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IOBC-WPRS Bulletin Vol. 63, 2011


IOBC-WPRS Bulletin Vol. 63, 2011

Working Group "Multitrophic Interactions in Soil".
Proceedings of the meeting at Uppsala (Sweden), 11 - 13 June, 2009.
Editors: Welch, C., Fatehi, J., Finlay, R., Friberg, H., Funck Jensen, D., Hökeberg, N. and Steinberg, C.
ISBN 978-92-9067-238-8 [xvi + 79 pp.]


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Functional characterization of Trichoderma isolates antagonistic against Rhizoctonia solani
Muhammad Anees, Arne Tronsmo, Véronique Edel-Hermann, Linda Gordon Hjeljord, Cécile Héraud, Christian Steinberg

Abstract: The aim of the present study was to characterize sixteen isolates of Trichoderma
originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were
observed, for their ability to antagonize the phytopathogenic fungus R. solani AG 2-2 strain G6.
Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic
resistance in plant host were investigated using in vitro and in vivo tests in disinfested and nondisinfested
soils. This functional approach revealed the intraspecific diversity as well as
biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for
different strains. The most antagonistic strain, T30 was identified as T. gamsii.


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Soil microbial community changes under oilseed rape grown in shortened rotations
Amanda J. Bennett, Sally Hilton, Gary Bending, David Chandler, Peter Mills

Abstract: Microbial communities were analysed from bulk soil and rhizosphere samples
collected from a rotational field trial investigating the effect of shortened rotations on yield of
oilseed rape (OSR). Total DNA was extracted from the samples and Terminal Restriction
Fragment Length Polymorphism (TRFLP) analysis was carried out to obtain community profiles
for fungi and bacteria from selected rotational treatments. Cloning and sequencing were also
carried out to identify microorganisms that had dominant peaks in the TRFLP profiles. Changes
in the microbial communities were seen with the different rotations, and in particular the obligate
parasite Olpidium brassicae was found to be significantly more abundant in rhizosphere samples
from continuously cropped OSR than those from virgin OSR. Other fungi showing significant
differences in abundance with various rotations included Pyrenochaeta sp. and Verticillium
nigrescens. However, the involvement of these microorganisms in yield decline of OSR grown in
short rotation has not yet been elucidated.


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ACC deaminase-producing bacteria inhibit crown gall formation in tomato plants
infected by Agrobacterium tumefaciens or A. vitis

Natalia Dandurishvili,, Natela Toklikishvili, Naili Giorgobiani, Rusudan Keshelava, Marina Tediashvili, Ernő Szegedi, Bernard R. Glick, Alexander Vainstein, Leonid Chernin

Abstract: The plant hormone ethylene plays a key role in the development of crown galls, caused
by oncogenic Agrobacterium. Many plant growth-promoting bacteria (PGPB) produce the
enzyme 1-aminocyclo-propane-1-carboxylate (ACC) deaminase (ACCD) which can degrade
ACC, the immediate precursor of ethylene in plants, to α-ketobutyrate and ammonia and thereby
lower plant ethylene levels. In the present work, we have shown that the ACCD producing PGPB
strains Pseudomonas putida UW4 and Burkholderia phytofirmans PsJN as well as strain
Azospirillum brasilense Cd1843 (SP7) transformed by plasmid pRKTACC carrying the acdS
gene from UW4 under the tetracycline resistance (tet) promoter are able to significantly decrease
development of crown galls on tomato plants inoculated with strain A. tumefaciens Sh-1 or
A. vitis S4. The results suggest that ACCD provided by PGPB alter the balance of ethylene and
other phytohormones essential for tumorigenesis in plants transformed by pathogenic


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Aspergillus flavus interactions with biofumigation related compounds in soil
Stefania Galletti, Luisa Ugolini, Susanna Cinti, Pier Luigi Burzi, Stefano Cianchetta, Onofrio Leoni

Abstract: Biofumigation represents a low-impact alternative to chemical disinfestation aimed at
controlling soil-borne pathogens. This technique exploits the Brassicales defence system, which
releases biocidal compounds, mainly isothiocyanates, from glucosinolate hydrolysis by
myrosinase. The aim of our researches is to investigate the interactions of common nonpathogenic
soil fungi with compounds related to biofumigation. A soil isolate of Aspergillus
flavus was found to be able to enzymatically convert allyl glucosinolate into low toxicity
compounds, in liquid culture and in sterilised soil. This work is focused on investigating if
A. flavus enzymes are able to compete with Brassica carinata myrosinase during biofumigation,
preventing or reducing allyl isothiocyanate formation. In sterilised soil treated with Brassica
carinata seed meal, myrosinase prevailed on A. flavus enzymes and allyl isothiocyanate was
rapidly formed. However its concentration progressively decreased, faster than in the control or in
the presence of another fungal isolate (Trichoderma harzianum). The same was observed in vitro,
exposing the fungi to the vapours released by the wetted meal without a direct contact. This
method was adopted to screen among several fungal isolates, which showed differential
responses. The findings indicate that fungal enzymes cannot prevent the isothiocyanate formation
during biofumigation, but could cause a decrease of the active principle concentration, possibly
hampering biofumigation efficacy.


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Changes in root exudate composition mediated by pathogenic and mycorrhizal fungi
in tomato (Solanum lycopersicum L.)

Karin Hage-Ahmed, Vladimir Chobot, Wolfgang Postl, Andreas Voglgruber, Franz Hadacek, Siegrid Steinkellner

Abstract: Components in root exudates can act as signals and therefore play an important role in
triggering plant–microbe interactions, such as colonization by pathogenic or symbiotic fungi.
This work is based on a biological model system consisting of tomato (Solanum lycopersicum L.),
the soilborne tomato pathogen Fusarium oxysporum f. sp. lycopersici (FOL) and the arbuscular
mycorrhizal fungus (AMF) Glomus mosseae (GM) to assess metabolic changes in root exudation
caused by FOL and GM alone and in combination, respectively. In order to assess these
metabolic changes, HPLC-PDA and quadrupol GC-MS analyses were performed with special
focus on sugars, sugar alcohols, organic and amino acids, and secondary metabolites, such as
phenolic acids and flavonoids. Furthermore, the exudates were tested on the development of FOL
in vitro to give information about their influence on the initial behaviour and performance of


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Response of soil microbial community to compost amendment in an organic pear
orchard: preliminary data

Matteo Montanari, Gloria Innocenti, Claudio Ciavatta, Sante Scagliarini

Abstract: The effect of a municipal solid waste compost amendment at two rates (8 and 16t/ha x
year) on some soil microbial parameters was studied from 2006 to 2008 in an organic pear
orchard located in the Po Valley (Northern Italy). The compost amendment at the highest rate
significantly increased the total soil microbial activity measured by the FDA test. Also the soil
suppressiveness, assessed under controlled conditions using the pathosystem Pythium sp. – Beta
vulgaris, was improved by the highest rate of compost, even if statistically significant differences
were not found among treatments. The association of a specific species of Penicillium with
compost treatment was observed in 2007 and 2008.


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Identification of adaptive evolutionary changes in Trichoderma chitinase chi18-13
Magnus Karlsson, Katarina Ihrmark, Nashwan Asmail, Wimal Ubhayasekera, Petter Melin, Jan Stenlid

Abstract: Mycoparasitic fungi such as Trichoderma are used as biological control agents against
fungal pathogens on agricultural crops. Chitinases are hydrolytic enzymes that degrade fungal
cell walls and are important for the mycoparasitic attack. We here analyse the molecular
evolution of Trichoderma chitinase chi18-13, and show that changes in enzyme architecture are
preferentially localized to substrate- or product side of the catalytic cleft. These observations
suggest that mycoparasitic interactions can drive adaptive changes of enzymes. The approach can
be used to identify factors that are important for microbial interactions and to reveal relationships
between enzyme structure and function.


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Approaches to genetic stability, persistence and biological control of
Penicillium oxalicum strain 212

Inmaculada Larena, Gema Vázquez, Paloma Melgarejo

Abstract: This study focuses on the genetic stability, persistence and biological control of
Penicillium oxalicum strain 212 (PO212). In this study, we demonstrated that PO212 was more
effective for controlling Fusarium oxysporum f. sp. lycopersici (FOL) in tomato plants than 13
other P. oxalicum strains. When PCR using a specific primer pair for PO212 was combined with
serial dilutions of soil on semi-selective media (PoIM) PO212 can be identified and quantified
from peat in presence and in absence of tomato plant. Finally, this strain was genetically stable
after 2 years of long-term preservation.


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Exploring competitiveness and genetic variation in the nematophagous fungus
Pochonia chlamydosporia var. chlamydosporia and its significance for biological control

Rosa H. Manzanilla-López, Ian M. Clark, Simon D. Atkins, Penny R. Hirsch, Brian R. Kerry

Abstract: The fungus Pochonia chlamydosporia var. chlamydosporia is a potential biological
control agent of plant endoparasitic nematodes. Factors affecting control efficacy include fungusnematode
interactions, competition between isolates and intra-specific variation within
P. chlamydosporia. All require further investigation. PCR-based DNA fingerprinting techniques
can provide rapid means to examine the genetic variation of fungal isolates from various
geographical regions and nematode hosts, and also to monitor biocontrol agents after their release
into the soil. A series of experiments were conducted under glasshouse and in vitro conditions to
assess competitiveness for rhizosphere colonization and egg parasitism of selected Pochonia
isolates of the root-knot (Meloidogyne spp.) and cyst (Globodera spp., Heterodera spp.)
nematode biotypes as well as their compatibility for anastomosis. The abundance of Pochonia
isolates in two Portuguese sites under long-term cultivation of horticultural crops (tomato) was
determined by plating soil onto selective agar or quantitative PCR with species specific primers;
genetic diversity was assessed from ERIC-PCR profiles. Both sites showed a limited diversity of
the fungus but there were also different parasitic abilities. Isolate biotype selection and their
relevance to the management of nematode populations are discussed.


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Competitive interactions between the nematodes Aphelenchus avenae,
Aphelenchoides saprophilus and Aphelenchoides besseyi by cultivation on the
fungus Alternaria tenuis at different temperatures

Varvara Migunova, Alexander Shesteperov

Abstract: Interactions of three species of nematodes Aphelenchus avenae, Aphelenchoides
saprophilus and A. besseyi were investigated on potato dextrose agar in the presence of fungus
Alternaria tenuis at different temperatures. Competitive interaction between the nematodes was
measured as decrease in numbers of animals when they were grown in mixed cultures. A. avenae
was the best competitor compared with A. besseyi and A. saprophilus at temperatures of 15-25ºC
and 30ºC. The numbers of males of A. besseyi and A. saprophilus increased in the presence of
nematodes of other species. Temperature, reproduction rate and original properties of competitors
influenced interspecific interactions of the three nematode species.


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Effects of the arbuscular mycorrhizal symbiosis on biomass parameters of three
medicinal root drug plants

Monika Nell, Christoph Wawrosch, Siegrid Steinkellner, Horst Vierheilig, Chlodwig Franz, Johannes Novak, Brigitte Kopp, Karin Zitterl-Eglseer

Abstract: Due to an increase in phosphorus uptake root colonization by symbiotic arbuscular
mycorrhizal (AM) fungi often lead to beneficial effects on plant growth. Focussing the plant roots
this study was carried out to analyse whether the biomass growth of Valeriana officinalis
(valerian), Angelica archangelica (archangel) and Glycyrrhiza glabra (liquorice) could be
enhanced through the AM symbiosis. For that purpose two clones per species of in-vitro
propagated plants were tested with several AMF species. The AM root colonization of the
medicinal plants was high in all treatments inoculated with AMF. Generally the P nutrition of
AM plants was improved, but the effects on the root biomass were diverse.


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Stenotrophomonas rhizophila – a novel biocontrol agent for saline soils
Christoph Stephan Schmidt, Massimiliano Cardinale, Christin Zachow, Henry Müller, Gabriele Berg

Abstract: Stenotrophomonas rhizophila DSM 14405T promoted plant growth of a wide
variety of crops in saline soils of Uzbekistan. Contrary to its close relative S. maltophilia,
it is non-pathogenic to humans. Aim of this study is to elucidate mechanisms responsible
for the plant growth promoting effect and to identify environmental parameters (soil
salinity, accompanying rhizosphere flora, crop species) influencing the efficacy and
survival of this promising rhizosphere inoculant. In plant agar S. rhizophila builds up
high populations (108–109 per g plant) on all tested crop species (cotton, sweet pepper,
tomato, oilseed rape) irrespective of initial concentration (104 and 108 CFU/seedling).
Ecto- and endophytic growth in roots could be shown using dsred-labelled cells or FISH
by confocal laser scanning microscopy. Crops differed in response to the inoculant. At
low doses (104 cells per seedling), positive effects (increase in root tip number or length,
or increase in the number of secondary leaves) could be seen in cotton, tomato and sweet
pepper whereas high doses of 108 cells per seedling had negative effects in vitro. On the
contrary, oilseed rape did not show any significant response towards S. rhizophila at low
and high doses. S. rhizophila controlled seed borne diseases (Alternaria spec.) present in
the seed batches of cotton and sweet pepper. In non-sterile soil sweet pepper and tomato
showed the strongest positive response, whereas no effects were visible in cotton and
cucumber. The positive effects in non-sterile soil were more pronounced than in than in
gnotobiotic systems (plant agar and autoclaved soil). Neither in gnotobiotic systems nor
in non-sterile soil S. rhizophila alleviated salt stress directly. Our results indicate that S.
rhizophila either needs a factor present in soil for optimal plant growth promotion or
promotes the growth of plants indirectly via the inhibition of pathogens or deleterious
microorganisms in the rhizosphere.


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Functional aspects of biodiversity in relation to plant health
Christian Steinberg, Véronique Edel-Hermann, Hanna Friberg, Claude Alabouvette

Abstract: The most visible impact of human activities on biodiversity concerns species which are
part of our cultural heritage but a huge part of the species are still undescribed, mainly those in
the ocean, in extreme environments and of course in the soil where the damage caused by
anthropogenic activities are unknown. Although some estimation can be proposed, they are all
considered as under-evaluations of the putative number of taxa that could be present on the planet
Earth. Despite it is not yet known how, this biodiversity contributes to different services through
the functioning of ecosystems, and thus influences the sustainability of these ecosystems. At a
smaller scale, the one we can approach in the frame of this workshop, it is clear that biodiversity
is shaped by farmed ecosystems but in return, the organisms building up the community
contribute directly to the farmed ecosystems, via the regulation of parasites and diseases of crops
and the contribution to the fertility of the soils and to soil health. It seems therefore essential to
understand the nature of the interactions between biodiversity and agriculture to both preserve the
former and improve the latter. So far, none of the various indices used to measure the diversity
can be used as an indicator of the soil health, and the relation between diversity and soil
functioning is still difficult to understand. Sound use of agriculture practices should allow us to
control harmful organisms without eroding the biodiversity of these interacting components.


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A comparison of ecological elements of current safety assessments and regulation
of microbial and invertebrate biocontrol agents

Ingvar Sundh, Mark S. Goettel

Abstract: Current regulations and safety assessments for biological control agents (BCAs) differ
markedly among agents based on invertebrate animals (e.g. insects, mites, nematodes; IBCAs)
versus microorganisms (e.g. bacteria, fungi, viruses; MBCAs). Non-indigenous IBCAs are
regulated in several countries through quarantine legislation for prevention of introduction of
alien organisms, whereas indigenous IBCAs are not generally regulated. In contrast, all MBCAs
fall under legislation similar to chemical pesticides and need pre-market authorisation. Current
emphasis on ecotoxicological effects of MBCAs has resulted in unnecessarily complicated,
expensive and less relevant risk assessments and contributes to a very slow implementation of
MBCAs. The potential ecological hazards are similar between the two groups of BCAs, and the
environmental safety of both groups is therefore most pertinently evaluated according to
biological and ecological principles. Development of mutual systems for the regulatory oversight
of all living BCAs should be considered. One research area that needs to be further explored is to
what degree MBCAs persist and spread to surrounding areas. Another is the extent to which use
of MBCAs actually results in increased exposure of non-targets to microorganisms, compared to
“normal” background exposure. Finally, increased knowledge of the biogeography and microbial
ecology of representative MBCAs would facilitate determining whether a certain microbe is
indigenous or not. Native MBCAs should generally not need the same ecotoxicological oversight
as exotics, as is the present case with native IBCAs.


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Interactions between Clonostachys rosea IK726 and Pseudomonas chlororaphis PCL 1391
against tomato foot and root rot caused by Fusarium oxysporum f.sp. radicis lycopersici

Georgios D. Tzelepis, Anastasia L. Lagopodi

No abstract


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