Capsicum Chlorosis Virus: A New Viral Pathogen of Pepper in Greece

Abstract

Pepper (Capsicum annuum var. annuum) is an important vegetable crop in Greece cultivated both in open fields and greenhouses on more than 4,200 ha. On the island of Crete and more specifically in Ierapetra, pepper is an economically important crop, because for the last few years a high percentage of its production has been exported. In November 2017, virus-like symptoms similar to those caused by tospoviruses were observed in a greenhouse pepper crop (cultivar Areti) in Ierapetra. The symptoms included necrotic ringspots and mottling of leaves, and they appeared in low incidence of approximately 1%. To investigate the etiology of the disease, four samples were analyzed by tomato spotted wilt virus (TSWV)-specific primers TSWVF/TSWVR (Xiao et al. 2016) for the presence of TSWV, a tospovirus endemic in Greece for more than 50 years (Chatzivassiliou et al. 1996). Because the method gave negative results, the samples were also tested by a generic reverse transcription polymerase chain reaction (RT-PCR) assay against tospoviruses using the primer set gM410/gM870c (Chen et al. 2012). A DNA band of 500 bp corresponding to the NSm gene was amplified in all symptomatic samples. Two of the obtained amplicons were directly sequenced (accession nos. LT992917 to LT992918), and further nucleotide comparison revealed that the sequences were 97% identical to the isolates Qld-3432 and Ph of capsicum chlorosis virus (CaCV) from Australia and Thailand (accession nos. KM589494 and KC953854). Therefore, the same samples were also tested with the primers CaCF/CaCR (Krishnareddy et al. 2008) and CaCL1/CaCL2 (5′-ATTTATTCTATCWGTKGCTC-3′/5′-ATTGAYCAAATAAGGCARAGTGA-3′), which amplify a DNA fragment of approximately 850 bp from the nucleocapsid (N) gene and a fragment of 828 bp from the L protein gene of CaCV, respectively, as well as with antiserum against CaCV (DSMZ AS-864). Both RT-PCR and ELISA confirmed the presence of CaCV in all tested samples, and subsequent sequencing analysis of the two previous positive isolates (accession nos. LT993065 to LT993066 and LS992237 to LS992238) revealed 98% nucleotide identity of the N gene and 96% identity of the L gene with the isolate Qld-3432 (accession nos. KM589495 and KM589493). Mechanical inoculation of 10 plants each of cultivars Arlequin and Dovras using tissue from symptomatic pepper plants reproduced the original symptoms. Four to five weeks postinoculation, ringspots and mottling started to appear at the upper part in some of the pepper plants, whereas no symptoms appeared on noninoculated control plants. RT-PCR verified the presence of CaCV in 3 of 10 Arlequin and in 1 of 10 Dovras pepper plants. CaCV was initially reported in Australia (McMichael et al. 2002) and then in different countries of Asia and in Hoya calycina in Hawaii. To our knowledge, this is the first report of CaCV in this area and highlights its potential as a threat for pepper production in Greece and the Mediterranean basin. Even though so far its incidence is low, extensive surveys in other regions are required to unravel the spread of this new pathogen in pepper and other crops in Greece.