The objective of the study was to achieve a phytosanitary effect on fruiting bodies of
V. inaequalis in apple leaf litter by application of nutrient media, potential antagonists
and/or fungicidal plant extracts (saponins). The effects of leaf litter treatments on the
remaining ascospore potential were assessed in course of the primary season of the
pathogen. The principles and mode of action of the applied substances were
evaluated by microbiological and biochemical analyses. As an alternative method for
the isolation of saponins from plant materials an extraction with chloroform and
methanol was developed. The saponin extracts were cleaned with SPE cartridges
and for the first time with Hydrophobic Interaction Chromatography. Nearly all
ineffective substances could be removed from the extracts. The detection of
membranolytic/hemolytic fractions was performed with an agar diffusion assay
amended with sheep erythrocytes. Hemolytic fractions were further characterized by
HPLC analyses. In vitro all plant extracts showed a fungicidal effect on conidia
germination and mycelia growth of V. inaequalis. In seedling assays, a preventative
treatment with soap nut and chestnut extract (1%) reduced apple scab symptoms
nearly completely. A treatment 6 h after inoculation with conidia reduced scab
symptoms by more than 70%. Pathogen sporulation on infected leaves was reduced
by 98-100% in comparison to control groups. Ascospore potential could be reduced
by leaf litter treatments by up to 90%. The application of in vitro identified antagonists
on apple scab litter showed no effect on ascospore amount. Leaf litter treatments
with nutrient media in autumn/winter revealed a reduction in the ascospore potential
of more than 97% in four following apple scab seasons. The effect did not depend on
geographical location of the orchard site. The best results could be achieved with a
concentrated LEIBER yeast extract, which reduced the ascospore amount nearly
completely. The microbiological analyses of leaf litter showed an increase of the
number of organisms, including potential antagonists, from 10 up to 1.000 times. The
microbiological activity (biological oxygen demand) was up to three times higher and
leaf decay was enhanced compared to untreated litter. Bacterial and fungal leaf
colonizers were isolated and identified with MALDI-TOF or sequencing of ITS rDNA.
Pseudomonas proved to be the dominant bacterial species in apple leaf litter and
contributed 37 to 45% of bacterial isolates. In vitro an inhibition of mycelia growth of
V. inaequalis from 48 to 69% was observed. Cladosporium cladosporioides,
Aureobasidium pullulans and Epicoccum nigrum reduced mycelial growth by up to
57%. Each one of these species represented more than 10% of the fungal isolates.
In the majority of samples the microbial composition was changed mainly
quantitatively and a shift within the dominant species was not detectable. Peptides
≤ 3 kDa and amino acids could be identified as the effective constituents of the
media. Leaf litter treatments with nutrient media could be established as a new
phytosanitary strategy in organic fruit production and contribute to a replacement of
coppere1fungicides.
Aktualisiert: 2020-01-13
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