Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

A real-time PCR assay using a 3'-Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3'-Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain O population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain O cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64 x 10(2) and 1.64 x 10(5) cfu cm(-2) of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle- or large-scale experiments.     

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 biological control method against postharvest diseases of citrus fruits

Candida oleophila strain O was previously selected for its high and reliable antagonistic activity against Botrytis cinerea and Penicillium expansum, two important wound pathogens on post-harvest apples. The application of these antagonistic strains on wound pathogens of Citrus was more recently undertaken. The efficacy of yeast (applied at several concentrations from 10(5) to 10(8) CFU/ml) was assessed against P. digitatum and P. italicum inoculated after one hours (at a concentration of 10(5), 106 and 10(7) spores/ml) on 'Clementine' and 'Valencia late' varieties. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. The antagonistic activity of this strain was also dependent on the incubation time before pathogen inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Finally, the efficacy of biomass of C. oleophila strain O (produced at an industrial scale), and two different formulations of that biomass was assessed in comparison with fungicidal treatment (Thiabendazole) under semi-practical conditions against P. digitatum. This efficacy of strain O (whatever its formulation) was statistically comparable to that for TBZ at commercial dose, indicating that both formulations could be used as an alternative for conventional fungicide in postharvest treatments.     

Effects of Trichosporon sp. in Combination with Calcium and Fungicide on Biocontrol of Postharvest Diseases in Apple Fruits

The capability of yeast Trichosporon sp., an antagonist isolated from peach fruit, in biological control was evaluated in apple ( Malus domestica Borkh. cv. Fuji) fruits, when inoculated with different concentrations of Botrytis cinerea Pers. and Penicillium expansum (Link) Thom, as well as in combination with calcium and fungicide. The concentrations of the yeast cells and pathogen spores obviously influenced disease incidence and lesion development in apples. There was a significant negative correlation between concentrations of the yeast cells and infectivity of the pathogens. When the yeast cell suspensions reached the concentration of 108 colony-forming units (CFU)/mL, there was no infection caused by B. cinerea and P. expansum with spore concentrations below 106 spores/mL in apple fruits. The yeast at concentrations of 106-107 CFU/mL in combination with fungicide (iprodione at 50 μL/L) provided control of decay caused by B. cinerea and P．expansum better than separate application. Effect of controlling gray mould and blue mould rots was enhanced when Trichosporon sp., even at low concentration of 105 CFU/mL, was applied in the presence of 1%-2% CaCl2 in an aqueous suspension.     

Can the grey mould disease of the grape-vine be controlled by yeast?

Botrytis cinerea has been found to be highly pathogenic to 'Chardonnay' and 'Pinot noir' cultivars of the grape-vine producing the characteristic grey mould symptoms within 7 days of inoculation to the vitro-plants. The yeast Pichia anomala (strain FY-102), isolated from apple skin, was found to be antagonistic to B. cinerea as it completely inhibited the appearance of the grey mould symptoms when grown together. The yeast was responsible for morphological changes such as coagulation and leakage of the cytoplasm of B. cinerea. The pathogen, when applied together with P. anomala, failed to bring about the grey mould symptoms on the grape-vine, suggesting that the yeast could control the expression of this disease. An account of the interaction between B. cinerea and P. anomala, as well as the sequences of the complete ITS region of the ribosomal DNA of the yeast are described here.     

Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters

The adhesion and germination of conidia of nine fungal species were assessed on polycarbonate membranes or on the skin of apple fruit in sealed glass bottles injected or not injected with acetate esters. Adhesion was determined after dislodging conidia from surfaces using a sonication probe. Adhesion and germination of conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium citrinum, Penicillium claviforme, or Trichoderma sp. on membranes after 48 h were not increased in a 1.84 microg mL(-1) headspace of butyl acetate (BA), ethyl acetate, hexyl acetate, 2-methylbutyl acetate, pentyl acetate, or propyl acetate. Adhesion and germination of Botrytis cinerea, Penicillium expansum, and Penicillium roquefortii conidia were stimulated by all esters. Only conidia of B. cinerea and P. expansum exhibited increased adhesion and germination on the skin of apple fruit in bottles exposed to 0.92 microg mL(-1) of BA. Only conidia of B. cinerea and P. expansum produced decay in inoculated puncture wounds on fruit. Freshly made puncture wounds or 24-h-old puncture wounds in fruit were more adhesive than the unpunctured skin of fruit to conidia of B. cinerea or P. expansum. Fresh wounds were more adhesive to both fungi than 24-h-old puncture wounds. The skin and wounds of fruit were as adhesive to B. cinerea conidia as they were to P. expansum conidia. A 4-h exposure to 1.43 microg mL(-1) of BA increased adhesion of B. cinerea and P. expansum conidia in 24-h-old wounds. Results suggest that acetate-ester stimulation most likely is not a rare phenomenon in the fungi. For nutrient-dependent decay pathogens of apple fruit, acetate esters may be an alternative chemical cue used to maintain adhesion of conidia to wound surfaces.     

Selection of antagonists of postharvest apple parasites: Penicillium expansum and Botrytis cinerea

The objectives of this study were to constitute a collection of pathogenic agents of economic importance which cause losses of apple fruits after harvest namely Botrytis cinerea and Penicillium expansum and to select in vivo efficient antagonistic strains able to protect fruits against both pathogens at 5 degrees C (P. expansum) and 25 degrees C (P. expansum & B. cinerea). Twenty strains of P. expansum and ten strains of B. cinerea have been isolated from infected apple fruits. Potential antagonistic micro-organisms (thirty three isolates) belonging to yeast, bacteria and fungi have been isolated from apple surface. Six of them (strains Ach1-1, Ach2-1, Ach2-2 belonging to Aureobasidium pullulans (De Bary) Arnaud, and strains 1112-3, 1113-10 and 1113-5 belonging to Aureobasidium pullulans (de Bary) Am. v. pullulans) showed a high level of protection (more than 80%) at 25 degrees C. once inoculated with P. expansum or B. cinerea for 5 days. The highest level of protection against P. expansum (96%) was observed with the application of Ach 2-1. Six days after inoculation of B. cinerea, strains Ach 2-2 and Ach 2-1 insured 100% and 96% of protection, respectively. At lower temperature (5 degrees C), first symptoms of P. expansum appeared 13 days after its inoculation. Percentages of protection observed after apple treatment with one of the six antagonistic strains were ranged from 78% to 94% 20 days after P. expansum inoculation. Strains labelled Ach showed a protective level higher than 90% against this pathogen, followed by strain 1113-10 (90%), strain 1113-5 (89%) and strain 1112-3 (82%). At 26 days post-inoculation, levels of protection decreased but remained higher than 60% (more than 80% with strain Ach2-2 and strain 1113-5, 75% with strain Ach2-1 and 1113-10, 72% with ach1-1, 61% for the other strains). Strain Ach2-2 and 1113-10 were retained as the best antagonists for the subsequent studies.     

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.     

Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 10⁹ and 10⁸ cells ml⁻¹ inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH₄)₂SO₄, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 10⁸ cells ml⁻¹ followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.     

Different products for biological control of Botrytis cinerea examined on wounded stem tissue of tomato plants

For the moment the agents that are used against Botrytis cinerea, in glasshouses were tomatoes are cultivated, are from chemical origin. For reducing the use of chemical agents in the future it is important to search for effective biological control agents against the fight of Botrytis cinerae. The following biological products Vital pasta, Vital gel and Elot-Vis were examined in there possibility to control Botrytis cinerea. Elot Vis was tested out in experiments that were carried out in climate chambers were leafs of 3 week old tomato plants were artificially infected with Botrytis cinerea spores. Also the biological products of Vital were first investigated in experiments that were carried out in climate chambers. In stead off leafs of tomato plants it were stem wounds of tomato plants who were treated with the pasta or the gel that was spread over the wounded surface after this has been inoculated with a suspension of conidia of Botrytis cinerae. The results of these first tests that were executed in the climate chambers were the circumstances for Botrytis cinerea were ideal seemed promising. In the next step these products were tested out on large scale in glasshouses. For each plant 5 wounds were created by removing the leafs, these wounds were or first treated with the Biological product and thereafter artificially infected with Botrytis cinerea spores to check out if these products can be used as a preventive agent or the wounds were first inoculated with a suspension of Botrytis cinerea spores and thereafter treated with the product. For the product Elot-Vis a few plants were totally sprayed with an Elot-Vis suspension before leafs were removed and the wounds were inoculated with conidia of Botrytis cinerea to check out if this product was able to activated the induced systemic resistance pathway. The experiments that were executed in glasshouses showed different percentages of succeeded Botrytis cinerea infections. This is probable due to the different weather conditions during both days that the experiments were executed. For the wounds that were treated with pasta it was difficult to distinguish wounds were Botrytis cinerea succeeded to infect the plant, because these wounds frequently didn't show any sign of infection on the surface but when the wounds with the pasta were cut open it was possible to see Botrytis cinerea infections inside the stem.     

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.     

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.     

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.     

Biocontrol of Botrytis cinerea in apple fruit by Cryptococcus laurentii and indole-3-acetic acid

This study evaluated the effect of a yeast antagonist Cryptococcus laurentii and a plant regulator indole-3-acetic acid (IAA) on inhibition of Botrytis cinerea infection in harvested apple fruit. The results showed that the combined treatment with C. laurentii and IAA at 20 μg/ml was a more effective approach to reduce the gray mold rot in apple wounds than the C. laurentii alone. After 4 days of incubation, gray mold incidence in the combined treatment with C. laurentii and IAA was about 18%, which was a 50% reduction in incidence compared to the treatment with C. laurentii alone. Although IAA had no direct antifungal activity against B. cinerea infection when the time interval between IAA treatment and pathogen inoculation was within 2 h, application of IAA strongly reduced gray mold infection when IAA was applied 24 h prior to inoculation with B. cinerea in apple fruit wounds. Moreover, combination of IAA and C. laurentii stimulated the activities of superoxide dismutase, catalase and peroxidase with above 1.5-fold higher than that treatment with C. laurentii alone at 48 h. Therefore, combination of C. laurentii with IAA, which integrated the dual biological activity from the antagonistic yeast and plant regulator, might be developed to be a useful approach to control gray mold in harvested apple fruit.     

Improving Biocontrol Activity of Pichia guillermondii Against Post-harvest Decay of Oranges in Commercial Packing-houses by Reduced Concentrations of Fungicides

Three commercial tests were conducted in 2000-2001 in two commercial packing-houses (Muravera and Villacidro) located in Sardinia, Italy, to evaluate the efficacy of biological, chemical and integrated treatments against Penicillium digitatum and P. italicum on naturally inoculated orange fruit. Damage caused by the packing-house processing line was also assessed. Treating orange fruits with the yeast Pichia guilliermondii (strain 5A) in the processing line generally led to a significant reduction of post-harvest decay compared to the processed control, while the commercial product Aspire®, based on Candida oleophila , was ineffective in inhibiting the pathogen when applied alone. The integrated application of thiabendazole or imazalil with the biocontrol agents significantly improved the control of fruit decay. Using thiabendazole at concentrations of 0.1 and 1.2 g L -1 , led to similar inhibition of fruit decay in two trials. Both yeasts were equally able to colonize the fruit actively during storage. Passing fruits through the packing line caused a significant increase in fruit decay.     

The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea

BACKGROUND: Plants have evolved efficient mechanisms to combat pathogen attack. One of the earliest responses to attempted pathogen attack is the generation of oxidative burst that can trigger hypersensitive cell death. This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance. The HR is thought to deprive the pathogens of a supply of food and confine them to initial infection site. Necrotrophic pathogens, such as the fungi Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilize dead tissue. RESULTS: Inoculation of B. cinerea induced an oxidative burst and hypersensitive cell death in Arabidopsis. The degree of B. cinerea and S. sclerotiorum pathogenicity was directly dependent on the level of generation and accumulation of superoxide or hydrogen peroxide. Plant cells exhibited markers of HR death, such as nuclear condensation and induction of the HR-specific gene HSR203J. Growth of B. cinerea was suppressed in the HR-deficient mutant dnd1, and enhanced by HR caused by simultaneous infection with an avirulent strain of the bacterium Pseudomonas syringae. HR had an opposite (inhibitory) effect on a virulent (biotrophic) strain of P. syringae. Moreover, H(2)O(2) levels during HR correlated positively with B. cinerea growth but negatively with growth of virulent P. syringae. CONCLUSIONS: We show that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect plants against infection by the necrotrophic pathogens B. cinerea and S. sclerotiorum. By contrast, B. cinerea triggers HR, which facilitates its colonization of plants. Hence, these fungi can exploit a host defense mechanism for their pathogenicity.     

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.