Fruit differential protein patterns in strawberry cultivars susceptible or resistant to grey mould

The total proteins of four strawberry cultivars with differing resistance and/or susceptibility to grey mould were extracted by two different methods, phenol and sodium dodecyl sulphate (SDS). Compared to phenol extraction, SDS extraction produced higher protein yields but poor quality protein bands with a high degree of streaking indicating possible contamination. In contrast, the total proteins obtained by phenol extraction could be used in two-dimensional polyacrylamide gel electrophoresis (2-DE) because of their higher quality, and the protein spots were well separated for all tested genotypes. From all the cultivars studied in this research, we obtained 89 differential (unmatched) protein spots that may be related to fruit disease resistance or susceptibility. Using the SPSS 11.0 software, we obtained a cluster of the four strawberry cultivars. The cultivars “APF029-4” and “SJ8976-1” were in the same subcluster with the highest Pearson correlation coefficient (0.705). Afterwards, they were amalgamated into the second group with “Joliette”, a disease-resistant cultivar, which means that they might have a similar resistance to grey mould.     

Infection of strawberry flowers by Botrytis cinerea and its relevance to grey mould development

Corn stunt spiroplasma (CSS) multiplied in all injected Dulbulus maidis, reaching titres of over 1 × 10⁶ colony forming units (cfu)/insect and 1 × 10⁴ cfu/salivary gland of each insect. Spiroplasmas could be isolated from the haemolymph and from the salivary glands 1 h after injection and at any time subsequently. Insect extract at a concentration greater than the equivalent of 0.1 insects/ml was inhibitory to the growth of CSS in cultures. Helices could be seen in the haemolymph at any time after injection. However, distorted or partially deformed cells and small aggregates were not present until 2–3 wk after injection. The salivary gland cells of injected insects contained membrane-bound ‘pockets’ or ‘colonies’ packed with pleomorphic organisms, which included some filamentous forms. Intracellular colonies were always on the periphery of cells and were easily detectable by fluorescent microscopy. Both pleomorphic and filamentous forms were also seen intercellularly in the salivary glands. Following injection, transmission of CSS to maize and to sterile feeding solution were compared using 1 day feeding periods. A proportion of injected leafhoppers began to transmit to maize by the third day following injection (5%) and reached a maximum of 72% by day 14. By day 9 , 82% of the population had transmitted at least once to plants and by day 12 , 100% had transmitted. Similar insects transmitted through membranes to sterile feeding solution on day 4 (3%) reaching a maximum of 62% by day 14.     

Host-induced gene silencing of BcTOR in Botrytis cinerea enhances plant resistance to grey mould

Botrytis cinerea is the causal agent of grey mould for more than 200 plant species, including economically important vegetables, fruits and crops, which leads to economic losses worldwide. Target of rapamycin (TOR) acts a master regulator to control cell growth and proliferation by integrating nutrient, energy and growth factors in eukaryotic species, but little is known about whether TOR can function as a practicable target in the control of plant fungal pathogens. Here, we characterize TOR signalling of B. cinerea in the regulation of growth and pathogenicity as well as its potential value in genetic engineering for crop protection by bioinformatics analysis, pharmacological assays, biochemistry and genetics approaches. The results show that conserved TOR signalling occurs, and a functional FK506-binding protein 12 kD (FKBP12) mediates the interaction between rapamycin and B. cinerea TOR (BcTOR). RNA sequencing (RNA-Seq) analysis revealed that BcTOR displayed conserved functions, particularly in controlling growth and metabolism. Furthermore, pathogenicity assay showed that BcTOR inhibition efficiently reduces the infection of B. cinerea in plant leaves of Arabidopsis and potato or tomato fruits. Additionally, transgenic plants expressing double-stranded RNA of BcTOR through the host-induced gene silencing method could produce abundant small RNAs targeting BcTOR, and significantly block the occurrence of grey mould in potato and tomato. Taken together, our results suggest that BcTOR is an efficient target for genetic engineering in control of grey mould, and also a potential and promising target applied in the biocontrol of plant fungal pathogens.     

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     

The lre-miR159a-LrGAMYB pathway mediates resistance to grey mould infection in Lilium regale

Grey mould is one of the most determinative factors of lily growth and plays a major role in limiting lily productivity. MicroRNA159 (miR159) is a highly conserved microRNA in plants, and participates in the regulation of plant development and stress responses. Our previous studies revealed that lre-miR159a participates in the response of Lilium regale to Botrytis elliptica according to deep sequencing analyses; however, the response mechanism remains unknown. Here, lre-miR159a and its target LrGAMYB gene were isolated from L. regale. Transgenic Arabidopsis overexpressing lre-MIR159a exhibited larger leaves and smaller necrotic spots on inoculation with Botrytis than those of wild-type and overexpressing LrGAMYB plants. The lre-MIR159a overexpression also led to repressed expression of two targets of miR159, AtMYB33 and AtMYB65, and enhanced accumulation of hormone-related genes, including AtPR1, AtPR2, AtNPR1, AtPDF1.2, and AtLOX for both the jasmonic acid and salicylic acid pathways. Moreover, lower levels of H₂O₂ and were observed in lre-MIR159a transgenic Arabidopsis, which reduced the damage from reactive oxygen species accumulation. Taken together, these results indicate that lre-miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response.     

Biological control of grey mould in strawberry fruits by halophilic bacteria

Aims:  Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti- Botrytis metabolites and volatiles.
Methods and Results:  Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea . Thirty strains (20·2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20°C, from 50% to 91·66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui , B. subtilis , B. pumilus , B. licheniformis , Terribacillus halophilus , Halomonas elongata , Planococcus rifietoensis , Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites.
Conclusions:  Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries.
Significance and Impact of the study:  The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.     

Vapours from plant essential oils to manage tomato grey mould caused by Botrytis cinerea

Tomato grey mould has been a great concern during tomato production. The in vitro antifungal activity of vapours emitted from four plant essential oils (EOs) (cinnamon oil, fennel oil, origanum oil, and thyme oil) were evaluated during in vitro conidial germination and mycelial growth of Botrytis cinerea, the causal agent of grey mould. Cinnamon oil vapour was the most effective in suppressing conidial germination, whereas the four EOs showed similar activities regarding inhibiting mycelial growth in dose-dependent manners. The in planta protection effect of the four EO vapours was also investigated by measuring necrotic lesions on tomato leaves inoculated by B. cinerea. Grey mould lesions on the inoculated leaves were reduced by the vapours from cinnamon oil, origanum oil and thyme oil at different levels, but fennel oil did not limit the spread of the necrotic lesions. Decreases in cuticle defect, lipid peroxidation, and hydrogen peroxide production in the B. cinerea-inoculated leaves were correlated with reduced lesions by the cinnamon oil vapours. The reduced lesions by the cinnamon oil vapour were well matched with arrested fungal proliferation on the inoculated leaves. The cinnamon oil vapour regulated tomato defence-related gene expression in the leaves with or without fungal inoculation. These results suggest that the plant essential oil vapours, notably cinnamon oil vapour, can provide eco-friendly alternatives to manage grey mould during tomato production.     

Pantothenic acid in relation to the fat-forming mould: Penicillium lilacinum thom

Summary Penicillium lilacinum was grown in a synthetic medium highly conducive to fat formation. Calcium pantothenate was added to fungal cultures when 5 days old at 3 different concentrations, namely, 0.1, 1.0, and 2.5 mg/100 ml medium. Sugar uptake was suppressed at the three experimental concentrations. Growth was first suppressed but was later accelerated. Fat formation was also suppressed at first but later the fat content of the vitamin-supplied cultures exceeded the controls and a fat content amounting to 56% could be obtained with 1.0 mg vitamin/100 ml medium, compared with about 46% for the controls. It is assumed that, during suppression of growth, alcohol and other intermediate products may have accumulated and were later utilised in fat synthesis.     

Controlling grey and blue mould diseases of apple fruits using acetic acid vapours

Abstract

The effect of acetic acid vapours at three application times on grey and blue moulds on the incidence of apple fruits was studied. Acetic acid vapour at 6 µl/l caused complete inhibition of linear growth and spore germination of Botrytis cinerea and Penicillium sp. The most effective concentration was AA at 4 µl/l, which reduced spore germination and linear growth by more than 82.4 and 68.3% of both fungi respectively compared with un-fumigated fungi. On other trials the diameter of the inhibited growth zone of both fungi gradually increased as AA concentrations increased. The highest increase in inhibition zone diameter was obtained with AA at 12 µl/l followed by AA at 8 µl/l, meanwhile AA at 4 µl/l had a moderate effect. Apple fruits were divided into three groups, the first was fumigated at 24 h before inoculation, the second at zero time the and third at 24 h after inoculation and storage for 30 days. Results indicate that the higher reduction of grey and blue mould incidence was obtained with AA at 12 µl/l of three application times which reduced the disease incidence by more than 81.0, 81.0 and 73.5% before, at zero time and after inoculation, respectively. Acetic acid at 8 µl/l reduced the incidence of both diseases more than 52.0% with all application times. As for application times the most effective times were 24 before and at the zero time of inoculation which reduced both diseases more than 81.0 and 75.5 with AA vapours at 12 and 8 µl/l, respectively. Meanwhile treated apple fruits after inoculation was less effective. The same trend between disease incidence and percentage of rotted tissue parts was observed. It could be suggested that acetic acid vapours might be safely used commercially for controlling post-harvest disease of apple fruits during storage.     

Maackiain in Cicer bijugum Rech. f. associated with resistance to Botrytis grey mould

Resistance in Cicer bijugum Rech. f. a wild relative of chickpea, to Botrytis grey mould (BGM), caused by Botrytis cinerea Pers., was shown to be associated with high concentrations of maackiain when compared to three susceptible species. The two BGM resistant accessions of C. bijugum contained between 200 and 300 μg maackiain g⁻¹ of foliage whereas the BGM susceptible species C. arietinum, C. echinospermum and C. reticulatum contained less than 70 μg g⁻¹. Furthermore, the concentration of maackiain increased to more than 400 μg g⁻¹ in the resistant wild species after being inoculated with the pathogen whereas no significant increase was recorded in the susceptible species. The germination of spores of Botrytis cinerea, treated with maackiain, was inhibited in a dose dependent manner; less than 10% of spores germinated when treated with 500 μg ml⁻¹. The data indicate that maackiain may be an important component in BGM resistance in the wild chickpea C. bijugum and that the resistance is enhanced in the presence of the pathogen.     

Evaluation of fungal antagonists for grey mould management in early growth of pot roses

Several filamentous fungi can reduce disease incidence and suppress sporulation of Botrytis cinerea during early establishment and vegetative growth of pot roses. Two isolates of Ulocladium atrum were the most successful of those fungi tested, were consistently more effective than the fungicide, iprodione and not different from each other. Tween 80, added as a dispersant, had no effect on the efficacy of U. atrum. An isolate of Clonostachys rosea also gave better disease control than iprodione, although not to the levels given by isolates of U. atrum, nor was its behaviour consistent across experiments. Another isolate of C. rosea performed well in one trial, had no discernible effect in a second and was excluded from the third due to poor inoculum quality. Commercial Trichoderma harzianum preparations (Trichodex and Supresivit) failed to reduce disease incidence under the high disease pressure of these experiments, but did reduce sporulation of the pathogen relative to untreated controls.     

Harvesting practices and early Neolithic barley cultivation at el-Hemmeh,Jordan

Recent research strongly suggests polyphetic origins of multiple cultigens across Southwest Asia approximately 11,000 years ago during the Pre-Pottery Neolithic A period. The harvesting practices that contributed to the dedicated use of cultivation as a plant exploitation strategy remain largely unidentified. Archaeobotanical data from el-Hemmeh, a settlement site dating to ca. 10850 cal. b.p., provides an opportunity to examine in close detail the harvesting strategies that may have contributed to the development of domesticated forms. Initial analyses indicate a variety of wild plant foods including barley, lentils, vetch, Pistacia cf. atlantica and fig were exploited, while the presence of large predomesticated barley grains and potential weed species suggest cereal cultivation was also pursued at the site. Barley rachis internodes from el-Hemmeh typically possess a wild morphology, but 22% of specimens show evidence of a forced or “ripped” disarticulation. This suggests barley may have been harvested while ears were partially immature and required subsequent processing in order to disarticulate spikelets.     

Control of tomato grey mould on unheated crops using non- systemic fungicide sprays

High volume sprays of dichlofluanid (0.1 % a.i.) reduced total fruit numbers but gave better control of Botrytis cinerea infections of tomato stems, leaves and fruit than sprays at 0.05 or 0.025% a.i. Surface residues from the sprays at 0–1, 0.05 or 0.025% a.i. were respectively 5.4, 1.6, 0.7 μg/g fresh weight of ripe fruit at harvest. Tank-mixed zineb (0.12% a.i.) and captan (0.2% a.i.) were less effective and increasing the spray intervals from 1 wk to 2 and 3 wk reduced the fungitoxicity of captan and zineb more than that of dichlofluanid. Harvest residues on ripe fruit were 7.1, 2.8 or 2.4 μg/g when dichlofluanid (0.1 % a.i.) sprays were applied at 1, 2 and 3 wk intervals respectively. Good control of B. cinerea was achieved if the whole plant was sprayed initially with dichlofluanid as soon as the second truss was flowering and subsequent sprays were restricted to the upper section of stem including the four or five youngest trusses of buds, flowers and fruit. When used as a post-infection spray there was a period of c. 8 wk before dichlofluanid markedly reduced the incidence of grey mould. Tank-mixed zineb (0.12% a.i.) controlled Botrytis fruit spotting but not leaf and stem infections. Botrytis stem lesions extended more rapidly on zineb-sprayed plants than on unsprayed plants or on plants treated with captan or dichlofluanid.     

Isolation of bioactive antibiotic peptides from Bacillus brevis and Bacillus polymyxa against Botrytis grey mould in strawberry

Abstract

Grey mould caused by Botrytis cinerea is a devastating disease that results in extensive yield losses to strawberry. Bacillus brevis (Brevibacillus brevis) and Bacillus polymyxa (Paenibacillus polymyxa), which showed strong antifungal activity against B. cinerea, were isolated from the phyllosphere of strawberry plants. The advantage of using these bacteria is that the biochemistry and physiology of production of antibiotic peptides antimicrobial substances is well documented. A study was conducted to assess the activity of both Bacilli and their antibiotic peptides produced against B. cinerea in strawberry plants in vitro and in vivo. In vitro bioassay, both Bacilli have strongly inhibited pathogen germination, growth and extra-cellular enzyme production. Bacillus brevis was generally the most effective in reducing Botrytis growth. Gramicidin S and polymyxin B peptide antibiotics were extracted from culture filtrate of B. brevis and B. polymyxa, respectively, purified by silica thin chromatography and identified by high performance liquid chromatography. Germination, growth rate and production of extra-cellular enzymes were more sensitive to both antibiotics. Gramicidin S was the most active against B. cinerea with a minimal inhibitory concentration of 15 μmol/l. Polymyxin B also showed activity against B. cinerea at 25 μmol/l. Under controlled conditions (18 – 22°C, 90% relative humidity and 12 h photoperiod), strawberry plants were sprayed with pathogens (10⁵ spores/ml), antagonists (from 10⁵ to 10⁸ cells/ml) and antibiotic peptides (0 – 30 μmol/l) for reducing grey mould. Disease incidence was decreased in the presence of B. brevis. Both antibiotic peptides inhibited Botrytis growth that was observed by scanning electron microscopy. The plant leaves adsorbed significant amounts of antibiotics which reached from 46.1 to 67.5% of the original solution. Under natural field conditions, these biocontrol and antibiotic peptides at different concentrations were evaluated in 2003/2004 and 2004/2005 seasons against Botrytis grey mould. Treating plants with B. brevis exhibited a significant high activity against the development of Botrytis disease on strawberry. Gramicidin S showed a strong potential in reducing disease incidence, followed by polymyxin B, and acted as a fungicide to the pathogen growth. Inhibition of B. cinerea by both Bacilli was similar to equivalent levels of their antibiotics produced. In addition, these treatments significantly reduced the development of Botrytis and increased fruit yield. It can be suggested that B. brevis and B. polymyxa may be considered as potential biocontrol agents against Botrytis grey mould on strawberry based on the production of antifungal peptides. Therefore, gramicidin S and polymyxin B products are considered as biocontrol agents and may play a significant role in the future for practical applications in strawberry management systems.     

Nitrogen transformation processes in relation to improved cultural practices for lowland rice

Summary Inappropriate method and timing of N fertilizer application was found to result in 50–60% N losses. Recent nitrogen transformation studies indicate that NH₃ volatilization in lowland rice soils is an important loss mechanism, causing a 5–47% loss of applied fertilizer under field conditions. Estimated denitrification losses were between 28 and 33%. Ammonia volatilization losses from lowland rice can be controlled by i) placement of fertilizer in the reduced layer and proper timing of application, ii) using phenylphosphorodiamidate (PPD) to delay urease activity in flooded soils, and iii) using algicides to help stabilize changes in floodwater pH. Appropriate fertilizer placement and timing is probably the most effective technique in controlling denitrification at the farm level. The effectivity of nitrification inhibitors as another method is still being evaluated. With 60–80% of N absorbed by the crop derived from the native N pool, substantial yield gains in lowland rice are highly possible with resources already in the land. Extensive studies on soil N and its management, and an understanding of soil N dynamics will greatly facilitate the decrease in immobilization and ammonium fixation in the soil and the increase in N availability to the rice crop. Critical research needs include greater emphasis on N transformation processes in rainfed lowland rice which is grown under more harsh and variable environmental regimes than irrigated lowland rice.     

Application of iprodione by thermal fogging for the control of grey mould of tomato

A ready-to-fog' formulation of iprodione applied with themal foging equipment was a satisfactory alternative to high-volume (HV) sparys of iprodione for the control ofnatural infecton with grey mould (Botrytis cinerea) in an unheated tomato crop. There were no significant differences in numbers of droped fruit nor in the control of naturally-occurring lesions in stems or leaves, nor of ghost-spotting on fruit, between the HV sprays and fogs applied every fortnight. Except for ghost-spotting, control by fogs applied weekly was better than by eihter for nightly treatment. In contrast, the control of infection from B.cinerea inoculations on peitoles was significantly better by the fortnightly HV treatment than by either frequency of foging. Control of infection imporved with increase in the number of fungicide applications to the peitole before inoculation; two HV sprays gave complete control but six fog applications did not. The variation in the conrrol of infection by fogs and presumbly therefore in the distribution of fungicide within the 61 m² glasshouse compartments was small. Application of the iprodione fogging formulation significantly reduced the number of fruits see by about 5% compared with the HV treatment.