Infection of red raspberry styles and carpels by Botrytis cinerea and its possible role in post-harvest grey mould

The gynecium was selected for histological studies of infection of red raspberry (Rubus idaeus) flowers in the laboratory because it is one of several possible infection sites leading to fruit grey mould. Infection of emasculated flowers occurred when dry conidia were dusted on to receptive stigmas. The conidia germinated in the stigmatic fluid of both non-pollinated flowers and flowers pollinated before inoculation, their germ tubes then penetrated the stigma and hyphae grew intercellularly through the transmitting tissues of the style to enter the carpel within 7 days. The possible roles of stylar infections and endophytic mycelium are discussed in the context of grey mould development.     

Systemic infection of black currant flowers by Botrytis cinerea and its possible involvement in premature abscission of fruits

Emasculated flowers of several black currant cultivars were pollinated and then inoculated with dry conidia of Botrytis cinerea in the field and glasshouse. The infection of pistils was examined by U.V. fluorescence microscopy and the incidence of premature flower abscission recorded. Conidia germinated in the stigmatic fluid in all cultivars and hyphae spread symptomlessly throughout the style to infect the pericarp and ovules. Of six cultivars inoculated in the field, cv. Ojebyn was the most, and cv. Ben More the least resistant to flower shedding. Natural infection of stigmas by B. cinerea was common in the field and a high proportion of apparently healthy non-inoculated flowers which abscissed were found to contain infected ovules. Fewer flowers abscissed if inoculations were made 6 days after pollination. Symptomless or latent infection of black currant flowers by B. cinerea may be a contributory cause of premature abscission of developing fruits, or ‘running-off’, recorded in these experiments.     

Involvement of ethylene in the disease caused by Botrytis cinerea on rose and carnation flowers and the possibility of control*

Ethylene production by flowers, petals and leaves of rose was correlated with severity of grey mould. However, when the host became completely macerated, ethylene production diminished. Ethylene production by Botrytis cinerea grown on autoclaved flowers which were supplemented with methionine was negligible. Methionine spray, incubation with ethylene, or precooling of flowers at 4°C increased disease incidence considerably. Ethylene also induced susceptibility of carnation flowers to attack by B. cinerea. On the other hand, sprays of silver thiosulphate (STS) aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) decreased disease severity in rose petals and leaves inoculated with mycelial plugs or conidia. Treatment of cut rose flowers with STS (by dipping) or AOA (by spraying) significantly decreased disease incidence during subsequent incubation at 20 and 10°C. This suggests a treatment for reducing grey mould damage in flowers transported overseas.     

Factors affecting the development of Botrytis cinerea (grey mould) on linseed (Linum usitatissimum) buds, flowers and capsules

A key was produced to describe 10 stages of development of linseed buds, flowers and capsules. Botrytis cinerea conidia germinated more rapidly and germ tubes grew longer on linseed stigmas, petals and mature senescing capsules than on green leaves, sepals and immature capsules. The proportion of conidia which germinated increased and the germ tubes continued growing for longer in the presence of linseed pollen and flower petal extracts. In controlled environment and field experiments, the response of buds, flowers and capsules to inoculation with B. cinerea changed with stage of development; few pre‐flowering buds developed symptoms (brown lesions, then grey mould), but high proportions of flowering and post‐flowering buds did so. Few immature green capsules developed symptoms and the proportion of capsules which developed symptoms increased as they matured. The presence of linseed pollen decreased the incubation period from inoculation with spore suspensions to appearance of B. cinerea symptoms on buds. A disease cycle was produced to suggest the changes in susceptibility of linseed to infection by B. cinerea conidia during bud, flower and capsule development.     

Efficacy of plant essential oils on post-harvest control of rot caused by Botrytis cinerea on kiwi fruits

Antifungal activity of the essential oils of Carum carvi and Pimpinella anisum against Botrytis cinerea fruit rot of key kiwi fruit was studied. In vitro experiments, antifungal activities of essential oils were tested on potato dextrose agar media. Results of an in vitro experiment showed that these essential oils, at all applied concentrations, inhibited grey mould growth. Black caraway essential oil at concentrations of 600 and 800?μL?L^?1 inhibited germination spores of grey mould. Then, the fruits were artificially inoculated with a suspension at 1?×?10⁵?conidia/ml and then treated with different concentrations of these essential oils. The results of in vivo conditions showed that black caraway and anise essential oils applied at all concentrations were increasing the shelf life and inhibited the grey mould growth on kiwi fruits completely in comparison to control. The result showed that black caraway and anise oils at a concentration of 800?μL?L^?1 had higher total soluble solids, ascorbic acid, titrable acidity and antioxidant content compared to untreated fruits.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.     

Pollen transfer in fragmented plant populations: insight from the pollen loads of pollinators and stigmas in a mass‐flowering species

Pollinator and/or mate scarcity affects pollen transfer, with important ecological and evolutionary consequences for plant reproduction. However, the way in which the pollen loads transported by pollinators and deposited on stigmas are affected by pollination context has been little studied. We investigated the impacts of plant mate and visiting insect availabilities on pollen transport and receipt in a mass‐flowering and facultative autogamous shrub (Rhododendron ferrugineum). First, we recorded insect visits to R. ferrugineum in plant patches of diverse densities and sizes. Second, we analyzed the pollen loads transported by R. ferrugineum pollinators and deposited on stigmas of emasculated and intact flowers, in the same patches. Overall, pollinators (bumblebees) transported much larger pollen loads than the ones found on stigmas, and the pollen deposited on stigmas included a high proportion of conspecific pollen. However, comparing pollen loads of emasculated and intact flowers indicated that pollinators contributed only half the conspecific pollen present on the stigma. At low plant density, we found the highest visitation rate and the lowest proportion of conspecific pollen transported and deposited by pollinators. By contrast, at higher plant density and lower visitation rate, pollinators deposited larger proportion of conspecific pollen, although still far from sufficient to ensure that all the ovules were fertilized. Finally, self‐pollen completely buffered the detrimental effects on pollination of patch fragmentation and pollinator failure. Our results indicate that pollen loads from pollinators and emasculated flowers should be quantified for an accurate understanding of the relative impacts of pollinator and mate limitation on pollen transfer in facultative autogamous species.     

Pollination intensity and potential seed set in Passiflora vitifolia

Summary Initial seed set and fruit set were pollen-limited in a Costa Rican population of Passiflora vitifolia, a self-incompatible species with 200–350 ovules per flower. Pollination intensity was measured by counting the number of allogamous pollen grains on stigmas of the large one-day flowers. Hand-pollinations demonstrated that 25–50 pollen grains are required for fruit set, and >450 are needed for maximum seed set, with a pollen:seed ratio of about 1.6:1.0. Hummingbirds (Phaethornis superciliosus) delivered sufficient allogamous pollen for maximum seed set to only 28% of the flowers examined. Naturally pollinated flowers yielded fewer fruits and fewer seeds per fruit than those pollinated by hand. Most pollen transferred by humming-birds was self-incompatible; emasculated flowers yielded higher seed set than flowers with intact anthers. Visitation rates did not provide a good index of effective pollination.There were significant differences in ovule number, maximum seed set, and maximum per cent seed set among individual vines. More than half of an individual's flowers failed to set fruit, whether pollinated by birds or by hand. In this population, maximum reproductive potential may be limited by maternal resources for fruit development, but seed set varies with pollination intensity. Pollen-limited seed set may be a disadvantage of self-incompatibility, especially in species with many-seeded fruits.     

In vitro spore germination and infection of cultivars of Rubus and Rosa by downy mildews from both hosts

The cardinal temperatures for in vitro germination of conidia of imported and indigenous isolates of downy mildew from hosts in the genera Rubus and Rosa were similar. A high percentage of conidia germinated above 2°C and germination remained between 80% and 90% up to 15°C or 20°C, depending on the isolate. The highest incidence of disease on leaf disks of Tummelberry (blackberry × red raspberry) inoculated with an isolate of Peronospora rubi occurred at c. 15°C, with infection over a range from 2°C to 28°C. Tests on leaf disks in vitro, and leaflets of primocane and lateral shoots in plastic tunnels, with three hybrid berry (blackberry x red raspberry), six blackberry and nine red raspberry cultivars showed the hybrid berries to be most susceptible. In a plastic tunnel infected drupelets of red raspberry fruits developed more slowly and failed to ripen evenly compared with uninfected drupelets. Similar malformation of infected fruits occurred in a plantation of Tummelberry. An isolate of P. rubi attacked severely both Tummelberry and rose cv. Can Can. Fluorescence microscopy after staining with aniline blue showed that leaf disks of Tummelberry were extensively colonised by intercellular mycelium of P. sparsa isolated from rose, even though sporulation was sparse or absent. This supports the view that P. rubi and P. sparsa may be conspecific. Oospores of P. rubi were found routinely within leaf disks of Rubus cultivars inoculated in vitro and once in naturally infected leaflets of Tummelberry.     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

The phenylpropanoid pathway affects apple fruit resistance to Botrytis cinerea

Apple fruits are rich in phenolic compounds that may enhance resistance to grey mould disease caused by Botrytis cinerea. Using Malus domestica Borkh. cultivars Fuji and Qinguan, we analysed the contents of total phenols, total flavonoids, eight individual phenolic compounds, H₂O₂ and O₂^.? as well as the activities of key enzymes in the phenylpropanoid pathway in the flesh of control and B. cinerea‐inoculated fruits. Chlorogenic acid contents increased for a short period in the less susceptible cultivar Qinguan fruits, but decreased in the disease‐susceptible Fuji fruits. Additionally, ferulic acid production was induced in both cultivars in response to B. cinerea. Furthermore, the activities of phenylalanine ammonia lyase, cinnamate 4‐hydroxylase, 4‐coumarate:coenzyme A ligase and cinnamyl alcohol dehydrogenase were differentially induced between the two apple cultivars. Remarkably, the contents of H₂O₂ and O₂^.? as well as the activities of enzymes in phenolic metabolism tested in this study were always higher in Qinguan fruits than in Fuji fruits. Our data imply that phenylpropanoid metabolism is closely associated with apple fruit resistance to grey mould disease. These findings may be useful for characterizing the mechanism(s) underlying plant resistance to B. cinerea, with potential implications for the screening of grey mould disease‐resistant apple varieties in breeding programmes.     

Harvesting and bedding practices in relation to grey mould of strawberries

Relationships of cultural and harvesting practices in strawberries to epidemics of grey mould, caused by Botrytis cinerea, were examined in field plots in 1983 - 84 and 1984 - 85. The strawberries were grown in matted rows or solid beds and harvested by hand or by machine. Foliage cut by the machine was removed by hand-raking. Potential sporulation of B. cinerea on dead strawberry leaves, the principal inoculum source, was estimated by quantifying sporulation on samples of the leaves incubated in humidity chambers. During May, the incidence and density of sporulation, and total spore production per unit area of strawberry bed, usually were lower in plots harvested by machine in the preceding year than in those harvested by hand. Machine harvesting in the preceding year also suppressed incidence of grey mould on the fruits in June. Potential sporulation on dead leaves was usually less in solid beds than in matted rows in 1984 but not in 1985. Bed type usually did not affect incidence of grey mould fruit rot. No relationship was found between dry weights of dead leaves from sampling quadrats and potential sporulation on the leaves. It was postulated that the long-term impact of machine harvesting on grey mould epidemics was related to removal of the majority of the foliage and berries from the plots during harvesting operations.     

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.     

A method for in vitro production of conidia of Elsinoe veneta and the inoculation of raspberry cultivars

Conidia of two isolates of Elsinoe veneta were consistently produced in vitro by a 3-stage culture system in which sparsely sporulating microcolonies produced on corn meal agar were used to ‘seed’ Czapek-Dox V8 juice agar. E. veneta then sporulated on this medium if dises were floated mycelium uppermost on sterile distilled water for 24 h at 25°C. Both isolates also produced microconidia in vitro. Only the growing apices of raspberry canes were susceptible to infection. Of 12 genotypes inoculated in the glasshouse, a red raspberry derivative of Rubus coreanus, EMRS 2769/9, was the most resistant and the black raspberry selection SCRI 815286 and cv. Malling Delight were the most susceptible. Of four red raspberry cultivars inoculated in the field cv. Malling Orion was the most susceptible. Inoculated flowers and developing fruits produced malformed fruits because infected drupelets were smaller or slower to ripen than uninfected ones.     

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.     

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.     

POLLEN FLOW WITHIN A TRISTYLOUS SPECIES: LYTHRUM SALICARIA

Lythrum salicaria L. is a classic example of tristyly. Pollen flow within a natural population of this species was studied by analyzing pollen loads on stigmas of both intact and emasculated plants. The data obtained indicated that a significant amount of self pollination occurs within flowers but that between flowers or plants pollinations are predominantly legitimate, i.e. between stamens and styles of equal length. It is concluded that while the multiallelic system excels in reducing frequency of incompatible pollinations between plants, the heteromorphic system is relatively more effective in reducing self pollinations within a plant.     

Status of self-pollen in bee pollination efficiency of white clover (Trifolium repens L.)

Flowers of white clover (Trifolium repens L.) are hermaphrodite and self-incompatible; their cross-pollination depends entirely on insect visitors, mainly bees (Apoidea). Because self-pollination of white clover occurs before flower anthesis, we determined whether selfing affected the pollination efficiency of a honeybee visit. We compared pollen deposition in emasculated and intact flowers following (1) a single honeybee visit, (2) open-pollination for a day and (3) enclosure in a cloth bag to prevent insect visits. In emasculated flowers, open-pollination resulted in more pollen deposited than after one visit (+30%) which is consistent with flowers being visited more than once by pollinators during the course of a day. On intact flowers, saturation of the stigma was achieved after the first visit of a honeybee (near 280 grains) because of self-pollination. Additional visits did not increase pollen deposits, but they improved pollen efficiency in terms of numbers of pollen tubes reaching the ovules. In such a context of easily saturated stigmas, self-pollen does not inhibit cross-pollen activity, but represents a constraint for pollination which demands multiple bee visits to each flower to achieve maximum fertilization. Received: 20 May 1997 / Accepted: 25 October 1997