Isolation of Microbial Antagonists for Biocontrol of Grey Mould Disease of Strawberries

A screening programme is described for the assessment of the potential of biocontrol agents to control grey mould of strawberries caused by Botrytis cinerea. Bacteria were isolated from strawberry fruits, leaves and flowers from a commercial field site and screened for antagonism towards B. cinerea using two in vitro and one in vivo screening techniques. From 559 microorganisms isolated, 108 inhibited pathogen growth on agar plates and 27 of these prevented spore germination on Cellophane membranes. The ability of these 27 isolates to inhibit infection of young strawberry leaves by B. cinerea on whole plants under glass was then tested. Seven isolates reduced grey mould development and were subsequently assessed in a field trial. Two isolates, one of Bacillus pumilus and one of Pseudomonas fluorescens, were as effective or more effective than standard dichlofluanid sprays and may therefore be of potential value as antagonists of B. cinerea.     

Ability of Moderately Halophilic Bacteria to Control Grey Mould Disease on Tomato Fruits

Tomato is one of the leading crops in Tunisia in terms of weight consumed (20 kg/per person/year). Preserving the quality of the fruit from field to consumer is essential to successful marketing. Grey mould rot induced by Botrytis cinerea is an important cause of postharvest loss depending on season and handling practices. We describe here the ability of halotolerant to moderately halophilic bacteria isolated from different Tunisian Sebkhas (hypersaline soils) to protect fresh‐market tomato fruits from B. cinerea. The tomatoes tested were at two different stages of ripening, (i) mature‐green and (ii) red. Six strains significantly reduced growth of the pathogens from 67% to 87%. The effectiveness of these antagonists was also confirmed on green tomatoes; in which the fruit rot protection rate ranged from 74% to 100%. The antagonists were characterized by morphological, biochemical and physiological tests as well as 16S rDNA sequencing. The halotolerant effective isolates were identified as belonging to one of the species Bacillus subtilis (M1‐20, J9) or B. licheniformis (J24). One effective moderately halophilic isolate (M2‐26) was identified as Planococcus rifietoensis. These strains are a source of hydrolytic enzymes such as chitinases, proteases, laminarinases, amylases, lipases and cellulases. For comparison, 12 halotolerant or moderately halophilic strains obtained from DSM culture collection were also evaluated for their antifungal activity against B. cinerea on tomato fruits. The most effective strains were Halomonas subglaciescola, Halobacillus litoralis, Marinococcus halophilus, Salinococcus roseus, Halovibrio variabilis and Halobacillus halophilus with a percentage of grey mould rot reduction ranging from 71% to 97%. Inoculation of mature‐green tomatoes by the bacterial antagonist of Halobacillus trueperi resulted in no disease development. Our results indicate that the use of halotolerant to halophilic micro‐organisms should be helpful in reducing grey mould disease of stored tomatoes.     

Botrytis cinerea: the cause of grey mould disease

Introduction:  Botrytis cinerea (teleomorph: Botryotinia fuckeliana ) is an airborne plant pathogen with a necrotrophic lifestyle attacking over 200 crop hosts worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi.
  Taxonomy:  Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botryotinia.
  Host range and symptoms: Over 200 mainly dicotyledonous plant species, including important protein, oil, fibre and horticultural crops, are affected in temperate and subtropical regions. It can cause soft rotting of all aerial plant parts, and rotting of vegetables, fruits and flowers post-harvest to produce prolific grey conidiophores and (macro)conidia typical of the disease.
  Pathogenicity:  B. cinerea produces a range of cell-wall-degrading enzymes, toxins and other low-molecular-weight compounds such as oxalic acid. New evidence suggests that the pathogen triggers the host to induce programmed cell death as an attack strategy.
  Resistance:  There are few examples of robust genetic host resistance, but recent work has identified quantitative trait loci in tomato that offer new approaches for stable polygenic resistance in future.
  Useful websites:  http://www.phi-base.org/query.php , http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html , http://urgi.versailles.inra.fr/projects/Botrytis/ , http://cogeme.ex.ac.uk     

Antagonistic Properties of Two Bacterial Biocontrol Agents of Grey Mould Disease

Bacillus pumilus NCIMB 13374 and Pseudomonas fluorescens NCIMB 13373 inhibit the growth of Botrytis cinerea , the cause of grey mould of strawberries. Both antagonists increased the pH of the growth medium from pH 6 to pH 8-8.5 and both produced antifungal agents. The compound(s) produced by B. pumilus had a fungicidal effect during conidial germination, whereas the compound(s) produced by P. fluorescens had a fungistatic effect. There was no evidence for the production of inhibitory volatile compounds. Both isolates also showed the ability to inhibit other strawberry fungal pathogens, and have potential for a wider range of biocontrol of plant diseases.     

Activity of the Yeasts Cryptococcus laurentii and Rhodotorula glutinis Against Post-harvest Rots on Different Fruits

More than 200 yeasts were selectively isolated from microbial populations on the surface of different fruits. Fifty of these isolates were tested against blue mould ( Penicillium expansum ) on wounded apples. Isolates LS-11 of Rhodotorula glutinis and LS-28 of Cryptococcus laurentii were the most effective antagonists. They were further evaluated at 20IC on different fruits (apples, pears, strawberries, kiwi fruits and table grapes) against several of the main post-harvest pathogens ( Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer and Aspergillus niger ) and at 4IC on apples inoculated with P. expansum . At 20IC the antagonists significantly reduced rot incidence and showed a wide range of activity on different hostpathogen combinations; isolate LS-28 exhibited a higher and more stable activity than LS-11. Both yeasts were also effective against P. expansum in cold storage conditions. Populations of the two yeasts were assessed on wounded and unwounded surfaces of apples kept at both 20 and 4IC. At either temperature, isolate LS-28 reached greater densities in wounded tissues than LS-11, but had a lower ability to colonize unwounded apple skin. The two yeasts were able to grow in culture at temperatures ranging from 0 to 35IC. In assays performed in vitro at 24IC, the antagonists showed low sensitivity towards several fungicides commonly applied on fruits and vegetables.     

Mycofumigation with Oxyporus latemarginatus EF069 for control of postharvest apple decay and Rhizoctonia root rot on moth orchid

Aims:  To characterize the volatile antifungal compound produced by Oxyporus latemarginatus EF069 and to examine in vitro and in vivo fumigation activity of the fungus.
Methods and Results:  An antifungal volatile-producing strain, O. latemarginatus EF069 inhibited the mycelial growth of Alternaria alternata , Botrytis cinerea , Colletotrichum gloeosporioides , Fusarium oxysporum f. sp. lycopersici , and Rhizoctonia solani by mycofumigation. An antifungal volatile compound was isolated from the hexane extract of wheat bran–rice hull cultures of O. latemarginatus EF069 by repeated silica gel column chromatography and identified as 5-pentyl-2-furaldehyde (PTF). The purified PTF inhibited mycelial growth of R . solani in a dose-dependent manner. The mycofumigation with solid cultures of EF069 also reduced effectively the development of postharvest apple decay caused by B. cinerea and Rhizoctonia root rot of moth orchid caused by R. solani .
Conclusions:  Oxyporus latemarginatus EF069 showed in vitro and in vivo fumigation activity against plant pathogenic fungi by producing 5-pentyl-2-furaldehyde.
Significance and Impact of the Study:  Oxyporus latemarginatus EF069 producing an antifungal volatile compound may be used as a biofumigant for the control of fungal plant diseases.     

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.     

Biological Control of Botrytis cinerea Pers. ex Fr. in Strawberry by Paenibacillus polymyxa (Isolate 18191)

Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea , the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 10⁶, 1 × 10⁷, 1 × 10⁸) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans. Heat treatment was also used to bias this selection towards spore-forming bacteria. Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus . Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P. mamillatum when screened using dual culture analysis. Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B. subtilis . In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea , demonstrated the production of antifungal compounds by these four Bacillus antagonists. With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions. The antifungal compounds produced by these isolates were shown to be heat-stable. Their identification, production and release require further study for exploitation as biocontrol systems.     

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea

* Botrytis cinerea is a necrotrophic fungus that causes grey mould on a wide range of food plants, especially grapevine, tomato, soft fruits and vegetables. This disease brings about important economic losses in both pre- and postharvest crops. Successful protection of host plants against this pathogen is severely hampered by a lack of resistance genes in the hosts and the considerable phenotypic diversity of the fungus. * The aim of this study was to test whether B. cinerea manipulates the immunity-signalling pathways in plants to restore its disease. * We showed that B. cinerea caused disease in Nicotiana benthamiana through the activation of two plant signalling genes, EDS1 and SGT1, which have been shown to be essential for resistance against biotrophic pathogens; and more interestingly, virus-induced gene silencing of these two plant signalling components enhanced N. benthamiana resistance to B. cinerea. Finally, plants expressing the baculovirus antiapoptotic protein p35 were more resistant to this necrotrophic pathogen than wild-type plants. * This work highlights a new strategy used by B. cinerea to establish disease. This information is important for the design of strategies to improve plant pathogen resistance.     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

Detoxification of cruciferous phytoalexins in Botrytis cinerea: spontaneous dimerization of a camalexin metabolite

Phytopathogenic fungi are able to overcome plant chemical defenses through detoxification reactions that are enzyme mediated. As a result of such detoxifications, the plant is quickly depleted of its most important antifungal metabolites and can succumb to pathogen attack. Understanding and predicting such detoxification pathways utilized by phytopathogenic fungi could lead to approaches to control plant pathogens. Towards this end, the inhibitory activities and metabolism of the cruciferous phytoalexins camalexin, brassinin, cyclobrassinin, and brassilexin by the phytopathogenic fungus Botrytis cinerea Pers. (teleomorph: Botryotinia fuckeliana) was investigated. Brassilexin was the most antifungal of the phytoalexins, followed by camalexin, cyclobrassinin and brassinin. Although B. cinerea is a species phylogenetically related to the phytopathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary, contrary to S. sclerotiorum, detoxification of strongly antifungal phytoalexins occurred via either oxidative degradation or hydrolysis but not through glucosylation, suggesting that glucosyl transferases are not involved. A strongly antifungal bisindolylthiadiazole that B. cinerea could not detoxify was discovered, which resulted from spontaneous oxidative dimerization of 3-indolethiocarboxamide, a camalexin detoxification product.     

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.     

Effect of essential oils on postharvest decay and quality factors of tomato in vitro and in vivo conditions

For increasing the shelf life and control of devastating fungal pathogen grey mould (Botrytis cinerea), tomato fruits during storage were applied different concentrations of ammi (Carum copticum) and anise (Pimpinella anisum) essential oils. First, antifungal activities of essential oils were tested on artificial growth media. The growth of grey mould was completely inhibited by ammi and anise essential oils at relatively higher concentrations. In second stage, fruits were infected artificially by grey mould spore and then treated with different concentrations of these essential oils. The results of in vivo conditions showed that ammi and anise essential oils applied at all concentrations were increasing the shelf life and inhibited the grey mould growth on tomato fruits completely in comparison to control. Fruits treated with these essential oils had significantly higher total soluble solids (TSS), ascorbic acid, β-carotene and lycopene content compared to control fruits.     

Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera co-cultured with plant growth-promoting rhizobacteria

The potential of a plant growth-promoting rhizobacterium, Pseudomonas sp. (strain PsJN), to stimulate the growth and enhancement of the resistance of grapevine (Vitis vinifera L.) transplants to gray mould caused by Botrytis cinerea has been investigated. In vitro inoculation of grapevine plantlets induced a significant plant growth promotion which made them more hardy and vigorous when compared to non-inoculated plantlets. This ability increased upon transplanting. When grown together with B. cinerea, the causal agent of gray mould, significant differences of aggressiveness were observed between the inoculated and non-inoculated plants. The presence of bacteria was accompanied by an induction of plant resistance to the pathogen. The beneficial effect from this plant-microbe association is being postulated.     

The Fungal and Bacterial Flora of Stored White Cabbage

1. The predominant organisms isolated from the outer wrapper leaves of freshly harvested white cabbages were: bacteria, yeasts, Alternaria spp., Aureobasidium pullulans, Botrytis cinerea, Cladosporium spp. and Penicillium spp.
2. Few qualitative or quantitative changes were seen in the leaf surface flora during storage at 2°C for up to 33 weeks.
3. Numbers of bacteria, particularly fluorescent and pectolytic pseudomonads, were considerably higher on cabbages drenched with fungicide or water than on corresponding undrenched cabbages.     

Antifungal activity of thyme oil against Geotrichum citri-aurantii in vitro and in vivo

Aims:  To investigate antifungal effect of thyme oil on Geotrichum citri-aurantii arthroconidia germination and germ tube elongation, to reveal effects of thyme oil on morphological structures on fungal hyphae and arthroconidia and to assess potential bio-control capacities of thyme oil against disease suppression in vivo conditions.
Methods and Results:  Thyme oil controlled the growth of G. citri-aurantii effectively. Arthroconidia germination and germ tube elongation in potato dextrose broth was greatly inhibited by thyme oil. At 600 μl l⁻¹, it inhibited the germination of about 94% of the arthroconidia and the germ tube length was only 4·32 ± 0·28 μm. Observations using light microscope, scanning electron microscope and transmission electron microscope revealed ultrastructural modifications caused by thyme oil that included markedly shrivelled and crinkled hyphae and arthroconidia, plasma membrane disruption and mitochondrial disorganization. Thyme oil applied to 'Satsuma' mandarin oranges that had been artificially wounded and inoculated with G. citri-aurantii reduced sour rot from 78·1% among untreated control fruit to 14·1% after 5 days at 26°C. Thyme oil applied to intact fruits reduced the decay from 76% among untreated control fruit to 35% after 30 days at 20°C. Thyme oil treatment did not harm 'Satsuma' mandarin oranges when they were examined after treatment and storage at 20°C for 30 days.
Conclusions:  Thyme oil may provide an alternative means of controlling postharvest sour rot on citrus fruit.
Significance and Impact of the Study:  The use of such essential oil may constitute an important alternative to synthetic fungicides. They can be exploited in commercial production and applied under storage and greenhouse conditions.     

Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea

Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo. Cell-free filtrates showed activity against B. cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB). Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B. cinerea. An assays based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA. The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter. The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves. Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps. antimicrobica were applied to the leaf tissue with the conidia. In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps. antimicrobica. These tests indicate that Ps. antimicrobica would be a suitable biocontrol agent for the control of B. cinerea.