First Report of Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum luteum Associated with Canker and Dieback of Grapevines in Tunisia.

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 µm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 µm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 µm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia. References: (1) M. Chattaoui et al. Plant Dis. 96:905, 2012. (2) H. S. Fawcett. Calif. Citrogr. 16:208, 1931. (3) B. T. Linaldeddu et al. J. Plant Pathol. 91:234. 2009. (4) B. T. Linaldeddu et al. Phytopathol. Mediterr. 47:258, 2008.

First Report of Agrobacterium vitis as Causal Agent of Crown Gall Disease of Grapevine in Tunisia.

Since October 2011, a serious outbreak of crown gall disease was observed on 1- and 2-year-old grapevines (Vitis vinifera L.) cv. Superior Seedless in several vineyards located in the region of Regueb in the center of Tunisia. Fifty isolates of Agrobacterium were isolated on a tartrate medium from galls of affected plants. To prepare template DNA, cell suspensions were lysed in 0.25% sodium-azide (NaN3) buffer prepared in 1% Triton X-100 by heating the samples at 95°C for 10 to 15 min (1). The strains were differentiated using a multiplex PCR assay with a combination of VIRFF1/VIRFR2 and VIRD2S4F716/VIRD2S4R1036 primers (2), which detect regions of virF and virD2 genes, respectively, in A. vitis strains carrying octopine or nopaline Ti plasmids and A. vitis vitopine strains. In order to differentiate A. vitis strains from A. tumefaciens strains, PGF/PGR (4), a polygalacturonase specific primer set, was added to the mixture in multiplex PCR. The isolates segregated into three main groups. The first group carries octopine type Ti plasmids, the second carries vitopine type Ti plasmids, and the third group carries both octopine and vitopine type Ti plasmids. The polygalacturonase gene sequence from 10 isolates showed 94 to 97% identity to the sequences of A. vitis previously deposited in the NCBI GenBank database (Accession No. CP000633.1gb). The biochemical test results corresponded to the results of genetic analysis. The ability to aerobically convert lactose to 3-ketolactose was tested by spotting bacteria onto medium containing lactose and flooding plates with a layer of Benedict's reagents after incubation at 28°C for 48 h. Acid production from glucose was tested by spotting bacterial strains onto potato dextrose agar (PDA) medium supplemented with CaCO3. Alkali production from L-tartrate was tested by streaking bacteria on AB minimal medium supplemented with L-tartrate and growth in salt medium was tested by streaking on nutrient broth supplemented with 2% NaCl. All isolates except one were negative in 3-ketolactose. They were negative in acid clearing on PDA-CaCO3, grew in 2% NaCl, and produced alkali from tartarate. Pathogenicity of all 50 strains was tested on 1-month-old tomato plants (Lycopersicum esculentum cv. Riograndi). Plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as control treatment. Plants were grown for 4 weeks at 23 ± 3°C and symptoms were recorded. Typical tumors developed at the inoculation sites and no symptoms were observed on the control plants. In Tunisia, crown gall disease was observed only on stone fruit trees and only A. tumefaciens Biovar 1 have been reported and assigned to four genomic species G4, G6 G7, and G8 basically on the recA sequencing (3). To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Tunisia. References: (1) A. Abolmaaty et al. Microbios 101:181, 2000. (2) F. Bini et al. Vitis 47:181, 2008. (3) D. Costechareyre et al. Microb. Ecol. 60:862, 2010. (4) E. Szegedi and S. Bottka. Vitis 41:37, 2002.

A preliminary study of the use of peripheral quantitative computed tomography for investigating root canal anatomy.

AIM: To evaluate the use of peripheral quantitative computed tomography (pQCT) for qualitative and quantitative analysis of root canal anatomy and for assessing the extent of canal enlargement during root canal instrumentation. SUMMARY: The volume variation achieved by S1 ProTaper instruments in the coronal third of the root canals was analysed using peripheral computed tomography. The tooth was scanned in the horizontal plane producing 36 consecutive cross-sectional images. All images were the result of 360 projections with a section thickness of 250 microm, a distance between slices of 0.5 mm and an in-plane pixel size of 70 x 70 microm. The evaluation was completed before and after S1 ProTaper instrumentation (with or without circumferential filing) of one root canal of a freshly extracted maxillary first premolar tooth. The acquired images were realigned geometrically and processed using a 3D visualization software. pQCT scanning allowed 3D reconstruction of the root canal anatomy and the assessment of the extent of canal enlargement during root canal instrumentation with lateral displacement of canal walls and hence volume change being greater than the coefficient of variation. The densitometry evaluation showed uniform density along the root canal wall. KEY LEARNING POINTS: * pQCT scanning allowed 3D reconstruction of the root canal anatomy and the assessment of the extent of canal enlargement during root canal instrumentation. * pQCT shows promise for allowing qualitative and quantitative analysis of endodontic procedures.