Molecular Approaches to Assist the Screening and Monitoring of Postharvest Biocontrol Yeasts

Nineteen yeast isolates obtained from the surface of several fruits and vegetables grown in Southern Italy and Israel were compared by molecular analysis using arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA technique (RAPD-PCR). Genetic analysis made it possible to distinguish between closely-related genitically different strains which had the same morphological characteristic, and to discard isolates which were genetically identical. Following PCR characterisation, 6 isolates were selected and tested for their biocontrol activity against major postharvest pathogens (Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato). All the isolates showed a good biocontrol efficacy on both wounded and non-wounded fruits. Furthermore, the preharvest application of the most effective antagonist (LS15) on table grape resulted in a significant reduction in grey mold ranging from 28.3% to 38.2% compared to the untreated control. The RAPD-PCR technique was also useful for identifying and monitoring the survival of the antagonist after field application.     

Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests

The occurrence of Bacillus entomopathogenic bacteria on a Tunisian biodynamic farm was determined by examining 75 samples from olive tree (Olea europaea L.) habitats. A total of 40 Bacillus isolates were characterized according to their phenotypic, physiological and biochemical parameters. Isolates of the species Bacillus subtilis, Bacillus mycoides, Brevibacillus brevis, Paenibacillus polymyxa, Bacillus licheniformis, Bacillus sp. (1), Bacillus sp. (2) and a standard strain Btk HD-1 were used separately in feeding bioassays on fresh artificial diet against larvae of lepidopterans Prays oleae (Bernard) and Palpita unionalis (Hübner) and coleopterans Hylesinus oleiperda (F.) and Phloeotribus scarabaeoides (Bernard), which are olive tree pests. Larvae were successfully reared on an artificial diet with 25 g powdered olive tree leaves. Compared to the control data, only Btk and the isolates of B. licheniformis, P. polymyxa and B. brevis were entomopathogenic. Larval mortality assessed 7 days post-treatment showed high mortality rates with Btk to lepidopteran larvae (86.6% for P. oleae and 80.9% for P. unionalis) and low mortality against coleopteran pests. B. brevis isolates showed high mortality rates against P. oleae (up to 67.9%). B. licheniformis isolates caused up to 59.2% larval mortality for P. oleae and 43.6% for P. unionalis. Highest coleopteran mortality was achieved by P. polymyxa isolates (up to 55%). According to the 16S rDNA results, isolates of each of the three entomopathogenic strains were similar. Proteins in the strain supernatants were toxic to P. oleae larvae with LC50 values of 10.0 (B. brevis), 12.5 (B. licheniformis) and 37.6 μg/ml (P. polymyxa). Also, P. polymyxa showed an LC50 of 12.4 mg/l against P. scarabaeoides. Our results suggest that entomopathogenic Bacillus present locally in the biodynamic farm could be used in biological control programmes of olive tree pests.     

Superior bio-efficacy of a combined formulation of carbendazim and mancozeb in inducing defense responses in chilli seedlings against Sclerotium rolfsii Sacc. in comparison with methyl jasmonate

The objective of this work was to investigate the efficacy of a prepackaged combined formulation, Companion (carbendazim 12% + mancozeb 63% WP), sole application of carbendazim 50% WP, manozeb 75% WP and methyl jasmonate (MeJA), an inducer of systemic acquired resistance on disease severity and their role in post-infectional defense responses in chilli seedlings against Sclerotium rolfsii. Seeds were treated for 8 h with MeJA (2.5 mM and 5.0 mM) and each of fungicides (500 ppm), and were sown in pots containing soil and fungal inocula (95:5 w/w). At 15 days after sowing maximum defense against fungal infection was exhibited by Companion comparably followed by the sole application of carbendazim and mancozeb. MeJA reduced percent mortality of S. rolfsii-infected chilli seedlings significantly as compared to the inoculated control. Assessment of peroxidase (POX) and esterase (EST) at 15 days after sowing revealed the increased activity under inoculated condition. Highest POX activity in MeJA treatments (5 mM > 2.5 mM) was followed by the Companion treatment. Highest EST activity was registered in Companion treatment. The zymogram of POX isozymes showed over-expression of POX 2 and POX 4 isoforms, and induction of POX 1 isoform in inoculated treatments. On the other hand, that of EST isozymes showed induction of EST 1 isoform in Companion, carbendazim and MeJA treatments. All EST isoforms were over-expressed in Companion-treated seedlings. Both fungicides and MeJA showed significant effects on disease severity, induction of defense enzymes and isozyme pattern in S. rolfsii-infected chilli seedlings. Contact and systemic fungicides under the experiment demonstrated differential responses. The combination formulation was superior in disease control to application of the fungicide components individually. They compared favourably with MeJA in induction of defense-related enzyme activities. All these findings are new with respect to the chilli-S. rolfsii host–pathogen interaction system, S. rolfsii representing the sclerotial basidiomycetes in particular.     

Prevalence and incidence of bovine trypanosomosis in an agro-pastoral area of southwestern Burkina Faso

The present study was conducted to determine the prevalence and incidence of trypanosomosis and to investigate some factors influencing them in an agro-pastoral area of southwestern Burkina Faso. A total of 363 crossbred cattle (Baoule-zebu peul), which were bred under natural trypanosomosis challenge, were monitored monthly for parasitaemia, packed cell volume (PCV) and serological analyses over 2 years. The parasitological prevalence estimated at the beginning of the survey using the buffy coat technique (BCT) was 7.54%. As much as 66.7% of all trypanosome infections were due to Trypanosoma vivax, 23.8% due to Trypanosoma congolense and 9.5% due to T. vivax/T. congolense mixed infections. The monthly serological incidence varied from 0.29% to 19.29%.The season was the most important factor influencing the serological prevalence and incidence and the animal PCV. The dry hot season is associated with increasing seroprevalences and incidences and consequently a decreasing average of PCV. In addition, an important spatial heterogeneity was observed.     

Induction of Resistance to Penicillium digitatum in Grapefruit by β-Aminobutyric Acid

-Aminobutyric acid (BABA), an inducer of pathogen resistance in plants, induced disease resistance in reproductive parts of the plant, such as grapefruit peel tissue. Application of BABA to specific wound sites on the fruit peel surface induced resistance to Penicillium digitatum, the main postharvest pathogen of citrus fruit, in a concentration-dependent manner, being most effective at 20mM, and rather less effective at either higher or lower concentrations. The effect of BABA in inducing resistance to P. digitatum in the fruit peel surface was local and limited to the vicinity (within 1–2cm) of the BABA-treated site. In addition to inducing pathogen resistance, increasing concentrations of BABA (from 1 to 100mM) also exhibited direct antifungal activity and inhibited P. digitatum spore germination and germ tube elongation in vitro. The induction of resistance to P. digitatum by BABA was accompanied by the activation of various pathogen defense responses in grapefruit peel tissue, including activation of chitinase gene expression and protein accumulation after 48h, and an increase in phenylalanine ammonia lyase (PAL) activity after 72h.     

Feeding deterrent and growth inhibitory properties of limonoids from Khaya senegalensis against the cotton leafworm, Spodoptera littoralis

Three rearranged phragmalin-type limonoids, khayanolide A, khayanolide B and 1-O-acetylkhayanolide B, and a mexicanolide-type limonoid, khayalactol, have been isolated from the stem bark of Khaya senegalensis (Desr) A Juss (Meliaceae). The antifeedant and growth-inhibitory activities of the isolated compounds were evaluated on Spodoptera littoralis (Boisduval). When added to an artificial diet, khayanolide A, khayanolide B and 1-O-acetylkhayanolide B showed antifeedant activity in a concentration-dependent manner. Khayalactol exhibited strong antifeedant activity without significant differences at all of the tested concentrations (7.5-100 mg kg(-1)). Khayanolide B was the most potent antifeedant with an ECso of 2.19 mg kg(-1). The results also revealed that the isolated compounds caused marked larval growth inhibition on S littoralis after 7 days of feeding on treated diet; this effect was concentration-dependent. Khayanolide B was the most active growth inhibitor among the isolated compounds, with an EC50 of 6.96 mg kg(-1).     

EST-SSR cross-amplification and genetic similarity in <Emphasis Type="Italic">Onobrychis</Emphasis> genus

EST-SSR from Medicago truncatula Gaertn. and Glycine max (L.) Merr. were tested for transferability in various species of Onobrychis (O. pyrenaica Sennen, O. argentea Boiss. and O. viciifolia Scop.). Repeatable amplification was obtained for 81% of the microsatellites and 52% were polymorphic. Six selected SSRs from M. truncatula were used to fingerprint and estimate the genetic similarity of a set of 23 accessions of O. viciifolia. PCA analysis discriminated among the different Onobrychis species and the sainfoin accessions were clustered in a single major group. This grouping is discussed in terms of the history of cultivation of sainfoin in Spain. The selected SSRs will allow fingerprinting and genetic studies in Onobrychis species, solving the lack of available SSR markers in this species.