Ali Rhouma

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens.

Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis

Aims:  To investigate the genetic diversity of Pseudomonas savastanoi pv. savastanoi strains and to look whether these strains were distributed to geographical location.

Influence of Olive Mill Wastewater (OMW) Spread on Carbon and Nitrogen Dynamics and Biology of an Arid Sandy Soil

Olive mill wastewater (OMW) contains large amounts of organic substances, including phenols, that can have negative environmental consequences when applied to soil. Because of the high concentrations of organic matter and mineral nutrients, OMW holds promise as a fertilizer source for cropping systems. The effects of the addition of 0.5, 1, or 2% OMW to a soil on carbon (C) and nitrogen (N) dynamics and microbiological characteristics of the soil were investigated. Mineral N presented a significant negative correlation with the amount of OMW. The total number of soil bacteria and fungi increased. However, the nitrifying population was sensitive to 2% OMW during the first month. The increase in microbiological activity accelerated the loss of soil organic carbon (SOC). However, the treated soil recovered the SOC, and the total C gains were 2.1, 1.9, and 2.4 g kg−1 for the three doses, whereas the SOC loss was 1.1 g kg−1 for the control.

First Report of Fruit Rot of Olives Caused by Botryosphaeria dothidea in Tunisia

During the summer of 2010, unfamiliar symptoms of fruit rot were frequently observed on different Tunisian olive (Olea europaea) cultivars. These symptoms appeared to be associated with the damage caused by the olive fruit fly (Bactrocera oleae). At first, infected olives showed a brown color and then fruits begin to depress until they become completely mummified and fall immaturely. This problem was more pronounced on table olive cultivars (Ascolana, Meski, and Picholine) in the northern Tunisian regions (Nabeul), with an infection incidence of 65%. Infected Ascolana olives were disinfected with 70% ethanol for 2 min, rinsed in sterile distilled water, and air dried. Several pieces were cut and placed on acidified (2.5 ml of a 25% [vol/vol] solution of lactic acid per liter of medium) potato dextrose agar medium (PDA). All plates were incubated at 25°C for 4 days under continuous fluorescent light. A fast-growing fungus with an abundant, aerial mycelium, which was gradually veering from white to dark gray, was constantly isolated. On the reverse side of the colonies, an olive green coloration spread to the edge and became darker from the center until the underside was completely black. Conidia produced on the PDA plate were hyaline, single or double cell, ellipsoid, with a subobtuse apex and a truncate base, and averaged 22.70 × 5.32 μm. Conidiophores were hyaline, cylindrical, smooth, branched at the base, with an average of 14 to 24 × 2 to 3 μm. Pathogenicity of an isolate was conducted by dipping 20 olives wounded by a sterilized scalpel in a conidial suspension (105 conidia/ml), covering inoculated olives with moisture filter paper, and incubating them in a polyethylene bag under darkness at 25°C. Controls however, were wounded and dipped in sterile distillated water. Seven days after the inoculation, olives showed a brown color covering half of the fruit. Later (15 days after), this browning was accentuated and several black pycnidia were observed. Forty days after inoculation, fruits were completely dried out and the kernel was already appearing. Controls, however, remained totally healthy. Kochs postulates was then verified and showed that pure cultures were obtained after reisolations from inoculated olives, whereas the controls were free of the fungus. BLAST analysis of the internal transcribed spacer region (ITS) of rDNA of one isolate showed 99% identity with the ITS sequence of Botryosphaeria dothidea (GenBank Accession No. FM955381.1). Species of the family of Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants (3). Drupe rot problem caused by B. dothidea was reported on olives in Greece (4) and southern Italy (2). It was reported that the fungus invades the drupes through the wounds caused by the olive fruit fly and may even be transmitted by it (1), and recently Moral et al. (3) suggested that the olive fruit fly is essential for the initiation of the disease on the fruit. To our knowledge, this is the first report of fruit rot of olives caused by B. dothidea in Tunisia. References: (1) N. González et al. Bol. San. Veg. Plagas 32:709, 2006. (2) C. Lazzizera et al. Plant Pathol. 57:948, 2008. (3) J. Moral et al. Phytopathology 100:1340, 2010. (4) A. J. L Phillips et al. Mycopathology 159:433, 2005.

Characterization of a Colletotrichum population causing anthracnose disease on Olive in northern Tunisia

To phenotypically, physiologically and molecularly characterize the causal agent of olive anthracnose in the northern Tunisia and to study its genetic variability and pathogenicity.

Biocontrol of olive knot disease by Bacillus subtilis isolated from olive leaves

We evaluated the biocontrol efficacy of a Bacillus subtilis strain isolated from symptomless olive leaves against the olive knot disease pathogen Pseudomonas savastanoi pv. savastanoi. Bacillus subtilis F1 and F-4 displayed antibacterial activity against the pathogen. In planta, only B. subtilis F1 significantly reduced the weight of knots caused by P. savastanoi pv. savastanoi IVIA 1628 and Aw9, whereas copper treatment was effective only when using strain Aw9, thus a reduction of 50% was obtained. Preliminary characterisation of the active compound produced by B. subtilis F1 showed that it was proteinaceous in nature. These results suggest that B. subtilis strain F1 could be used to control, and prevent infection by, the causal agent of olive knot disease.

Evaluation of susceptibility of an olive progeny (Picholine x Meski) to olive leaf spot disease caused by Fusicladium oleagineum

Forty five descendants from a crossing between olive cultivar Picholine de Languedoc and the local cultivar Meski were evaluated for their susceptibility to olive leaf spot (Fusicladium oleagineum) in field and laboratory studies. Disease incidence varied greatly among the descendants and was correlated with the severity of leaf symptoms. Based on field observations and laboratory tests, the hybrids were classified into five groups: 1) very susceptible to the disease: Meski and 17H, 2) susceptible to the disease : 20J, 8I, 11I, 21I, 14H, 16H, 23H, 6J, 8J and 23J, 3) moderately resistant to the disease : 15I, 16I, 22I, 23I, 8K, 13H, 18H, 22H, 9J, 10J, 11J, 12J, 14J, 16J, 18J, 4) resistant to the disease: 21J, 12I, 13I, 18I, 19I, 9K, 10K, 12H, 15H, 19H, 15J and 22J, and completely resistant to the disease : cultivar Picholine and hybrids 6I and 19I. The analysis of anatomical parameters showed that the polyphenol content and composition were not correlated with disease incidence. However, the cuticle thickness and the trichome density are very important physical parameters that can determine the degree of susceptibility of hybrids to the disease. The assessment method may be useful to screen olive cultivars and hybrids for Fusicladium resistance.

First report of Botryosphaeria obtusa as causal agent of olive tree branch dieback in Tunisia.

A branch dieback of olive trees (Olea europaea L. cv. Manzanilla de Sevilla) was observed in 2010 in an orchard (50 ha), located in the Testour region of northern Tunisia. More than 50% of trees were severely damaged by the disease. Symptomatic trees presented dead branches and wilted leaves, which remained attached to the shoots, and the affected tissues appeared abnormally dark compared with the inner bark of healthy branches. Numerous pycnidia were observed on the surface of the infected branches. For diagnosis, symptomatic stems were collected and small pieces of discolored tissues were excised from lesion margins, surface sterilized in 0.5% sodium hypochlorite for 1 min, rinsed and dried on sterilized filter paper, then placed on acidified Difco potato dextrose agar plates (APDA; 2.5 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 4 to 5 days, and hyphal tips from developing fungal colonies were transferred to PDA and placed under fluorescent light (12 h/day). A fastgrowing, pycnidia-producing fungus was consistently isolated, with conidia exuding onto the agar surface of 10-day-old cultures. On the basis of colony characteristics, isolates were identified as Botryosphaeria obtusa (3). Conidia were large, dark brown, aseptate, rounded at both ends or truncate at base, and 25 to 26.8 × 10.5 to 12.03 μm. Pathogenicity tests were performed on detached stems of cv. Manzanilla by 7-mm diameter mycelial plugs cut from actively growing cultures of the fungus. Stems (30 cm length) were cleaned, surface sterilized with sodium hypochlorite (0.25% for 2 min), and wounded with a sterilized scalpel. Mycelial disks were placed over wounds and wrapped with Parafilm to prevent desiccation. Control stems were mock inoculated with sterile agar plugs. Inoculated and control stems were placed in polyethylene boxes and incubated at 25°C. After 45 days, inoculated stems developed brown discoloration, and small dark pycnidia appeared on stem surfaces. Controls remained healthy. Kochs postulates were verified by isolating the fungus from symptomatic stems. To confirm the identification, DNA of one isolate was extracted and the fungal primers ITS1 and ITS4 (4) were used to amplify the internal transcribed spacer region of rDNA. Purified amplicons were sequenced and a BLAST search of the GenBank database revealed 99% homology with B. obtusa isolate HO166525.1. The anamorph of the fungus, Diplodia seriata, has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback (2). To our knowledge, this is the first report of a canker disease of olive trees caused by B. obtusa in Tunisia. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. Taylor et al. Australas. Plant Pathol. 34:187, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

IDENTIFICATION AND SCREENING OF BACTERIAL ISOLATES FROM SAHARAN WEEDS FOR VERTICILLIUM DAHLIAE CONTROL

Five of fifty bacteria isolated from Saharan weeds (Xanthium spinosum, Desmazeria rigida and Anacyclus clavatus) growing in the south of Tunisia showed in vitro antagonistic activity against Verticillium dahliae, thus were further evaluated for their ability to suppress Verticillium wilt of young olives. Three of the isolates, related to known members of the genera Pseudomonas and Achromobacter, induced a significant decrease in the rate at which the disease occurred in V. dahliae-inoculated plants. The active compounds of the isolates were further investigated in terms of their inhibitory effects on the growth pattern of pathogenic fungi. The active compound produced by Pseudomonas sp. and Achromobacter xylosoxidans was not a protein. Overall, the findings presented in the current study indicate that the three bacterial strains isolated from the root of Saharan weeds hold promising potential for future application as biocontrol agents for the mitigation and prevention of Verticillium wilt of olive trees.

First Report of a Branch Dieback of Olive Trees in Tunisia Caused by a Phoma sp.

From 2007 to 2008, a new dieback of branches of olive trees was observed in several orchards in central and southern Tunisia. The appearance of this new syndrome coincided with warm temperatures and frequent rainfall from February to April 2007. Affected trees were observed in seven commercial orchards; disease incidence ranged from 1 to 9% and affected trees were randomly distributed in each orchard. Symptoms included abundant dead branches and wilted leaves remained attached. Distinct brown areas appeared on the bark of current-year shoots as well as on larger branches. Cankers on branches that were >2 years old were difficult to detect but were conspicuous in current-year shoots. To determine the etiology of this new syndrome, a study was carried out on samples of affected branches collected from 2007 to 2008 from different areas of the country. Unidentified species of Chaetomium and Phoma were isolated from the margins of the cankers. Kochs postulates were performed with one isolate each of a Chaetomium sp. and a Phoma sp on 2-year-old olive trees, cv. Chemlali, grown in 13-cm-diameter pots containing a sand/lime/peat mixture. Stems were inoculated by placing 10 μl of conidial suspension (106 conidia/ml) on 1-cm-long longitudinal stem wounds that had been made with a sterile scalpel. Control plants were wounded, but inoculum was replaced with sterile distilled water. Three sets of 10 plants each were wound inoculated with each of the fungi on the same day. Inoculated plants were covered with a polyethylene plastic bag to retain moisture and incubated for 2 months at 30°C with a 12-h photoperiod. After 45 days, only branches inoculated with the Phoma isolate showed brown discoloration areas at the inoculation sites. A Phoma sp. was recovered from necrotic bark from each of the 10 inoculated plants. Conidia were hyaline, unicellular, slightly ellipsoidal, and 4.8 to 6.3 × 1.8 to 2.2 μm. To confirm the identification, DNA extraction was done with hyphae collected from a 7-day-old culture on PDA after incubation at 25°C (1). Fungal primers ITS1 and ITS4 (3) were used for amplification. Purified amplicons were directly sequenced using the ITS1 and ITS4 primers for the internal transcribed spacer region of rDNA. A BLAST search of the GenBank database revealed 96% homology with Phoma sp. isolate (AJ972865.1) and 98% homology with Phoma medicaginis isolate (DQ026014.1). P. incompta has been reported as responsible for branch dieback of olive tree in Italy (2). To our knowledge, this is the first report of a canker disease of olive caused by a Phoma sp. in Tunisia. References: (1) S. R. Tendulkar et al. Biotechnol. Lett. 22:1941, 2003. (2) L. Tosi and A. Zazzerini. Petria 4:161, 1994. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.