Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine leafroll associated virus-5 (GLRaV-5), two members of the genus Ampelovirus associated with grapevine leafroll disease, were transmitted by the mealybug Planococcus ficus and the soft scale insect Ceroplastes rusci from infected to healthy vines under experimental conditions. The efficiencies of transmission of GLRaV-3 and GLRaV-5 by P. ficus were 23.3 and 8.3%, respectively, and by C. rusci were 3.3 and 1.7%, respectively. Juvenile instars of P. ficus were more efficient in transmission of the viruses than adult females. This is the first report of the ability of C. rusci to transmit these viruses to grapevines.

Xylem vessels are essential pivotal organs in bulk hydraulic flow through the whole woody plant. However, environmental constraints generate disagreements in xylem structures, which are characterized by air emboli and occlusions formations, compromising water conductivity in grapevines. The aim of this work was to explore xylem morphology dynamics through the xylem sap flow of five Tunisian grapevine cultivars during the natural bleeding sap periods of 2019, 2021, and 2022. In fact, Sakasly, Khamri, Hencha, Razegui1, and Razegui2 rain-fed grapevine cultivars revealed differential responses towards xylem sap movement. The results demonstrated that the xylem sap flow was significantly more abundant in 2019 than 2021 and 2022 bleeding sap campaigns. A variation was revealed between the cultivars regarding the xylem sap flow. In fact, Sakasly gave the best xylem flow during the three campaigns. Razegui1 and Razegui2 registered approximately similar xylem sap flow, while Hencha and Khamri present the lowest sap fluxes during the three campaigns. Moreover, several vascular occlusions forms were identified from stem cross sections using environmental scanning electron microscopy (ESEM), including tyloses, gels, starch, and gum deposits. The highest occlusion number was observed in Sakasly, Razegui1, and Razegui2 cultivars. Among different biogenic calcium shapes, several were observed for the first time in grapevine, including multi-faceted druse, cubic, crystalline sand, styloids, spherical, or drop-like structures. Considering their lower flow and totally blocked vessels, both Hencha and Khamri confirmed their susceptibility to environmental constraints. However, Sakasly, Razegui1, and Razegui2 cultivars presented higher tolerance according to their sap flow and xylem morphology.

Grapevine leafroll disease is one of the most important diseases that occurs in cultivated grapevines in the world. So far, nine serologically distinct viruses of the family Closteroviridae have been isolated from diseased vines (3). A previous study (4) has shown that Grapevine leafroll-associated viruses (GLRaV) -1, -2, and -3 are present in Tunisian grapevines and GLRaV-3 is the predominant virus associated with leafroll disease. A survey was conducted in table grapes to identify other viruses associated with this disease. Samples of dormant canes were collected and screened by indirect Biotin Steptavidin ELISA with specific antibodies to GLRaV-5 (Bio-Rad, Sanofi, France) according to the manufacturer's instructions. Serological analysis revealed that nearly 47% of the samples were infected with GLRaV-5. To confirm GLRaV-5 identification and identify other leafroll viruses, vines with severe leafroll symptoms were collected and total RNA extracts were obtained from six samples and tested at Waite Diagnostics (University of Adelaide, Australia) by reverse transcription (RT)-PCR using primers for GLRaV-5 (2), LR5-1F 5′-CCCGTGATACAAGGTAGGACA-3′ and LR5-1R 5′-CAGACTTCACCTCCTGTTAC-3′ with a resulting amplicon size of 690 bp and primers for GLRaV-9, LR9F 5′-ACAGTGGTCGGCATAAGAAAAG-3′ and LR9R 5′-ACACAAACATGCAGGCCAAAG-3′ with a resulting amplicon size of 250 bp. Results showed that 1 of 6 and 5 of 6 of the samples were infected with GLRaV-5 and GLRaV-9, respectively, by RT-PCR and comparable results were obtained by ELISA. Amplicons were cloned and sequenced to confirm the identification of GLRaV-5 and GLRaV-9. The obtained sequences showed 99.1% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-5 (GenBank Accession No. AF233934) and 97.6% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-9 (GenBank Accession No. AY297819). The occurrence of GLRaV-9 has previously been reported in California and Australia (1). To our knowledge, this is the first report on the occurrence of GLRaV-5 and -9 in Tunisian grapevines. The widespread occurrence of GLRaV-5 and -9 is probably due either to the presence of their putative vectors, Planococcus ficus (Signoret) and Planococcus citri (Risso), or by propagation using infected local source material. Further studies are in progress to verify the implication of indigenous mealybugs in the spread of these viruses. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) X. Good and J. Monis. Phytopathology 91:247, 2001. (3) P. Gugerli. ICVG, Extended Abstracts 14:23, 2003. (4) N. Mahfoudhi et al. EPPO Bulletin 28:197, 1998.

Isolates (PV006687-PV006694) revealed 100% nucleotide identity with the W11 strain (MF198463.1) from the USA and the GA/8 strain (OR419772.1) from Turkey. These findings emphasize the need for enhanced surveillance and biosecurity measures to prevent the spread of viroids in pistachio crops, particularly in trees grafted onto P. atlantica rootstocks. To the best of our knowledge, this is the first report of CBCVd-pis infectingP. atlantica in Tunisia.