resistant. This necrotic reaction was also observed in sorne accessions which were hornozygous for the allele Cvy-12. This type of symptom has been observed in melon in interaction with other viruses. Two pathotypes ofZYMV have been described: the pathotype NF induces mosaic symptoms on 'Védrantais' and 'Doublon' while the pathotype F induces mosaic symptoms on 'Védrantais' and wilting and necrosis on 'Doublon' (Lecoq and Pitrat 1984). One dominant gene (Flaccida necrosis, symbol Fn) present in 'Doublon' controls this phenotype (Risser et al. 1981). A similar situation has been described with Moroccan watermelon mosaic virus (MWMV) and the gene Nm (Quiot-Douine et al. 1988). The interaction with PRSV-W is more similar to what is observed with CVYV At the locus Prv, the allele Prv' controls resistance to a11strains of PRSV and the allele Prv' controls resistance to sorne strains and systemic necrosis with other strains (Pitrat and Lecoq 1983).

The phenotype of partial resistance with very light symptoms has also been identified in only one accession and is controlled by one récessive allele cvy-2. Up to now only a few recessive genes have been described in melon for virus resistance: cab-l and cab-2 for Cucurbit aphid borne yellows virus (CABYV), nsv for Melon necrotic spot virus (MNSV) and QTLs for Cucumber mosaic virus (CMV). The practical interest of the gene cvy-2 in association with Cvy-I' or alone for the level and/or the durability of resistance has to be evaluated.

Abouthalf ofthe accessions presented severe yellowing and stunting symptoms. This type of symptom cannot be related to geographical origin or botanical types. It is controlled by one dominant allele Cvy-3. Surprisingly, this allele is present in the resistant accession PI 164323 with Cvy-I' or in the necrotic accession HSD 93-20-A with Cvy-V But the allele is not necessary for the phenotype of resistance as plant with the [Cvy-Jl Cvy-3+] genotype are resistant. Nevertheless its interaction with the locus Cvy-l for the durability of resistance should be investigated. Indeed, in preliminary tests, CVYV isolates from other geographical origins could induce necrotic or stunting symptoms on the PI 164323 inbred line described in the paper.

  The genotypes of the five lines studied in this paper are the following: 'Védrantais' [Cvy-r cvy-2+ Cvy-3+], 'Ouzbèque 2' [Cvy-r cvy-2+ Cvy-3], PI 164323 [Cvy-Jl cvy-2+ Cvy-3], HSD 93-20-A [Cvy-P cvy-r Cvy-3] and HSD 2458 [Cvy-r cvy-2 Cvy-3+].

Fig. 1 .

 1 Fig. 1. Number of melon accessions according to the symptoms observed after inoculation with a strain of CVYV.

  Tablele): 11 tolerant plant vs 166 plants with necrosis, mosaic or severe mosaic (X2 = 0.0004, Prob = 98%). Védrantais' and HSD 93-20-A (data not shown), plants with mosaic or severe mosaic were observed. The "severe mosaic" symptom was dominant over the mosaic symptom. The segregation observed (Tablela) in the F 2 (X2 = 0.754, Prob = 39%), in the BC (X2 = 5.069, Prob = 2%) and the RILs (X2 = 1.684, Prob = 19%) fitted one dominant gene hypothesis. We propose the symbol Cvy-3 for this gene with the allele Cvy-3+ for mosaic symptom.The allele Cvy-3 is present in PI 164323, HSD 93-20-A and Ouzbèque 2 and the allele Cvy-3+ is present in Védrantais and HSD 2458 as shown (Table

	Inheritance of severe mosaic, yellowing and stunting
	ln the F?, BC and RILs between 'Védrantais' and PI 164323 (Table la), and _ s
	similarly in the F 2 between '

  Lecoq H, Dufour 0; Wipf-Scheibel C, Girard M,Cotillon AC, Desbiez C (2007) First report of Cucumber vein yellowing virus in melon in France. Plant Dis 91: 909 Marco CF, Aranda MA, Montoro T, Gômez-Guillamôn ML (2003) Evaluation of several accessions and wild relatives of Cucumis melo against Cucumber Vein Yellowing Virus (CVYV). Cucurbit Gen. Coop Rep 26: 7-8 Montoro T, Sanchez-Campos S, Camero R, Marco CF, Corella P, Gomez-Guillamon ML (2004) Searching for resistance to cucumber vein yellowing virus in Cucumis melo. ln Progress in cucurbit genetics and breeding research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding, (Lebeda A, Paris HS, eds), Palacky University in Olomouc, Olomouc (CZ), pp 197-202 Pico B, Sifres A, Martinez-Perez E, Leiva-Brondo M, Nuez F (2008) Genetics of the resistance to CVYV in cucumber. ln Modem variety breeding for present and future needs. Proceedings of the 18th EUCARPIA general congress, (Prohens J, Badenes ML, eds), Valencia (Spain), pp 452-456 Pitrat M, Lecoq H (1983) Two alleles for Watermelon mosaic virus 1 resistance in melon. Cucurbit Gen. Coop Rep 6: 52-53 Quiot-Douine L, Lecoq H, Quiot JB, Pitrat M, Labonne G (1988) Evidence for a biological and serological variability in a potyvirus infecting cucurbit: the Papaya ringspot virus (PRSV). ln EUCARPIAmeeting 'Cucurbitaceae 88', (Risser G, PitratM, eds),Avignon (FR), pp 35-42 Risser G, Pitrat M, Lecoq H, Rode JC (1981) Sensibilité variétale du melon au virus du rabougrissement jaune du melon et à sa transmission par Aphis gossypii Glov. Hérédité de la réaction de flétrissement. Agronomie 1: 835-838

Table 1 .

 1 Number of plants observed with different types of symptoms in progenies between different lines of .ç,. melon (HSD 2458, HSD 93-20-A, Ouzbèque 2, PI 164323 and Védrantais) following inoculation with cvvv.

		Progeny	Symptoms
			No symptom	Necrosis	Mild mottle	Mosaic	Severe mosaic
	a	Védrantais				50
		PI 164323	43	2
		FI Védrantais x PI 164323		34
		F 2 (Védrantais x PI 164323) (±)	30	237		23	77
		Bes (Védrantais x PI 164323) x Védrantais	1	85		33	54
		Ber (Védrantais x PI 164323) x PI 164323	53	116			1
		RILs Védrantais x PI 164323	44			15	23
	b	Ouzbèque 2					7
		HSD 93-20-A	4	12
		FI Ouzbèque 2 x HSD 93-20-A		12
		F2 (Ouzbèque 2 x HSD 93-20-A) (±)	10	147		4	63
	c	HSD 2458	1		50
		PI 164323	65	2
		FI HSD 2458 x PI 164323	4	51
		F 2 (HSD 2458 x PI 164323) (±) sc, (HSD 2458 x PI 164323) x HSD 2458	106 1	287 44	28 37	44 Il	62 2
		Ber (HSD 2458 x PI 164323) x PI 164323	29	70		1
	d	HSD 2458			20
		Védrantais	1			19
		Ouzbèque 2					25
		FI Védrantais x HSD 2458			13	7
		FI Ouzbèque 2 x HSD 2458			1	34
		F 2 (Védrantais x HSD 2458) (±)	2		57	180
		F 2 (Ouzbèque 2 x HSD 2458) (±)	3		101	175	149
	e	HSD 2458			10
		HSD 93-20-A		20
		FI HSD 2458 x HSD 93-20-A		5
		F2 (HSD 2458 x HSD 93-20-A) (±)		110	1-1	22

ACKNOWLEDGEMENTS

This work was partly supported by a grant from the French Ministry of Agriculture and by the participation of the following seed companies: ASL, Clause, Gautier, Monsanto, Rijk Zwaan, Syngenta and Takii.

Literature cited

Cohen S, Nitzany FE (1960) A whitefly transmitted virus of cucurbit in Israel.