Tunisian Vineyards Research Articles

Acquisition and transmission of Grapevine fanleaf virus (GFLV) by Xiphinema index and Xiphinema italiae (Longidoridae).

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Influence of Phytosanitary Practices in Tunisian Vineyards on the Presence of Pesticide Residues in Winegrapes

Influence of Phytosanitary Practices in Tunisian Vineyards on the Presence of Pesticide Residues in Winegrapes

Identification of phytoplasmas and Auchenorryncha in Tunisian vineyards

To verify the risk that plant pathogens such as phytoplasmas could have been introduced with infected propagation material, a preliminary investigation for the presence of phytoplasmas and their insect vectors in the main Tunisian viticultural regions was carried out. To identify phytoplasmas and leafhopper fauna several survey have been carried out on grapevine and spontaneous plant species in the main grapevine growing regions of Tunisia, i.e. Rafraf, Baddar, Mraissa, Belli, Gobba, and Bousalem. A total of 1,109 insect specimens were collected and 30 species were identified, 25 Cicadomorpha and 5 Fulgoromorpha. The 82.58% of the leafhopper species recorded are reported either as vectors of viruses or of phytoplasmas or direct damaging pests, while the 17.49% remaining do not have an important role in field management. In some of the collected grapevine materials aster yellows phytoplasmas (16SrI-B) were identified, confirming previous reports.

Isolation, identification and in vitro characterization of grapevine rhizobacteria to control ochratoxigenic Aspergillus spp. on grapes

Aspergillus spp. are fungal pathogens that attack the grape and that are known for their secretion of mycotoxins, in particular, ochratoxin which is very toxic. A total of 39 bacterial strains isolated from the rhizosphere in Tunisian vineyards were identified using 16S rDNA and rpob gene sequencing: 35 were Bacillus spp. strains, 2 were Brevibacterium spp., 1 was Paenibacillus sp. and 1 strain was Microbacterium oxydans. Biochemical and microbiological screenings revealed that those 39 strains (i) metabolized differently carbon sources, (ii) possessed antibiotic genes and (iii) produced siderophores. Based on their PGP traits, 21 strains were selected and tested in vitro for their antagonistic effect against two fungal pathogens, Aspergillus ochraceus and A. carbonarius. All the tested antagonists were able to reduce the growth of A. ochraceus, with Bacillus amyloliquefaciens being the most efficient; and A. carbonarius, in particular Bacillus pumilus. In vitro screening using detached berries showed the potential of B. pumilus strain G3AX for inhibiting contaminations by Aspergillus spp., that are OTA-producing fungi. At the berry surface, the efficacy of the bacterial strains strongly depended on the presence or absence of wounds.

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors.

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. ‘Hencha’ were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1−1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1−1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% ‘Hencha’ somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

Assessment of the performance of some new insecticides for the control of the vine mealybug Planococcus ficus in a Tunisian vineyard

Mealybugs (Hemiptera: Pseudococcidae) cause major economic losses in many Tunisian grapegrowing areas. In an attempt to improve management strategies for the Vine Mealybug (VM) Planococcus ficus (Signoret), three insecticides, imidacloprid, Prev-Am® and spirotetramat, were evaluated for their effect on this insect on vine, with reference to methidathion. The systemic insecticide spirotetramat (Movento® 150 OD) provided the greatest control performance of the VM populations, compared to the contact insecticide methidathion, the systemic insecticide imidacloprid applied through furrow irrigated system, and to Prev-Am®, a new contact biopesticide. Three weeks after treatment, VM eggs and adult females were missing from spirotetramat-treated vines. Additionally, spirotetramat supplied a long-residual activity against VM populations and prevented further spread of these insects on vine leaves. Therefore, this new systemic insecticide could be incorporated in an Integrated Pest Management program for VM control in Tunisian vineyards. Despite its short-residual activity, Prev-Am® was shown to be more effective than both methidathion and imidacloprid, mainly on VM eggs and L3 nymphs, and resulted in the highest level of VM L1-L2 nymph decrease on vine trunks. Hence, this biopesticide might prove useful for VM management in vineyards.

Pheromone-mediated mating disruption of Planococcus ficus (Hemiptera: Pseudococcidae) in Tunisian vineyards: Effect on insect population dynamics

AbstractThe mating disruption technique using Checkmate

Endophytic bacteria with antagonistic traits inhabit the wood tissues of grapevines from Tunisian vineyards

Vineyards throughout the world, including Tunisia, are being attacked by Grapevine Trunk Diseases (GTDs) such as Esca and Botryosphaeriae dieback. In this study, the bacterial microflora colonizing the non-necrotic and necrotic wood tissues of Tunisian mature grapevines (cv Muscat d’Italie) was investigated. Both types of tissues were studied in order to decipher microbial communities associated with them and to find a suitable BCA that can be applied to the Tunisian terroir. Single-Strand Conformation Polymorphism (SSCP) analyses showed that complex bacterial communities specifically colonized both types of wood tissues. The 19 most abundant cultivable strains, selected on their morphology, were isolated from plant samples and assigned to Pantoea, Pseudomonas, Curtobacterium and Bacillus species based on the 16S rRNA and rpoB genes. Biochemical and microbiological screenings revealed that those 19 strains (i) metabolized differently carbon sources, even within the same species, (ii) possessed antibiotic genes, (iii) produced siderophores and solubilized phosphates and (iv) had an in vitro antagonistic effect against 3 fungal pathogens (Lasidiodiplodia pseudotheobromae, Neofusicoccum parvum and Schizophyllum commune) involved in GTDs. One strain, Bacillus subtilis “B6”, had a positive effect on young vines of a cultivar, Muscat d’Italie, frequently planted in Tunisia, by reducing the size of the wood necrosis caused by N. parvum, showing its potential to counteract infection caused by this GTDs agent.

Occurrence of Agrobacterium Vitis Carrying Two Opine-Type Plasmids in Tunisian Vineyards

Fifty DNA samples were recovered from infected wood tissue of 1 and 2-year-old grapevine (Vitis vinifera L.) cultivars collected from several vineyards located in the Center of Tunisia. The strains were differentiated using a Multiplex PCR assay with combination of pehA, virF and virD2 gene-specific primers which amplified the corresponding fragments from nearly all strains and distinguished clearly between Agrobacterium vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. The isolates were segregated into three main groups, the first group carries octopine type Ti plasmids; the second vitopine type Ti plasmids and the third group both octopine and vitopine type Ti plasmids. The polygalacturonase gene sequence from 10 isolates showed 94-97% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. Obtained nucleotide sequences were submitted to Genbank under accession numbers from JX946285 to JX946294.

OCCURRENCE AND WIDESPREAD DISTRIBUTION OF GRAPEVINE VIRUS D IN TUNISIAN GRAPEVINES

Rugose wood is a complex disease affecting grapevine worldwide. At present, at least six different viruses belonging to the family Betaflexiviridae are known to be associated with the disease (Saldarelli, 2014). Previous studies had shown that Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting associated virus (GRSPaV) are common in Tunisian grapevines (Martelli, 2014), whereas no information is available on the presence of Grapevine virus D (GVD). Therefore, a total of 140 samples from several cultivars, were collected from major Tunisian viticultural regions and tested for the presence of this virus by RT-PCR using the GVD-specific primers CP471C and CP7V (Abou-Ghanem et al., 1997). A 474 bp product corresponding to a fragment of the coat protein gene was amplified from 58 samples, accounting for an infection rate of 41.42%. The virus was present in all the surveyed areas and grape cultivars tested. The infection rate ranged from 11.7% (cv. Red Globe) to 88.8% (cv. Muscat RafRaf). To confirm the identity of the target virus, three RT-PCR products were cloned and sequenced. The Tunisian isolates shared 86-91% common nucleotides with the other GVD isolates from GenBank, and 72-73%, 60-62% and 53-55% nucleotide identity with GVA (JF754577), GVB (NC003602.1) and GVE (JX402759.1), respectively. To our knowledge, this is the first report on the occurrence of GVD in Tunisian grapevines. The infection rate of GVD (41.42%), which is close to that of GVA (47.2%) in the same samples, suggests the presence of common vectors for both viruses in Tunisian vineyards.

OCCURRENCE OF CROWN GALL OF THE GRAPEVINE IN TUNISIA AND CHARACTERIZATION OF TUNISIAN AGROBACTERIUM VITIS AND A. TUMEFACIENS STRAINS

The occurrence of crown gall disease in Tunisian vineyards was investigated. Crown gall symptoms were detected in several vineyards in the northern and central regions of the country but with low incidence (9.72%). Different Agrobacterium species were isolated and char- acterized. Morphological characterization, molecular as- says and pathogenicity tests were conducted to identify Agrobacterium isolates. A set of 380 isolates with mor- phologically Agrobacterium-like colonies on MG medi- um was obtained from different parts of diseased vines and rhizospheric soils. Eighty out of them were chosen for detailed study in comparison with Agrobacterium reference strains obtained from the French CFBP col- lection. Biovars 1, 2 and 3 were first distinguished on the three semi-selective media 1A, 2E and 3AN respec- tively. Further, the isolates were differentiated by BOX- PCR analysis. It revealed that 11 Tunisian isolates could be identified as A. tumefaciens and four as A. vitis. The assignment of six A. tumefaciens strains to genomic species was achieved using the housekeeping gene recA. Results confirmed those of the BOX-PCR analysis and showed 99% similarity of three strains with the A. tumefaciens G4 strain (CFBP7128) and a comparable similarity (99%) of two strains was recorded with the A. tumefaciens G4 strain (CFBP7126). A non pathogenic strain was identified as A. tumefaciens G7 with 98% similarity to CFBP 7129. Two strains were confirmed to belong to the A. vitis by analysis of their 16S genes. The virulence and tumourigenicity of these Tunisian isolates were analyzed by vir-PCR and pathogenicity tests on carrot disks and indicator plants. The study revealed a relatively low percentage of highly tumourigenic isolates in Tunisia. Only 16% of the 300 isolates tested on car- rot disks proved to be highly tumourigenic (++) and to form typical tumours around the cambium of carrot slices, whereas 36%, induced not clearly discernible tu- mours (+). The rest of the strains (48%) were negative (-). Of the 80 isolates tested on indicator plants, only two elicited tumours on tomato and tobacco plants. The vir-PCR test of 10 strains revealed the pathogenicity of five of them.

A morphological and molecular characterization of vine mealybug populations (Hemiptera, Pseudococcidae) from Tunisia

Some vine mealybug, <em>Planococcus ficus </em>(Signoret) populations in Tunisian vineyards have been morphologically and genetically characterized. The morphological examination was based on the main distinctive characteristics of species of <em>Planococcus</em>, namely the number and distribution of the multilocular disc pores and tubular ducts on the adult female. This showed the existence of two different vine mealybug populations in Tunisia. Likewise, in the molecular analyses, two separate clades were revealed in the neighbour-joining phylogenetic tree, supporting the morphological studies and suggesting that there are two distinct populations of <em>P. ficus</em> on grapevine in Tunisia.

A Survey of Scale Insects (Hemiptera: Coccoidea) and Tending Ants in Tunisian Vineyards

In 2009, two mealybug species Planococcus ficus (Signoret) and Planococcus citri (Risso) and an armoured scale species, Hemi- berlesia lataniae (Signoret) were recorded throughout Tunisian table-grape vineyards. P. ficus was by far the most widespread scale insect species within the investigated grape growing areas and was also recorded on fig trees. Among 31 inspected vineyards, 8 were non-infested, 16 slightly to moderately infested, and 7 highly infested by mealybugs. Therefore, the latter are principal pests in vine- yards and should be sustainably managed to avoid major crop losses on grapevine. Six ant species were found in Tunisian mealybug- infested table-grape vineyards. They are Tetramorium semilaeve Andre, Plagiolepis schmitzii (Forel), Tapinoma nigerrimum (Nylander), Crematogaster schmidti (Mayr), Pheidole pallidula (Nylander) and Pheidole teneriffana Forel. The four latter species were reported tending mealybugs on grapevines. T. nigerrimum was found to be the ant most associated with the pseudococcid mealybugs. Our findings are new data for Tunisian grape-growing areas and will contribute to enhance Integrated Pest Management programs in vineyards.

Imidacloprid Applied Through Drip Irrigation as a New Promising Alternative to Control Mealybugs in Tunisian Vineyards

Imidacloprid Applied Through Drip Irrigation as a New Promising Alternative to Control Mealybugs in Tunisian VineyardsMealybugs are serious insect pests in Tunisian vineyards where they can cause major production losses. Thus, a management program of these insects is a priority for grape growers. A summer pesticide trial was conducted in a vineyard, located in the Cap-Bon Region of Tunisia. The trial was carried out to assess the use of imidacloprid, a systemic insecticide, against mealybugs on vine. Imidacloprid was applied through the drip irrigation system for each vine and was then compared to methidathion, a contact insecticide. Imidacloprid was found to be more effective than methidathion on all mealybug developmental stages. In addition to its outstanding, up to 100% efficiency, imidacloprid provided an interesting long-term control of mealybugs. No significant difference was found between the two imidacloprid rates (1 and 2 ml/vine). Methidathion generated an overall low to intermediate efficacy on mealybugs and was more effective on both first instar nymphs and adult females than on the other mealybug developmental stages. Thus, imidacloprid applied through a drip irrigation system is a new promising option to control mealybugs in vineyards. For this reason it can be employed in an integrated management program against these pests in the Tunisian grape-growing area.

Isolation and characterization of ochratoxin A and aflatoxin B1 producing fungi infecting grapevines cultivated in Tunisia

A survey was conducted to access mycotoxin-producing fungi and to evaluate the ochratoxin A (OTA) and the Aflatoxin B1 (AFB1) potential production for fungal strains contaminating table grapes in different Tunisian vineyards. Among 100 Aspergillusisolates, Aspergillus niger aggregate were the most frequent (70%) followed byAspergillus carbonarius (7%) and Aspergillus flavus (23%). The mycotoxigenic capacity of the isolates was tested in culture media revealed that the highest levels of OTA production were obtained with strains of A. carbonarius (80% of them) whereas only 5% of A. niger aggregate were OTA producers. Furthermore, 39% of the A. flavus produced aflatoxin B1 (between 21 and 54 µg/g). Finally, we demonstrated, for the first time, that 3% of the Penicillium isolates (especially Penicillium expansum) were OTA producers in grapes. Key words: Aspergillus, Penicillium, grape, ochratoxin A, aflatoxin B1.

Survey of Mealybugs (Hemiptera: Pseudococcidae) and their Natural Enemies in Tunisian Vineyards

A survey of mealybugs and their associated natural enemies occurring in vineyards was conducted in 2003 and 2004 in different Tunisian grapevine-growing regions. Two mealybug species, the citrus mealybug, Planococcus citri (Risso), and the vine mealybug, Planococcus ficus (Signoret), were found in all inspected vineyards. Parasitoids were recorded more frequently than predators as natural enemies of the two Planococcus species. Four encyrtid primary parasitoids, Anagyrus pseudococci (Girault), Leptomastix dactylopii (Howard), Leptomastidea abnormis (Girault) and Coccidoxenoides peregrinus (Timberlake), were recorded from mummified mealybugs collected in the field. Anagyrus pseudococci, with a rate of parasitism of 80.3 %, was the most abundant parasitoid, followed by L. abnormis (12.1 %), C. peregrinus (4.5 %) and finally L. dactylopii (3 %). Two coccinellids, Rhyzobius lophanthae and Scymnus sp., were found associated with mealybugs.

Incidence and Distribution of Grapevine Leafroll‐associated viruses in Tunisian Vineyards

AbstractSurveys were carried out in Tunisian table grape vineyards for assessing the occurrence and distribution of grapevine leafroll‐associated viruses (GLRaVs). Leafroll symptoms were commonly observed in most of the surveyed vineyards. Samples were randomly collected from 712 individual vines for laboratory testing. Enzyme‐linked immunosorbent assay (ELISA) tests showed that 81.5% of the vines were infected by one (35.7%) or more (45.8%) viruses. GLRaV‐3 was the most widespread virus (76.3%), followed by GLRaV‐5 (38.5 %), GLRaV‐6 (13.2%), GLRaV‐1 (9.1%), GLRaV‐2 (6.3%), and GLRaV‐7 (0.9%). GLRaV‐3 and GLRaV–5, two mealybug‐transmissible Ampeloviruses, were present in mixture in 35.9% of samples. The highest infection rate was found in Cape Bon region (81.7 %), where cv. Italia had an infection rate of 79.5%. Superior seedless, the main cultivar in Sidi Bouzid, had 75% infection. GLRaV‐6 and ‐7 were detected for the first time in Tunisia.

Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts ( n = 24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 μg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.

Occurrence of pathogenic fungal species in Tunisian vineyards

Mycotoxins are secondary metabolites produced by filamentous fungi detected in food, such are grapes. OTA was evaluated in ten handle musts from different Tunisian vineyard. This mycotoxin was found at levels 1.1 μg/L to 4.3 μg/L. A survey was conducted to assess the contamination of the Tunisian vineyard with pathogenic fungal species, in particular those responsible of the OTA production. The results were evaluated for the first time in parcels cultivated in the North, in the Centre and in the South of the country. Italia Muscate and Superior Seedless varieties were concerned at three developmental stages of the berry, setting, veraison and maturity. Carigon variety was used as positive control for musts contaminating by OTA. The main fungal species isolated were Aspergillus spp. (33.32%), Botrytis cinerea (23.32%), Alternaria spp. (12.80%), Cladosporium spp. (10.59%) and Penicillium spp. (8.3%). The isolates of the Aspergillus genus were identified as Aspergillus niger aggregate (77%), Aspergillus carbonarius (15%) and Aspergillus flavus (8%). Their presence was characterized by a significant decrease in the Centre during the veraison and a slight increase in the North and the South during the maturity stage. Furthermore, when comparing Superior Seedless and Italia Muscate cultivated in the same area, the aspergilli were particularly less abundant at the setting stage in the case of Superior Seedless. There is no correlation between the OTA amount in musts and the contamination by Aspergillus species in different vineyards and for grape varieties studied.

Detection and characterization of two strains of Grapevine fanleaf nepovirus in Tunisia

The nematode‐borne Grapevine fanleaf nepovirus (GFLV) causes severe degeneration of grapevines in vineyards worldwide. In a recent survey of the sanitary status of grapevine plants in north Tunisian vineyards, we were interested to study the polymorphism of GFLV. Purified virus, from mechanically inoculated Chenopodium quinoa, was used to produce anti‐GFLV antiserum, which specifically recognized GFLV in different Tunisian grapevine samples using the DAS‐ELISA technique. Positive samples were subjected to oligoprobe‐RT‐PCR to amplify a 606 bp region of the viral coat protein sequence. PCR products used for RFLP analysis after digestion with endonuclease AluI produced 3 restriction profiles. RFLP data allowed clear distinction of two GFLV strains in Tunisia. The nucleotide sequence of the PCR‐generated amplicons from each strain was determined showing 93.4% identity at the nucleic acid level and 97.5% similarity at the aminoacid sequence level compared to the previously characterized GFLV‐F13 French isolate. This paper is the first report on molecular variability of GFLV in Tunisia.