Plant and Soil (2005) 268: 75–87
DOI 10.1007/s11104-004-0228-1
© Springer 2005
Negative feedback on a perennial crop: Fusarium crown and root rot of
asparagus is related to changes in soil microbial community structure
C. Hamel1,2,5 , V. Vujanovic3 , R. Jeannotte2 , A. Nakano-Hylander2,4 & M. St-Arnaud3
1 Current
address: Environmental Health / Water and Nutrients, Agriculture and Agri-Food Canada, 1 Airport
Road Box 1030, Swift Current (SK) S9H 2X3 Canada. 2 Natural Resource Sciences, Macdonald Campus of
McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue (QC) H9X 3V9 Canada. 3 Institut de recherche en
biologie végétale, Université de Montréal and Jardin botanique de Montréal, 4101 Sherbrooke est, Montréal (QC)
H1X 2B2 Canada. 4 Current address: Microbial Ecology, Ecology Building, Lund University, SE-223 62 Lund,
Sweden. 5 Corresponding author∗
Received 22 January 2004. Accepted in revised form 26 April 2004
Key words: Asparagus officinalis, arbuscular mycorrhizal fungi, crop management, disease development, phospholipid fatty acid profile, plant-microbe interaction, soil microbial community structure
Abstract
The dynamic equilibrium of an ecosystem is driven by mutual feedback interactions between plants and soil microorganisms. Asparagus exerts a particularly strong influence on its soil environment through abundant production
of persistent phenolic acids, which impact selectively soil microorganisms and may be involved in Fusarium crown
and root rot (FCRR) of asparagus. In a survey of 50 asparagus plantations of the province of Québec, we found
that FCRR was associated with a profound cultivar-specific, reorganization of the soil microbial community,
as revealed by phospholipid fatty acid (PLFA) profiling. According to PLFA indicators, microbial biodiversity
as well as bacterial and fungal abundance dropped sharply with the onset of FCRR in fields planted with the
cultivar Guelph Millenium. This drop was followed by a similar drop in the arbuscular mycorrhizal population.
Biodiversity and microbial population size then increased to finally reach a new equilibrium. Discriminant analysis
of PLFA profiles obtained from soil samples also indicated a shift in soil microbial community structure associated
with FCRR development in fields planted with the cultivar Jersey Giant. Different soil biological conditions,
as indicated by microbial biomass C and N and soil enzyme activities, were associated with different cultivars.
Preceding crop, manure application, geographical location and tillage depth also influenced the structure of soil
microbial communities in asparagus plantations, as determined by PLFA profiling. If higher FCRR incidence is
a consequence of the soil microbial community reorganization, means to reduce FCRR incidence in asparagus
plantations may be found among practices such as soil organic fertilization, soil tillage and intercropping strategies
that would dilute the negative influence of asparagus on the soil microbial community. Finally, FCRR outbreaks
were generally promoted by a previous crop of maize. It seems that maize and asparagus host a F. proliferatum
teleomorph (Gibberella fujikoroi) of the same mating type.
Abbreviations: FCRR – Fusarium crown and root rot; PLFA – phospholipid fatty acid; FAME – fatty acid methyl
ester.
Introduction
A growing body of evidence indicates that soil organisms have a strong influence on plant community
dynamics (Bever, 2003). Some plant species typi∗ FAX No: +1(306)778-3188. E-mail: hamelc@agr.gc.ca
cally create self-stimulating biological conditions in
the soil they occupy. This enhances their growth in
a positive feedback reaction. In contrast, other plants
species create soil microbiological conditions that are
harmful to themselves. Such negative feedback-related
auto-inhibition of growth in the latter plants has been
76
related with the accumulation of pathogen in the soil
of their rooting zone (Klironomos, 2002).
The reason why some plants species accumulate
pathogens is unclear. Negative feedback has been related to the production of allelochemicals by plants
(Souto and Pellissier, 2002). While most allelochemicals would not build to toxic levels under natural conditions, allelopathy could be a problem in perennial
monoculture of agricultural plants expressing negative
feedback dynamics.
Asparagus (Asparagus officinalis L.) is a perennial
plant that releases allopathic compounds in soil. Asparagus stems, root and old litter extracts can inhibit
asparagus seed germination (Young and Chou, 1985).
Experiments in which concentrations of inhibitory
compounds were found in asparagus rhizosphere soil
extracts and root exudates revealed that they are rootproduced, and may inhibit the growth of a variety
of plants in addition to being auto-toxic (Young and
Chen, 1987). These inhibitory compounds were identified as phenolic acids (Young and Chen, 1987) and
saponins (Lake et al., 1993). Some inhibitory materials were also located in the amino acid/carbohydrate
fraction of asparagus root extracts (Lake et al., 1993).
Asparagus stands decline through years, a syndrome that was attributed to the activity of pathogens.
In particular, asparagus is parasitized by a complex
of Fusarium spp. namely, Fusarium proliferatum (T.
Matsushima) Nirenberg [synonym: F. verticilloides
(Sacc.) Nirenberg previously known as F. moniliforme
Sheldon (Seifert et al., 2003)], F. culmorum (Wm.G.
Sm.) Sacc., F. oxysporum Schlenchtend.: Fr. f. sp. asparagi S.I. Cohen & Head (Elmer, 2000, 2001) and
F. redolens Wollenw. f. sp. asparagi Baayen (Baayen
et al., 2000). These Fusarium spp. cause Fusarium
crown and root rot (FCRR) in asparagus. Depopulated
areas in asparagus plantations are typical indications
that FCRR has occurred (Elmer, 2001).
Pathogenic Fusarium spp. are ubiquitous in asparagus plantations (Blok and Bollen, 1996b; Vujanovic et al., 2004), but FCRR is only expressed
sometimes in pathogenic Fusarium infected plants
(Blok and Bollen, 1996b; Elmer, 2000). The erratic
nature of FCRR expression could be explained by a
requirement for some facilitating conditions, in addition to the presence of a pathogenic Fusarium strain in
the plant environment.
The possibility that some soils would be conducive to FCRR expression was recently raised (Fravel
et al., 2003). FCRR facilitating conditions may have
physicochemical and biological components. First,
asparagus is auto-toxic. Inhibitory compounds may
accumulate in soil with time and increase the susceptibility of asparagus itself to FCRR as the plantation
ages. Furthermore, root extract of this plant were
found to be inhibitory to several soil fungi, but not
to Fusarium spp. (Blok and Bollen, 1996c). In particular, ferulic, caffeic and methylenedioxy cinnamic
acids, have been shown to negatively affect arbuscular
mycorrhizal fungi (Pederson et al., 1991). In contrast,
formononetin, a molecule that stimulates arbuscular
mycorrhizal fungi, has reduced the percentage of asparagus root lesions caused by Fusarium spp. when
compared to a non-treated control (Elmer, 2002). Arbuscular mycorrhizal fungi are well known as wide
spectrum biocontrol agents (Dehne, 1982; St-Arnaud
et al., 1995), and were associated to reduce severity of FCRR in asparagus seedlings (Elmer, 2002)
and in commercial asparagus plantations (Matsubara
et al., 2001). Higher FCRR incidence was also associated with reduced populations of Mn-reducers, mostly
fluorescent pseudomonads, among which some had
biocontrol activity (Elmer, 1995a, 2003). Therefore,
the incidence of FCRR in asparagus plantations might
be the expression of a negative feedback created by
the alteration of the soil microflora following phenolic
acid release in soil.
Asparagus could produce good yields for 15 years
or more, but FCRR chronically affects plantations, reducing the productivity and profitability of the crop.
The development of crop management strategies to reduce FCRR incidence requires a better understanding
of the ecological basis of the disease. The objective
of this study was then to describe the soil microbial
communities that exist at different levels of FCRR expression, within the commercial asparagus plantations
of the province of Québec, Canada. This article reports
on the reorganization of soil microbial communities
accompanying FCRR development. A companion paper reports on the ecological conditions associated
with FCRR development (Hamel et al., unpublished),
and an in depth description of the mycodiversity and
mycogeography of the Fusarium spp. isolated from
the soil and plants of this study is also reported
elsewhere (Vujanovic et al., 2004).
77
Materials and methods
Experimental approach
Fifty commercial asparagus plantations located in the
four asparagus growing regions of the province of
Québec were characterized, in an attempt to find relationships between indicators of soil microbial community structure and function, and FCRR incidence.
The FCRR incidence in plantations was evaluated
visually and scored on a percentage scale. Eighteen continuous variables were used to describe soil
microbial quality. They were: activity of dehydrogenase, phosphatase and β-glucosidase, soil microbial biomass C and N, percentage of asparagus roots
harbouring arbuscular mycorrhizal colonization (Giovannetti and Mosse, 1980), total fungi and Fusarium in rhizospheric soil, in roots, in crown and in
stems, the abundance of fungal-, arbuscular mycorrhizal fungi- and bacterial phospholipids fatty acid
(PLFA) indicators extracted from soil, and Shannon
Weaver biodiversity index calculated from PLFA profiles. Management practices that were presumed to
influence the quality of the soil microbial community in asparagus plantation were monitored. These
were: Fertilizers (mineral fertilizers, compost, manure of rabbit, chicken, cow, or mixture of cow and
chicken manure), time of organic fertilizer application (pre-planting, in spring, after harvest, or in fall),
preceding crops (strawberry, maize, green manure, cereal crops, and other infrequently used species), time
of soil tillage (end of summer, in spring, in fall or
only before planting), tillage tools used (disc harrow
or a rotovator), cultivars (Guelph Millenium, Jersey
Giant, Viking KB3, Jersey Knight, SYN-456, Mary
Washington, and Lucullus) and plantation age, which
spanned from 1 to 21 years old.
Data collection and sample analysis
The information on the crop management practices
used was collected from plantation owners through a
survey form filled by owners with the assistance of
local agronomists. The soil and plants of two microplots, defined within the 50 asparagus fields, were
sampled during the two last weeks of June 2001, a
period corresponding to the end of the asparagusharvesting season. The two micro-plots, one with
asymptomatic and the other with symptomatic asparagus plants, covered a triangular area with approximately 10 m between the points of the triangle.
In total, six whole plants (three diseased and three
asymptomatic plants) were taken from each field. A
100 g samples of the 0–15 cm soil layer were collected in proximity of each plant using a soil probe.
The three soil samples were combined and mixed thoroughly to obtain one representative soil samples of
300 g per micro-plot. Soil and plant samples were
placed in paper bags and air-dried. Soil samples were
split in two parts. One part was kept at 4 ◦ C and
used within a few days to determine potential dehydrogenase, phosphatase and β-glucosidase activities
(Tabatabai, 1982), for microbial biomass C and N determination, using the fumigation extraction method
(Voroney et al., 1993) and for Fusarium counts.
Fusarium species were isolated from soil using dilution (10−2 ) series made using 3-g sub-samples from
each of the soil samples taken. Plant tissues were
treated with tap water to remove soil particles, surface sterilised for 3 min in 2.5% sodium hypochlorite
containing 0.1% Tween 20, and rinsed three times in
sterile distilled water, before isolation of microorganisms from plant roots, crowns and stems. Each plant
tissue was cut into small sections with a sterile scalpel.
Five sections (approximately 1 cm2 of plant tissues)
were plated in each 9-cm Petri dish containing either a
Fusarium selective medium, Myclobutanil agar (Vujanovic et al., 2002), or a general (PDA) medium.
The myclobutanyl medium allows both the enumeration and identification of Fusarium spp., at least to
the genus level. Soil dilutions and plant tissues from
each of the three sampling points of each micro-plot
was plated in five replicates (i.e., in five dishes). Petri
dishes were incubated at 22 ◦ C in the dark and observed after 7 days. The total fungal colony forming
units (CFU) counts were averaged to yield one total
fungi CFU count and one Fusarium spp. CFU count
for each of soil, crown, root and stem of each experimental unit, the micro-plot. Fusarium colonies were
examined microscopically to confirm the identity of
species using standard literature (Burgess et al., 1988;
Gerlach and Nirenberg, 1982; Nelson et al., 1983).
Soil microbial community structures were determined by PLFA. Soil samples had been stored at
−10 ◦ C prior to phospholipids fatty acid (PLFA) extraction. Total soil lipids were extracted from six to
eight grams of freeze-dried soil sample using an Accelerated Solvent Extractor ASE 200 (Dionex Corporation, Sunnyvale, CA, USA) according to the method
of Macnaughton et al. (Macnaughton et al., 1997). The
extraction mixture was chloroform: methanol (1:2)
heated at 80 ◦ C and maintained under a pressure of
8.22 kPa for 5 min before filtration. This extraction
78
cycle was repeated three times on each sample. Lipid
class separation on silicic acid gel column and fatty
acid methyl ester (FAME) transformation followed the
method of White and Ringelberg (White and Ringelberg, 1998). Individual fatty acids have been used as
signatures for various taxonomic groups of microorganisms (Bardgett et al., 1999; Bossio et al., 1998;
Grayston et al., 2001; Pankhurst et al., 2002; Yao et al.,
2000). We chose C12:0, C13:0, 2OH-C12:0 and 3OHC12:0, C14:0, 2OH-C14:0 and 3OH-C14:0, 14-methC15:0, C15:0, C:16, 15-meth-C16:0, c9,10-methC16:0, C17:0, C18:0, C18:1ω9c, c9,10-meth-C:18,
and C20:0 to represent bacterial PLFAs. The PLFA
C18:2ω6 was taken as indicator of fungal biomass
(Frostegard and Baath, 1996) and C16:1ω5, as indicator of extraradical mycorrhizal hyphae and spores
(Olsson, 1999). Peak identification was based on retention time as compared to standards (Supelco 37
Component FAME Mix #47885-U, Supelco Bacterial
Acid Methyl Esters #47080-U, and 11-hexadecenoic
acid (C16:1ω5) methyl ester constructed from its
fatty acid (Matreya/BioLynx cat. No. 1208) converted
into methyl ester with BF3/methanol. The arbuscular
mycorrhizal fungal biomarker (C16:1ω5) was tested
against lipid extracts from Glomus intraradices hyphae and spores produced in vitro on Agrobacterium
rhizogenes transformed carrot roots (St-Arnaud et al.,
1996) prior to analysis. Shannon Weaver biodiversity index was calculated as H = pi ln pi, where
pi = peak area of i th peak over the area of all peaks.
For this calculation, all peaks associated to fungal and
bacterial indicators were utilized.
Data analysis
A principal component analysis was conducted to
interpret the data expressing soil microbial activity,
microbial abundance, disease symptom severity as related to cultivars and age of plantations. This analysis
did not considered PLFA profiles, which were more
appropriately analyzed by discriminant analyses.
Discriminant analysis was used to assess differences in soil microbial community structure at different FCRR incidence levels, and the impact of different
management practices on the soil microbial community structure. The combination of all identified PLFAs
was used in these analyses. Plantation FCRR levels
where grouped into classes prior to analysis (0–1%,
2–5% and more than 5%, for Guelph Millenium plantations, and 0–2%, 3–4%, 7–11% and more than 11%,
for Jersey Giant). Grouping was based on the rela-
tionship found between FCRR incidence level, and
biodiversity index and PLFAs levels. The range of
FCRR incidence levels present in the Guelph Millenium and Jersey Giant datasets was also considered.
Only functions with Eingenvalues ≥1 were retained in
models. Significant canonical scores were plotted to
illustrate how treatments clustered. Mahalanobis distances, which are a measure of the distance between
points and their group means (centroids), were calculated to insure that PLFAs profiles were correctly
classified within groups. The associated probabilities were used to detect significant separations among
FCRR groupings.
The relationship between FCRR incidence levels
within each field and the abundance of the PLFA
marker of arbuscular mycorrhizal fungi was assessed
by regression analysis. Analyses were conducted on
the entire dataset and on two subsets that were either
related to the cultivar Guelph Millenium or to Jersey Giant, as the other cultivars were not planted into
enough fields to allow meaningful analyses.
All the statistical analyses were conducted using the software Systat (v. 9.1, Systat Software Inc.,
Richmond, CA, USA).
Results
Biological influence in the plantations system
Cultivars behaved differently in the asparagus plantation system studied (Figure 1). In general, the more
ancient cultivars were associated with FCRR incidence. Plantation age also had a large impact, as the
most recently released cultivars are preferentially used
when a field is planted. This is expressed in Figure 1
where cultivars are placed along an age gradient going
from Guelph Millenium, with an average plantation
age of 3.2 years old in the upper left corner of the
graph, to Lucullus, SYN-345 and Mary Washington
plantations, with average ages of 10, 10.4 and 13 years
old, respectively, in the lower right area of the graph.
Jersey Knight and Jersey Giant plantations, with average ages of 6.3 and 7.5 years old, are at intermediate
locations in the graph. Viking KB3 plantations, with
an average age of 16.8 years old, deviate from the
general pattern; they are the oldest plantations, but
appeared at an intermediate location in the graph. This
reflects that is spite of their old age, the FCRR incidence of Viking KB3 was low (9%), ranking third
only after Jersey Knight (4.9%) and Guelph Millenium
(5.5%).
79
Figure 1. Factor loading plot constructed with factor 1 and factor 2 of a factorial analysis showing relationships between asparagus cultivars
(cvGM, Guelph Millenium; cvJK, Jersey Knight; cvVi, Viking KB3; cvJG, Jersey Giant; cvLu, Lucullus; cvSy, SYN-456; cvMW, Mary
Washington), and soil biology related variables i.e., Fusarium crown and root rot incidence (FCRR), soil enzyme activity (β-glucosidase, Gase;
acid phosphatase, Pase; and dehydrogenase, Dase), soil microbial abundance (Mb-C, soil microbial biomass carbon; Mb-N, soil microbial
biomass nitrogen), total cultivable fungi in plant stem (FTstem ), crown (FTcrown ), root (FTroot ) and rhizosphere soil (FTsoil ), and total Fusarium
in plant stem (Fusstem ), crown (Fuscrown ), root (Fusroot ) and rhizosphere soil (Fussoil ). Factor 1 and factor 2 explained 30.4% of the variation
encountered in the data set.
Interestingly, FCRR incidence showed little relationship with the abundance of Fusarium and total cultivable fungi in asparagus tissues and in rhizosphere
soil. Rather, it appeared inversely related to soil
enzyme activity and overall soil microbial biomass.
Microbial community structure analysis
The dataset was examined both as a whole and split
by cultivars. Only the data subsets related to Guelph
Millenium and to Jersey Giant contained enough observations for meaningful analyses. Close examination
of the Guelph Millenium dataset revealed a 2-phase
relationship between FCRR incidence and soil microbial biodiversity indicators including bacterial, fungal
and arbuscular mycorrhizal fungi PLFA indicators values (Figure 2). At a FCRR level of 0%, microbial
biodiversity and abundance of bacterial, fungal, and
arbuscular mycorrhizal fungi PLFA indicators were
high. Microbial biodiversity, bacterial PLFA and fungal PLFA abundance decreased sharply to reach a
minimum at 3–4% of FCRR incidence (Figures 2A,
B and C). Beyond this point, the Shannon Weaver
biodiversity index and abundance of the fungal PLFA
indicator increased up to their initial levels, while bacterial PLFA indicators also increased, but to a lower
level. A similar trend in arbuscular mycorrhizal fungal PLFA occurred, although it spanned over a longer
range of FCRR incidence (Figure 2d). The relationship
between arbuscular mycorrhizal fungi PLFA indicator
and FCRR incidence could be described by a second
degree polynomial regression equation.
Examination of the Jersey Giant data did not reveal clear trends of relationship between FCRR and
80
Figure 2. Relationships found between Fusarium crown and root rot incidence, and (A) the Shannon Weaver Biodiversity Index, (B) bacterial
PLFA indicators, (C) fungal PLFA indicator, and (D) arbuscular mycorrhizal fungi PLFA indicator, in Guelph Millenium planted fields.
81
Figure 4. Canonical score plots constructed with factor 1 and factor 2 of analyses on the impact of (A) organic amendments (1. Compost;
2. Rabbit manure; 3. Chicken manure; 4. Cow manure; 5. Cow and chicken manure; and 6. No organic amendment), (B) tillage depth in Guelph
Millenium plantations (1. <10 cm; 2. >10 cm; 3. no tillage), (C) field geographical location (1. North of Montreal; 2. Québec; 3. St-Hyacinthe;
4. Trois-Rivières), and (D) preceding crops (1. Cereal; 2. Green manure; 3. Maize; 4. Strawberry), on microbial community structure in the soil
of asparagus plantations of six years of age or less. The whole dataset was used for these analyses unless otherwise stated.
soil microbial Shannon Weaver biodiversity index
or microorganisms-indicating PLFAs. However, Discriminant Analysis conducted on the whole microbial
PLFA profiles revealed the occurrence of distinct microbial community structure (Wilk’s lambda = 0.001,
P = 0.0003) under different FCRR incidence classes,
in Jersey Giant planted fields (Figure 3a). As similar analysis of the cultivar Guelph Millenium PLFA
profile subset also indicated the occurrence of distinct
soil microbial community structure (Wilk’s lambda
<0.001, P = 0.002) in fields of different FCRR incidence classes (Figure 3b). Mahalanobis distances
confirmed that all microbial communities were classified correctly in their assigned group. The PLFA C:13
was the most useful to discriminate soil microbial
communities in the Guelph Millenium planted fields,
while C:14, C:17 and 14-meth-C:15 were the most
important PLFAs to discriminate communities in the
Jersey Giant fields. Discriminant Analysis of the entire
dataset, in contrast, did not show any difference in soil
microbial community structures in fields with different percentage of FCRR incidence (data not shown).
This apparent discrepancy in results when all cultivars
are simultaneously considered may indicate that the
relationship between FCRR incidence and microbial
community reorganization is cultivar specific.
The crop management related variables were examined in an attempt to pinpoint those that influence
soil microbial community structure and, hence, that
might be manipulated to reduce FCRR progression.
Amongst the discrete variables recorded in this study,
soil organic amendment (Wilk’s lambda < 0.0001,
P < 0.0001) and geographic localization (Wilk’s
lambda = 0.20, P < 0.0001) influenced soil microbial community structure (Figures 4a and c). In the
later case, there was a distinct soil microbial community structure in the Québec region as compared
to the North of Montreal and St-Hyacinthe regions,
as shown by their distinct clustering in the canonical
score plot, while Trois-Rivières had an intermedi-
82
Figure 5. Impact of preceding crops on the percentage of field area with damage due to Fusarium crown and root rot in fields of six years of
age or less. Bars represent standard error of the means; n = 20 for cereal, 12 for green manure, 8 for maize, and 10 for strawberry.
ate community structure (Figure 4c). Tillage depth
(Wilk’s lambda = 0.022, P < 0.0001) also influenced soil microbial community structure, but only
in Guelph Millenium fields (Figure 4b). The effect of
preceding crops on soil microbial community structure
was significant (Wilk’s lambda = 0.005, P < 0.0001)
when the data related to plantation of 0 to 6 years
of age were considered (Figure 4d), but not when all
fields were included in the analysis, suggesting that
the effect of a previous crop disappears with time. Soil
microbial community structure was not influenced by
drainage, tillage equipment, the method of harvest or
the source of plants (data not shown). Examination of
the data revealed that notwithstanding plantation age
or cultivars, in this study, 6 year old or younger asparagus plantations preceded by a crop of maize had
more than twice as much field area affected by FCRR
as those preceded by other crops (Figure 5).
Discussion
Soil microbial population shifts and FCRR incidence
This study reveals that a shift in soil microbial community structure in commercial asparagus fields was
concurrent to early FCRR development. This shift was
occurring within fields rather than being associated
to diseased plants, as indicated by the similarity of
biological conditions in the vicinity of symptomatic
and asymptomatic plant that differed only in Fusarium
CFU counts (higher in symptomatic plant tissues; Vujanovic et al., 2004), and in potential dehydrogenase
activity (lower in symptomatic micro-plot soil; Hamel
et al., unpublished). In contrast, fields planted to different cultivars had largely different soil biological
characteristics. Replant problems in fields previously
planted with asparagus (Blok and Bollen, 1996a) as
well as asparagus fields decline are worldwide problems (Elmer, 2000; Reid et al., 2002; Seefelder et al.,
2002; Warncke et al., 2002) suggesting that the shift
in soil microbial community structure we observed,
translates into the degradation of the soil biological
conditions. Asparagus plants appear to create a selfinhibitory soil environment (Young and Chou, 1985).
The replant problem, decline syndrome and FCRR incidence in asparagus as well as the observed reorganization of the soil microbial community in plantations,
all point to the occurrence of negative feedback reaction, as it was described for natural plant populations
(Bever et al., 1997). Negative feedback observed in
some plants has been related to the accumulation of
deleterious organisms in the saprobe/pathogen fraction of the soil microbial community, in particular,
to that of pathogens (Klironomos, 2002). The fungi
most commonly found in the root zone of these plants
were Verticillium, Fusarium and Cylindrocarpon spp.
which, in all cases, formed infections in plant roots
upon re-inoculation. In the asparagus fields surveyed,
16 Fusarium spp. were recovered, and of these, only
three species are known as pathogenic on asparagus
(Vujanovic et al., 2004).
We can speculate on the nature of the shift in the
soil microbial community that occurs early in the progression of Fusarium-related field depopulation, but
83
Figure 3. Canonical score plots constructed with factor 1 and factor 2 of analyses on the impact of the level of damage due to
Fusarium crown and root rot found in asparagus plantations on
soil microbial community structure. (A) In Jersey Giant plantations, distinct community structures were found for the disease
level classes (1) 0–2%, (2) 3–4%, (3) 7–11% and (4) >11% of
field area depopulated, and (B) in Guelph Millenium plantations,
distinct community structures were found for the disease level
classes (1) 0–2%, (2) 3–4%, (3) 5–6% and (4) >7% of field area
depopulated.
it appears clearly that the change encountered in soil
microbial populations was profound. We observed a
30% reduction in microbial biodiversity early in the
Fusarium-related field depopulation process, followed
by a recovery in the level of biodiversity correlated
with further increase in field area affected by FCRR.
This observation underscores that biodiversity in itself might not be a sufficient condition for a soil to
be disease-suppressive and concurs with others on the
importance of the composition of the soil microbial
community (Reeleder, 2003).
The transient reduction in soil microbial diversity
was concurrent with a 70% drop in the abundance of
bacteria PLFA indicators that was followed by a recovery in bacteria abundance. Bacteria-indicating PLFAs
abundance increased up to approximately 60% of its
initial level, when the percentage of field area affected
progressed further. The drop in the arbuscular mycorrhizal fungi PLFA indicator was even more dramatic
with an 83% reduction. This state of low arbuscular
mycorrhizal fungi abundance, which we interpret as a
transition period where soil microbial populations are
getting reorganized, spanned over a longer range of
FCRR incidence percentages.
Arbuscular mycorrhizal fungi spores are typically
large and produced in very low amounts, as compared to spore production in other fungal species, and
while they are commonly carried over short distance
by soil animals, they are only accidentally carried
by wind along with soil particles during windstorms.
Evidence for succession of arbuscular mycorrhizal
fungi in asparagus plantations was found in Michigan
(Wacker et al., 1990). Chlamydospores of Glomus and
Acaulospora spp. were prevalent in young plantations,
while Gigaspora spp. predominated in asparagus plantations over 12 years of age. The slow dispersal of
arbuscular mycorrhizal fungal propagules and the low
biodiversity of this group of fungi in soil could explain
the dramatic decrease in abundance of these fungi
during initial disease development.
Mycorrhizal fungi were associated to reduced
severity of FCRR (Elmer, 2002; Matsubara et al.,
2002). Some arbuscular mycorrhizal isolates were
found more effective than others, in this regard (Matsubara et al., 2001). We do not know if the arbuscular
mycorrhizal populations present before and after the
shift occurs are equally effective against FCRR. If
they are equally effective, this transient inhibition of
the arbuscular mycorrhizal population should translate into a 3-phase pattern of disease development in
asparagus plantation; first there would be a lag period with slow development of FCRR, followed by an
acceleration of the rate of bare patch development in
asparagus plantations and, then, the rate of increase
in the area affected would level off as the arbuscular
mycorrhizal population is restored, presumably with
different species or strains. Agronomists observed in
Québec asparagus plantations that, typically, FCRR
is practically absent or present at very low levels
in the first four to five years of an asparagus stand,
84
but develop rapidly between the fifth and tenth year
(Jean-Guy Tessier, personnal communication). The
same development pattern was also observed in Northeastern United State where decline is expected within
five to ten years (Elmer, 1996).
If FCRR outbreak in asparagus plantations is due
to the transition phase during which arbuscular mycorrhizal fungi are practically absent, a simple way
to reduce losses due to FCRR could be to inoculate
asparagus seedlings with the arbuscular mycorrhizal
species that are selected for in asparagus plantations,
right at the propagation stage. It would be important, however, to first test early and late arbuscular
mycorrhizal fungal species for their impact on asparagus growth, in case deleterious arbuscular mycorrhizal
fungi were selected by asparagus plants. Yield decline
under continuous corn and soybean cropping was attributed to the selection of less mutualistic arbuscular
mycorrhizal fungal populations by these crop plants
when monocultured (Johnson et al., 1992). In his study
on the mechanism driving negative feedback reaction
in some plants, Klironomos, tested and rejected the
hypothesis of accumulation of deleterious arbuscular
mycorrhizal fungal spp. under self-inhibitory plants.
In all cases, the arbuscular mycorrhizal fungi isolated
from the soil environment of the plants, enhanced
rather than inhibit plant growth when they were reinoculated on the latter. The inoculation of asparagus
seedlings with arbuscular mycorrhizal species that are
selected by asparagus plants may be one way to reduce
the impact of FCRR on plantation. This hypothesis
remains, however, to be tested.
According to the literature on asparagus (Pederson
et al., 1991; Walters, 2003), FCRR plantations would
stem from the release of phenolic acids by asparagus
plants. Phenolic compounds produced by asparagus
were shown to be associated with asparagus decline,
with reduced asparagus root colonization by arbuscular mycorrhizal fungi (Pederson et al., 1991), with
reduced abundance of microorganisms with biocontrol
activity (Walters, 2003), and with increased number of
pathogenic Fusarium (Elmer, 1996). Other asparagusrelated influences may also induce a transitory shift in
the soil microbial community with a transitory negative impact on the plant health. Plants are known to
change their soil microbial environment (Bever, 2003).
The negative impact of phenolic acid in FCRR of
asparagus strangely appears as a unique case. Phenolic
acid production is usually seen as an expression of
disease resistance in plants. Enhanced production of
phenolic acids was associated to resistance to Fusar-
ium oxysporum f.sp. albedinis root infection in date
palm (El Modafar and El Boustani, 2000, 2001) and
to Fusarium wilt of banana (de Ascensao and Dubery, 2003). Resistance to Pythium aphanidermatum
was also associated to enhanced production of phenolic compounds (Ongena et al., 2000), and the severity
of Gibberella ear rot expression was negatively correlated with diferulic acid content in maize grain tissues
(Bily et al., 2003). It is unclear how phenolic acids
would enhance disease expression in asparagus, but
would reduce it in other plants. Conclusions derived
from laboratory experiments may become erroneous
when plant-pathogen interactions are taken into the
complexity of the soil environment.
Although it is known that plant species influence
the make up of soil microbial community (Eom et al.,
2000; Fang et al., 2001; Hedlund, 2002) and that the
practice of crop rotation is precisely aimed at preventing adverse soil microorganisms population build-up
(Paulitz et al., 2002), to our knowledge, this is the
first report describing profound soil microbial community reorganization in relation to disease spread, in
a perennial monoculture. The changes in soil microbial community structure observed in the fields studied
were occurring throughout the entire microbial communities as evaluated by PLFA indicators of large
microbial taxonomic groups and went well beyond
mere pathogen-antagonist interactions. If profound
changes in soil microbial community structure are determinant for FCRR expression, the utilization of crop
management strategies that impact on soil microbial
community structure may delay or even prevent FCRR
outbreak in asparagus plantations.
Management factors influencing soil microbial
community structure
The choice of cultivar could be a factor influencing
the rate of soil microbial community degradation in
asparagus plantations. The fact that relationships between soil microbial community structure and FCRR
incidence were found only within groups of fields
planted with a single cultivar (Guelph Millenium or
Jersey Giant) and not when all cultivars were confounded further suggests that different cultivars develop distinct soil microbial communities. It is also
possible that the changes induced by different cultivars, although similar in nature, would occur at
different rate, thus creating a variability that occults
any FCRR- soil microbial community relationship.
85
Little difference in the soil physicochemical and
microbiological conditions were found in micro-plots
with symptomatic and asymptomatic plants (Hamel
et al., unpublished; Vujanovic et al., 2004). The observation of differences in soil microbiology related
variables associated with different cultivars, in this
study, suggests the potential for a cultivar influence
on FCRR. The Viking KB3 plantations surveyed had
persisted for 16.8 years, on average, and resisted to
FCRR particularly well. While higher FCRR incidence was expected in older plantations, we observed
lower average FCRR incidence in Viking KB3 than in
all other cultivars except Guelph Millenium and Jersey Knight, which were located in the youngest fields
(3.2 and 6.3 years old, respectively). All this suggests
that Viking KB3 is relatively tolerant to FCRR. The
seemingly larger tolerance of Viking KB3 remains to
be confirmed in controlled experiments.
Biological control agents, in particular nonpathogenic Fusarium, have reduced FCRR incidence
(Blok, 1997; Reid et al., 2002). Disease suppression
after NaCl application was associated with increases
in the densities of fluorescent pseudomonads and Mnreducing bacteria in rhizosphere soil (Elmer, 2003).
This suggests that a solution to the problem of FCRR
in asparagus may be found in the management of
soil microorganisms. We observed that the addition of
organic amendments to soil, tillage depth, geographical location and preceding crop, could all influence
soil microbial community structure on the long-term.
The preceding crop was particularly influential. Maize
increased disease severity markedly, suggesting that
maize may be a host for asparagus pathogens or
favours their survival. FCRR is thought to be caused
by a specific Fusarium population (Elmer, 2001), but
maize and asparagus may have a pathogenic Fusarium in common. Maize seedling blight is caused by
a complex of Fusarium species including F. oxysporum Schlechtend., F. verticilloides [previously known
as F. moniliforme (Seifert et al., 2003)] (Munkvold
and O’Mara, 2002), a similar species assemblage
as in asparagus. The teleomorph of F. verticilloides
and F. proliferatum is Gibberella fujikuroi, which
has sexually distinct mating populations termed ‘A–E’
(Leslie, 1991). Isolates of F. proliferatum from asparagus belong to the ‘D’ mating population (Elmer,
1995b). In a survey conducted in the maize growing area of Slovakia, the organisms most frequently
isolated on maize ear rot samples were strains of
F. verticillioides, F. proliferatum and F. subglutinans,
belonging to mating populations ‘A, D and E’ of
the teleomorph G. fujikuroi (Srobarova et al., 2002).
The mating population ‘D’ of G. fujikuroi (anamorph:
F. proliferatum) was also found on maize ear rot in
Argentina (Chulze et al., 2000). It seems, therefore,
that one of the causing agents of maize ear rot could
also cause FCRR in asparagus.
No relationship between soil organic amendment
and the percentage of FCRR was observed in this
study. Nevertheless, it can not be concluded that organic amendments have no impact on FCRR; there
was little consistency in the application of organic
amendments in the plantations surveyed. A range of
organic materials were used and their application rate
and frequency varied from site to site. A few studies have examined the effect of organic materials on
FCRR. Compost, chitin and phosphoramidadate, a nematicide, had no effect on yield of asparagus (Elmer
et al., 1999), but coconut charcoal and coffee residues
have reduced the severity of FCRR (Matsubara et al.,
2002).
Conclusion
This study revealed three research avenues that could
lead to the control of FCRR in asparagus. We observed
that a profound reorganization of the soil microbial
community was parallel to the process FCRR infection
development in asparagus plantations. This may indicate that asparagus induces its own decline through
soil microorganism-mediated negative feedback. If
this is true, manipulation of soil microbial community
through cropping practices that influence soil microorganisms could provide means to reduce the impact
of FCRR. Second, the impact of cultivars on soil
microbial populations and activity, and the higher tolerance to FCRR in Viking KB3, suggests a potential
for the development of asparagus cultivars tolerant to
pathogenic Fusarium strains or FCRR suppressive. Interestingly, some plant species were found to have
fungitoxic effects on F. udum (Singh and Rai, 2000).
Some plant species might similarly be toxic on the
Fusarium spp. pathogenic to asparagus. Finally, we
also observed that maize as a preceding crop favours
FCRR incidence, presumably by sharing a common
F. proliferatum teleomorph (Gibberella fujikoroi) of
the same mating type. Although this remains to be experimentally confirmed, our results indicate that maize
should not precede the establishment of an asparagus
plantation.
86
Acknowledgements
This research, was financially supported by the Conseil des recherches en pêches et agroalimentaire du
Québec (CORPAQ) and by the Natural Science and
Engineering Research Council of Canada (NSERC).
We thank Jean-Guy Tessier for his invaluable help
in planning the survey and the other agronomists of
Québec Ministry of Agriculture who help us collect
the crop management data. Thanks are also expressed
to Richard Reeleder who critically reviewed the manuscript and to Stéphane Daigle for assistance with
statistical analysis.
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