A collection of 200 bacterial isolates recovered from citrus plants (Citrus limon, Citrus sinensis, and Citrus reticulata), Medicago truncatula and Laurus nobilis, was established. In vitro screening indicated that 28 isolates exhibited an inhibitory activity against the vascular pathogens Phoma tracheiphila and Verticillium albo-atrum. Isolates were screened according to their hydrolytic activities, plant growth-promoting bacteria (PGPB) abilities, as well as for the presence of nonribosomal peptide synthetase (NRPS) genes responsible of the lipopeptide biosynthesis. The results were positive for 16 isolates which exhibited at least two PGPB activities and a single NRPS gene. Genetic diversity of the selected isolates was studied using random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP) tools that showed clustering of strains into three major groups (I, II, and III) (i, ii, and iii), respectively. Clustering was further confirmed by the 16S rDNA sequencing that assigned nine isolates to Bacillus velezensis, four isolates to Bacillus methyltrophicus, one isolate to Bacillus amyloliquefaciens, and two isolates to Bacillus mojavensis. Organ-bacterial genotype interaction as well as positive correlation with NRPS genes are discussed.
Surveys of yellowing viruses under nonheated and geothermal heated plastic tunnels and in open field crops of melon (Cucumis melo), cucumber (C. sativus), zucchini (Cucurbita pepo), squash (C. maxima), watermelon (Citrullus lanatus), and ware cucurbit (Ecballium elaterium) were carried out year-round during 2000–2001, 2003, and 2004 in the major cucurbit-growing areas in Tunisia. Severe yellowing symptoms on older leaves of cucurbits were observed in open fields and under plastic-tunnel production systems. These yellowing symptoms and large populations of aphids (Aphis gossypii) on a diversity of cucurbit crops in Tunisia support the hypothesis of a viral cause of the disease. Virus identification using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), followed by reverse transcription–polymerase chain reaction (RT-PCR) and immunocapture (IC)-RT-PCR showed that Cucurbit aphid-borne yellows virus (CABYV) was largely distributed in melon, cucumber, zucchini, squash, and watermelon crops. Ware cucurbit (E. elaterium) and lettuce (Lactuca sativa) crops were identified as potential CABYV reservoirs. The RT-PCR-amplified partial coat protein (CP) and P4 genes were cloned and sequenced from nine Tunisian CABYV isolates. CP and P4 gene nucleotide and amino acid sequence comparisons as well as phylogenetic reconstructions showed that the Tunisian isolates clustered into two major subgroups. Comparisons with CABYV sequences retrieved from GenBank showed high nucleotide and CP amino acid identities, and close relationships of the Tunisian isolates with Italian and French isolates.
Isolate TEB1 an antagonistic endophytic bacterium, obtained from citrus leaves and identified as Bacillus amyloliquefaciens by 16S rDNA sequencing, was used for the biological control of mal secco disease of Citrus aurantium seedlings caused by the mitosporic fungus Phoma tracheiphila. The isolate TEB1 exhibited a good in vitro activity against P. tracheiphila in dual cultures as well as with the well diffusion method. C. aurantium seedlings watered with a suspension of TEB1 cells showed a reduction of 53.61 and 48.63% in disease severity and incidence, respectively. A PCR test with specific primers was performed 365 days after inoculation and P. tracheiphila was detected along the whole stem in inoculated control plant while no amplification product was obtained in TEB1 treated seedlings. Molecular analysis of TEB1 revealed a positive amplification of fenD and ituC genes responsible of the biosynthesis of fengycin and iturin lipopeptides, respectively. Moreover, observations by optical microscope showed that TEB1 reduced by 55% the germination of P. tracheiphila conidia and exhibited a marked effect on mycelia structure. Data suggest that lipopeptides produced by the bacterium interact with the cytoplasmic membrane of the fungus causing pore formation. TEB1 appears a potential candidate for the biological control of citrus mal secco disease.
Chickpea chlorotic dwarf virus (CpCDV), a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the “hard fruit syndrome” of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS) techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR), CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the “hard fruit syndrome” of watermelon.
A lipopeptide-producing endophytic Bacillus methyltrophicus TEB1 strain exhibited potent antifungal activity against Phoma tracheiphila. Lipopeptide production started at the early growth phase plateaued after 36 h of culture where it reduced the mycelium growth by 80%. The crude lipopeptide extract harvested at the stationary phase efficiently inhibited the growth of P. tracheiphila mycelium and MIC values displaying 50 and 90% inhibition of conidia germination were around 47.5 and 100 μg ml(-1) , respectively. Increasing lipopeptide extract till 3 mg ml(-1) induced 10% swelling and 3% crumbling of P. tracheiphila conidia whereas 5 mg ml(-1) induced 40% swelling and 20% crumbling. Mass spectrometry analysis of the lipopeptide extract indicated that surfactin production took place from 12 to 20 h, iturin A from 16 to 72 h, and fengycin from 12 to 72 h and that the main active compound against P. tracheiphila was identified as C15 iturin A lipopeptide. Iturin A appeared as a potential biological control agent able to substitute the currently used chemical pesticides in agriculture.
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