Mohamed Jijakli

Characterization Of An Exo-Beta-1,3-Glucanase Produced By Pichia Anomala Strain K, Antagonist Of Botrytis Cinerea On Apples

ABSTRACT The exo-beta-1,3-glucanase (EC 3.2.1.58) activity of Pichia anomala strain K, an antagonistic yeast of Botrytis cinerea on postharvest apples, was studied in a synthetic medium supplemented with laminarin, a cell wall preparation (CWP) of B. cinerea, or glucose. The highest enzyme activity was detected in culture media containing a CWP of B. cinerea as the sole carbon source, whereas the lowest activity was observed in culture media supplemented with glucose. Exoglc1, an exo-beta-1,3-glucanase, was purified to homogeneity from culture filtrates of strain K containing a CWP. The molecular mass of exoglc1 was estimated to be under 15 kDa. Optimum activity of exoglc1 was recorded at 50 degrees C and pH 5.5. The exoglc1 K(m) value was estimated at 22.4 mg/ml. Exoglc1 showed in vitro a stronger inhibitory effect on germ tube growth of B. cinerea than on conidia germination and caused morphological changes such as leakage of cytoplasm and cell swelling. Exo-beta-1,3-glucanase activity was detected on apples treated with strain K and was similar to exoglc1 on the basis of activity on native gel. Moreover, the addition of a CWP to a suspension of P. anomala stimulated both in situ exo-beta-1,3-glucanase activity and protective activity against the pathogen, strengthening the hypothesis that exo-beta-1,3-glucanase activity is one of the mechanisms of action involved in the suppression of B. cinerea by P. anomala strain K.

Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K.

ABSTRACT The PaEXG2 gene, encoding an exo-beta-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-beta-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.

Separate and combined disruptions of two exo-beta-1,3-glucanase genes decrease the efficiency of Pichia anomala (strain K) biocontrol against Botrytis cinerea on apple.

The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.

Development of a SCAR marker and a semi-selective medium for specific quantification of Pichia anomala strain K on apple fruit surfaces

Abstract A washing procedure for apple fruit surfaces, and a semi-selective medium, were developed to assess the population dynamics of Pichia anomala strain K, a biocontrol agent against Botrytis cinerea and Penicillium expansum on fruit. The application of this plating technique allowed more than 99% recovery of strain K population on treated apples (by dipping them in a suspension of strain K at 10 7 cfu/ml). A strain K population decline of 51% was observed after 14 days of cold storage. To overcome the lack of specificity of the plating method, the RAPD technique was applied to a collection of 11 strains of P. anomala , including strain K. RAPD amplification with primer OPN13 produced a reproducible fragment of about 2000 bp, which was specific for strain K. Based on this DNA fragment, a SCAR marker of 262 bp was amplified with K1 and K2 primers for strain K as confirmed by Southern blot analysis, and was negative for a collection of 30 yeast strains including 21 P. anomala strains. A mixed monitoring method was developed and consisted of a combined plating technique on a semi-selective medium followed by a direct strain K-SCAR amplification without DNA extraction, on released DNA from resuspended white yeast colonies. This method was used on apples treated with strain K (10 7 cfu/ml) produced in Petri dishes, or in a bio-reactor (as a dry powder) with or without additives (2% CaCl 2 and 0.2% beta-1,3-glucan) previously identified to enhance the strain K efficacy against B. cinerea . The percentages of recovered white colonies identified as strain K with the use of the specific SCAR marker ranged between 91 and 100%. The population densities reached similar levels of 1.1×10 4 and 0.7×10 4 cfu/cm 2 on apples, 24 h after treatment with the powder formulation, without and with the stimulating agents, respectively. In contrast, a stimulating effect of glucan and CaCl 2 on strain K population density was observed in apples treated with fresh cells produced in Petri dishes. Whatever the treatment, population densities diminished 1 week after application in cold storage conditions.

A Box-Behnken design for predicting the combined effects of relative humidity and temperature on antagonistic yeast population density at the surface of apples.

The objective of this work was to develop models predicting the combined effects of relative humidity (RH, 75-98%), temperature (5-25 degrees C), and initial applied yeast concentration (10(4)-10(8) CFU/ml) on the apple-surface population densities of two biocontrol agents fused against postharvest diseases; the antagonistic yeasts Pichia anomala strain K and Candida oleophila strain O. Experiments were carried out according to a Box-Behnken matrix. Multiple regression analyses showed that both models yielded a good prediction of yeast density. The effect of relative humidity appeared greater than that of temperature. The number of yeast colony-forming units per square centimeter of apple fruit surface increased with increasing relative humidity, temperature, and initial applied yeast concentration. The models predict that under optimal growth conditions (25 degrees C, 98%), strains O and K should reach a density of 10(4) CFU/cm2 when applied initially at 2 x 10(7) (strain O) or 10(7) CFU/ml (strain K). The model results suggest that rainfall was likely the principal cause of the variability of yeast efficacy reported for previous preharvest orchard trials spanning two successive years. Temperature may also contribute to this variation. The models developed here are important tools for predicting population densities of both strains on the apple surface within the experimental limits. The use of these results should contribute to achieving yeast densities of 10(4) CFU/cm2 on apples by controlling yeast application and environmental factors such as relative humidity and temperature. The results of this study also confirm our previous in vitro findings that water activity has a greater effect than temperature on yeast population density.

Response surface methodology study of the combined effects of temperature, pH, and aw on the growth rate of Trichoderma asperellum

Aims:  To evaluate the influence of environmental parameters (water activity aw, temperature, and pH) on the radial growth rate of Trichoderma asperellum (strains PR10, PR11, PR12, and 659‐7), an antagonist of Phytophthora megakarya, the causal agent of cocoa black pod disease.

Virulence variation and RAPD polymorphism in African isolates of Phaeoisariospis griseola (Sacc.) Ferr., the causal agent of angular leaf spot of common bean

Fifty four isolates of Phaeoisariopsis griseola, the agent of common bean angular leaf spot disease from the Great Lakes Region of Africa, were characterised according to their virulence behaviour and their molecular patterns. Virulence properties were revealed through the inoculation of 29 genotypes of Phaseolus vulgaris, Phaseolus coccineus and Phaseolus polyanthus. Differences in reaction types revealed high variability among these isolates. Most of them, even when collected within the same location, showed differences in their respective reactions on many plant genotypes. For molecular typing, RAPD amplifications were performed for each isolate using five random primers. Isolates with different patterns were collected within one region. Simultaneously, similar molecular patterns were found in isolates collected at different sites. However, the average of molecular similarity, based on the percentages of shared bands for each isolates pair, was higher among isolates collected within one site. No direct correlation between molecular pattern and pathotype was observed.

Inter-laboratory evaluation of a duplex RT-PCR method using crude extracts for the simultaneous detection of Prune dwarf virus and Prunus necrotic ringspot virus

The operational capacity of a duplex RT-PCR method for simultaneous detection of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) has been established by nine European laboratories. A total of 576 samples from Prunus trees with known sanitary status, corresponding to 32 samples in two repetitions for each laboratory, were analysed. The level of sensitivity achieved by the method was 98.3% for PDV and 90.4% for PNRSV. The specificity was 87.4% for PDV and 94.3% for PNRSV. The unilateral 95% confidence intervals were calculated for all these values. Cohen’s Kappa coefficient of repeatability and reproducibility of the technique indicated a strong agreement between data. Likelihood ratios were 7.50 (positive) and 0.02 (negative) for PDV. For PNRSV, the positive likelihood ratio was 15.00 while the negative likelihood ratio was 0.11. In addition, post-test probabilities of infection were calculated to manage the risk associated with the routine use of this method. This allows an accurate test result interpretation to facilitate the integration of this new technique into a certification scheme.

Identification Of Differentially Expressed Genes By Cdna-Amplified Fragment Length Polymorphism In The Biocontrol Agent Pichia Anomala (Strain Kh5)

ABSTRACT cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was used to identify genes potentially involved in biological control, by strain Kh5 (Pichia anomala), of Botrytis cinerea, an important post-harvest pathogen on apples. Strain Kh5 was grown in yeast nitrogen base (YNB) plus glucose (G medium) or YNB plus cell walls of B. cinerea (B medium). Thirty-five primer pairs were used in AFLP amplifications, resulting in a total of more than 2,450 bands derived from the mRNA of strain Kh5 grown in B medium. Eighty-six bands (3.5%) corresponded to genes upregulated in B medium compared with G medium. Of these 86 bands, 28 were selected, cloned, sequenced, and subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) to confirm their differential expression. An appropriate housekeeping gene, G2, was selected and used to normalize the results of RT-PCR. Eleven genes presented an increased gene expression in the presence of B. cinerea cell walls (expression >1). Statistical analysis showed a significant increase for 5 of these 11 genes. The overexpressed genes show homologies to yeast genes with various functions, including beta-glucosidase, transmembrane transport, citrate synthase, and external amino acid sensing and transport. Some of these functions could be related to cell wall metabolism and potentially involved in mycoparasitic properties.

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims:  To determine the effect of water activity (aw = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715.