Philippe Lepoivre

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.

Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.).

Abstract. The utilisation of cryopreservation for the eradication of cucumber mosaic virus (CMV) or banana streak virus (BSV) from Musa spp. was investigated. Banana plants, cv. Williams (AAA, Cavendish subgroup), were mechanically infected with CMV or naturally infected with BSV, and proliferating meristems were produced from the infected plants. Excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The health status of regenerated in vitro plants was first checked by means of ELISA. The putative virus-free material was subsequently tested a second time following greenhouse acclimatisation. The frequency of virus eradication for CMV and BSV was 30% and 90%, respectively, following cryopreservation. In comparison, the frequency of virus-free plants regenerated directly from highly proliferating meristems, corresponding to a spontaneous eradication rate, reached 0% and 52% for CMV and BSV, respectively. The conventional meristem culture resulted in 0% CMV-free plants and 76% BSV-free plants, while the cryoprotective treatment resulted in 2% CMV-free plants and 87% BSV-free plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the structure of the meristem tips by light microscopy. The cryopreservation method used only allowed survival of small areas of cells located in the meristematic dome and at the base of the primordia.

Ultrastructural changes associated with cryopreservation of banana ( Musa spp.) highly proliferating meristems.

Abstract.Cryopreservation has been shown to improve the frequency of virus elimination – specifically cucumber mosaic virus and banana streak virus – from banana (Musa spp.) plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the ultrastructure of meristem tips at each step of the cryopreservation process. Excised meristematic clumps produced from infected banana plants belonging to cv. Williams (AAA, Cavendish subgroup) were cryopreserved through vitrification using the PVS-2 solution. We demonstrated that the cryopreservation method used only allowed survival of small areas of cells in the meristematic dome and at the base of the primordia. Cellular and subcellular changes occurring during the cryopreservation process are discussed.

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.

Molecular Evidence That The Whitefly-Transmitted Sweetpotato Mild Mottle Virus Belongs To A Distinct Genus Of The Potyviridae

SummaryComplementary DNA representing 2 108 nucleotides at the 3′ end of the genomic RNA of the whitefly-transmitted sweetpotato mild mottle virus (SPMMV) was cloned after PCR. Sequence analysis revealed an open reading frame of 1 797 nucleotides which codes for a protein of 599 amino acids, followed by a 3′ non-coding region of 311 nucleotides. Alignment of the deduced amino acid sequence with corresponding sequences of other members of thePotyviridae demonstrated that part of the presumptive RNA-dependent RNA polymerase and the coat protein coding regions of SPMMV are found at the 3′ end of its genome, in that order. Alignment of the amino acid sequence of the core of SPMMV coat protein with those of selected members of thePotyviridae showed limited identity, thus demonstrating — with phylogenetic analysis — that SPMMV belongs to a distinct genus of the familyPotyviridae.

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.

Effect of Juglone on Active Oxygen Species and Antioxidant Enzymes in Susceptible and Partially Resistant Banana Cultivars to Black Leaf Streak Disease

The black leaf streak disease (BLSD), caused by Mycosphaerella fijiensis, is the most destructive disease of bananas and plantains around the world. Breeding for resistance is the most promising strategy to fight this disease especially in small farmer plantations. Mycosphaerella fijiensis produces many phytotoxins such as juglone, which can be used, jointly with field and inoculations under controlled conditions, for screening banana cultivars for BLSD-resistance. This non-host specific phytotoxin has been shown to act on chloroplasts and disturbs the proton electrochemical gradient across the plasmalemma membrane. Moreover, an involvement of the oxidative burst during the interaction has been suggested. The present study was carried out using two cultivars that differed for either their juglone-responses or their resistance to BLSD (cv. Grande Naine susceptible to BLSD and juglone and cv. Fougamou partially resistant to BLSD and highly tolerant to juglone). The production of active oxygen species (AOS) and the enhancement of the enzymatic and/or non-enzymatic AOS-scavenging systems were investigated after treatment of the two cultivars with juglone. The time-course of AOS-production and AOS-scavenging was shown to be the key difference between these two tested cultivars after treatment with juglone. Thus, an early release of AOS (O2− radical and H2O2) and a quick stimulation of a preferment anti-oxidant system (superoxide dismutases, catalases, and peroxidases) was observed for cv. Fougamou as compared to cv. Grande Naine for which a late and weak generation of AOS accompanied by a late stimulation of the anti-oxidant systems were detected.

Yeast species for biocontrol of apple postharvest diseases: an encouraging case of study for practical use

Since early 1970’s, postharvest diseases of apple annually cause losses of 15–25 % despite modern storage facilities including controlled atmosphere (CA) or Ultra Low Oxygen (ULO) facilities (Bondoux, 1992). Factors that favour microbial growth, such as physiological senescence of fruits, mechanical injuries, as well as physiological disorders due to undesirable storage conditions can promote and explain these postharvest decays.

Use Of Mycosphaerella Fijiensis Toxins For The Selection Of Banana Cultivars Resistant To Black Leaf Streak

The results of our experiments suggest that toxin(s) of Mycosphaerella fijiensis would be involved neither in infection initiation, nor in the hypersensitive reaction in highly resistant cultivars but could serve at most as secondary determinant of the pathogenicity, contributing to the lesion expansion in cultivars exhibiting partial resistance to Black Leaf Streak disease. Moreover, the effects of toxin(s) on chlorophyll fluorescence, as well as preliminary electron microscopy observation, suggest that chloroplasts could be a precocious site of action of the toxin(s). Therefore, in vitro heterotrophic tissues would not be a suitable target to perform the screening with such toxin(s). The prospects and limitations of M. fijiensis toxin(s) for screening banana for resistance to Black Sigatoka are highlighted.

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.