Directory UMM :Data Elmu:jurnal:A:Applied Soil Ecology:Vol13.Issue3.Dec1999:
Applied Soil Ecology 13 (1999) 271±282
Effects of number of winter wheat crops grown successively
on fungal communities on wheat roots
G.L. Batemana,*, H. KwasÂnab
a
IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
Department of Forest Pathology, Agricultural University, ul. Wojska Polskiego 71c, 60-625 PoznanÂ, Poland
b
Received 13 April 1999; received in revised form 30 June 1999; accepted 5 July 1999
Abstract
Roots were taken from winter wheat plants sampled in early summer in each of three years. In each year, ®rst, third and
continuous (ninth or subsequent) wheat crops were grown on the same site so that epidemics of take-all disease (causal fungus:
Gaeumannomyces graminis var. tritici) were in, respectively, pre-build-up, build-up and decline stages. Fungi on pieces from
the upper parts of the roots, serially washed 20 times, were identi®ed by growing onto agar media and allowing them to
sporulate. Approximately 107 species of 50 genera were identi®ed, including some that were previously unrecorded in Britain
or on wheat. There was usually a trend (with statistically signi®cant differences only in one year) for more fungal isolations
per root piece with increasing number of successive wheat crops. Changes in populations of several fungi were associated with
number of wheat crops in one or more years but only Fusarium culmorum increased with increased number of crops in all
years. While individual fungi or the fungal community may be involved in take-all suppression or enhancement, there were no
clear relationships between either total numbers of fungal species or abundance of individual species and the stage of the takeall epidemic. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Wheat; Root fungi; Take-all; Gaeumannomyces graminis var. tritici
1. Introduction
Take-all disease, caused by the root-infecting fungus Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker (Ggt), often increases to
maximum severity during a sequence of susceptible
cereal crops such as wheat. Take-all decline sometimes follows severe disease, often in fourth or subsequent wheats. A break from the susceptible crop
*
Corresponding author.
E-mail address: geoff.bateman@bbsrc.ac.uk (G.L. Bateman)
usually breaks the disease cycle. Although changes in
the characteristics of the pathogen itself have been
associated with the progress of the epidemic (Cunningham, 1975; Bateman et al., 1997), it is usually
assumed that changes in populations of other rhizosphere microorganisms determine the course of a takeall epidemic by their in¯uence on the take-all fungus
or on the infection process. The course of an epidemic
is unpredictable, however, as it is in¯uenced by soil
and weather as well as by biotic factors.
Several reports have described bacterial populations
on wheat roots in relation to crop sequences and takeall epidemics, or to special cases of suppression other
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 4 0 - 2
272
G.L. Bateman, H. KwasÂna / Applied Soil Ecology 13 (1999) 271±282
than take-all decline. Brown (1981), sampling a ®eld
site at Rothamsted, found little evidence of consistent
associations between amounts of take-all in sequences
of wheat crops and rhizoplane populations of bacterial
groups suspected of being involved in take-all suppression. Fluorescent pseudomonads were thought to
be involved in take-all suppression in northwestern
USA (Cook and Rovira, 1976). In Montana, USA,
different causes of suppression, including pseudomonads, mycoparasitism and antagonistic fungi, were
thought to be involved, in different combinations,
on different sites (Andrade et al., 1994). In France,
antagonistic ¯uorescent pseudomonads were found to
be associated with the use of NH4+ fertilisers, the use
of which can lessen take-all in some circumstances
(Sarniguet et al., 1992). Although there have been
reports describing the fungal ¯ora of wheat roots (e.g.
MaÈkelaÈ and MaÈki, 1980; LemanÂczyk and Sadowski,
1997), studies on the occurrence of rhizosphere fungi
in relation to take-all epidemics and suppression have
usually concerned individual species. For example,
Trichoderma spp. were implicated in take-all suppression in Western Australian soils as they became
acidi®ed (Simon and Sivasithamparam, 1988).
This paper describes comparisons of fungal communities in the rhizosphere of wheat in crops in
different stages of take-all epidemics, including some
presumed to be in take-all decline. The times of
sampling were just before or immediately after
anthesis; early summer is likely to be a time when
suppression is operating during take-all decline
(Hornby, 1992). The fungal populations assessed
were principally those of the rhizoplane of the upper
part of the root system, which is usually the site of
the most damaging phase of take-all development
in late spring and early summer in the locality of
the experiments.
2. Materials and methods
2.1. Field sites
Two wheat ®elds on Rothamsted Farm, on silty
clay-loam with ¯ints, were sampled in 1996±1998.
Soil pH is maintained at ca. 6.5±7.0 by applications of
lime every sixth year. Fertiliser nitrogen was applied
as ammonium nitrate (34.5% N).
2.1.1. 1996
Samples were taken from a crop sequence experiment (coded CS/323) on West Barn®eld, described by
Hornby and Gutteridge (1995) and Hornby (1998). In
the 1995±1996 growing season, all plots were in
winter wheat after the completion of the main part
of the crop sequence experiment. Wheat cv. Mercia,
seed-treated with bitertanol + fuberidazole (Sibutol),
was sown at 380 seeds mÿ2 on 25 September 1995.
Plots (10 m 3 m) of three treatments from each of
three randomised blocks were used. The treatments
sampled were ®rst wheat after oats, third wheat after
oats, and ninth (continuous) wheat. Third wheat crops
were sampled because of visible evidence (moderate
root disease and little stunting of plants) that take-all
was increasing but had not reached its peak. Stunting
was more apparent in plots of fourth wheats, suggesting that peak take-all had been reached. Standard
inputs included fertiliser nitrogen at 30 kg haÿ1 on
7 March and 170 kg haÿ1 on 15 April.
2.1.2. 1997
Samples were taken from the long-term `Classical'
experiment on Broadbalk ®eld. Wheat cv. Hereward,
seed-treated with ¯udioxonil (Beret Gold) and fonofos
(Fonofos Seed Treatment), was sown at 380 seeds mÿ2
on 15 October 1996. Individual treatments (crop
sequences, fertilisers and other inputs) are not replicated in the Broadbalk experiment and so four replicate subplots (5 m 2 m) from the corners of each
main plot (23.2 m 6 m) were sampled for fungal
isolations. First, third and continuous (39th) wheats,
from sections 5, 7 and 9, respectively, were sampled.
Two plots with different amounts of fertiliser nitrogen,
applied on 11 April, were sampled from each section:
plot 7 (96 kg N haÿ1) and plot 9 (192 kg N haÿ1). The
®ve-year rotation in sections 5 and 7 was three successive wheat crops following oats and potatoes.
2.1.3. 1998
The Broadbalk experiment was sampled as in 1997.
Seed of wheat cv. Hereward was treated and sown as in
the previous year, but on 21 October 1997. In this year
the ®rst wheat was in section 3, the third wheat in
section 4 and the continuous (32nd) wheat in section 1.
Rotations in sections 3 and 4 were the same as in
sections 5 and 7. Only plot 9 (192 kg N haÿ1, applied
on 24 March) in each section was sampled.
273
G.L. Bateman, H. KwasÂna / Applied Soil Ecology 13 (1999) 271±282
2.2. Sampling
Plants for disease assessment were sampled on 1
July 1996 at growth stage (GS) 73 (Zadoks et al.,
1974), 9 July 1997 at GS 77 and 9 July 1998 at GS 75
by digging up ten 20-cm lengths of row (®ve for ®rst
wheats on Broadbalk), located in a W-pattern, from
each whole plot.
For assessment of fungal communities, a minimum
of 10 plants was dug from ®ve random positions in
each plot or subplot on 25 June 1996, when the plants
were at GS 69 (anthesis complete), on 2 June 1997, at
GS 53 (ears partly emerged), and on 1 June 1998, at
GS 51 (ear emergence just beginning). Most of the
length of the shoots was cut off and the remainder of
the plants stored in open plastic bags at 5oC for 10 days
(1996) or 1 day (1997 and 1998) before processing
was resumed.
2.3. Disease assessments
Take-all severity was assessed on the washed root
system of each plant as slight (75% blackened). A take-all rating
(TAR; 0±300) was calculated for each plot by the
equation TAR = 100 [(number of plants with slight
take-all) + (2 number of plants with moderate takeall) + (3 number of plants with severe takeall)] total number of plants (Dyke and Slope, 1978).
2.4. Identification of fungi on roots
After soaking and washing in running water, six to
eight randomly chosen 1-cm root pieces were cut from
the upper parts of the root system of each plant,
1.5 cm from their points of attachment. Each set
of root pieces was washed 20 times, for 3 min each
time, by shaking vigorously in 10 ml sterile distilled
water. Fresh sterile water, cooled to 5oC, was used for
each wash. This method is similar to that described by
Holdenrieder and Sieber (1992), who used ultrasonic
agitation rather than shaking. Twenty washes were
considered suf®cient to remove most detachable fungal fragments, the remaining fungi mainly representing those that have a vegetative existence on the root
surface (Harley and Waid, 1955). The root pieces were
dried in a sterile air ¯ow on sterile ®lter paper and each
was cut into two 0.5-cm pieces. One piece was put
onto potato dextrose agar (PDA; Oxoid) and one onto
low nutrient agar (SNA) containing KH2PO4 at
1 g lÿ1, KNO3 at 1 g lÿ1, MgSO47H2O at 0.5 g lÿ1,
KCl at 0.5 g lÿ1, glucose at 0.2 g lÿ1, sucrose at
0.2 g lÿ1 and Agar Technical (Oxoid) at 15 g lÿ1
(Nirenberg, 1976). Both media contained penicillin,
streptomycin sulphate and chloramphenicol. In 1997
and 1998, the positions of the two halves of each root
piece on the agar plates were recorded for subsequent
re-identi®cation. There were 60 root pieces per plot or
subplot on each medium. They were incubated for
three days at 20oC followed by two days or more at
5oC. The plates were examined microscopically at
intervals from three days to two months. Sporulating
fungi were identi®ed. Further subcultures onto PDA
(slants) and SNA were made as necessary. Sporulation
of some subcultures was encouraged by incubation
under near-ultraviolet light at 15o or 25oC, or in
daylight.
3. Results
3.1. Take-all severity
Take-all was absent from ®rst wheat crops and
slight to moderate in third and continuous wheat crops
(Table 1). TARs suggest that take-all had not begun to
build up in ®rst wheats, had begun building up but had
not reached peak severity in third wheats, and had
declined below peak severity in continuous wheats.
The mean TAR for fourth wheats in the experiment on
West Barn®eld in 1996 was only 109 (replicate plots
had TARs 64, 135 and 128), but the presence of
Table 1
Effects of number of successive wheat crops on take-all severity
No. of wheat crops Take-all rating (0±300)
1996
1
3
Continuous
SED (50 df)
p
0
47
91
26.5
Effects of number of winter wheat crops grown successively
on fungal communities on wheat roots
G.L. Batemana,*, H. KwasÂnab
a
IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
Department of Forest Pathology, Agricultural University, ul. Wojska Polskiego 71c, 60-625 PoznanÂ, Poland
b
Received 13 April 1999; received in revised form 30 June 1999; accepted 5 July 1999
Abstract
Roots were taken from winter wheat plants sampled in early summer in each of three years. In each year, ®rst, third and
continuous (ninth or subsequent) wheat crops were grown on the same site so that epidemics of take-all disease (causal fungus:
Gaeumannomyces graminis var. tritici) were in, respectively, pre-build-up, build-up and decline stages. Fungi on pieces from
the upper parts of the roots, serially washed 20 times, were identi®ed by growing onto agar media and allowing them to
sporulate. Approximately 107 species of 50 genera were identi®ed, including some that were previously unrecorded in Britain
or on wheat. There was usually a trend (with statistically signi®cant differences only in one year) for more fungal isolations
per root piece with increasing number of successive wheat crops. Changes in populations of several fungi were associated with
number of wheat crops in one or more years but only Fusarium culmorum increased with increased number of crops in all
years. While individual fungi or the fungal community may be involved in take-all suppression or enhancement, there were no
clear relationships between either total numbers of fungal species or abundance of individual species and the stage of the takeall epidemic. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Wheat; Root fungi; Take-all; Gaeumannomyces graminis var. tritici
1. Introduction
Take-all disease, caused by the root-infecting fungus Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker (Ggt), often increases to
maximum severity during a sequence of susceptible
cereal crops such as wheat. Take-all decline sometimes follows severe disease, often in fourth or subsequent wheats. A break from the susceptible crop
*
Corresponding author.
E-mail address: geoff.bateman@bbsrc.ac.uk (G.L. Bateman)
usually breaks the disease cycle. Although changes in
the characteristics of the pathogen itself have been
associated with the progress of the epidemic (Cunningham, 1975; Bateman et al., 1997), it is usually
assumed that changes in populations of other rhizosphere microorganisms determine the course of a takeall epidemic by their in¯uence on the take-all fungus
or on the infection process. The course of an epidemic
is unpredictable, however, as it is in¯uenced by soil
and weather as well as by biotic factors.
Several reports have described bacterial populations
on wheat roots in relation to crop sequences and takeall epidemics, or to special cases of suppression other
0929-1393/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 4 0 - 2
272
G.L. Bateman, H. KwasÂna / Applied Soil Ecology 13 (1999) 271±282
than take-all decline. Brown (1981), sampling a ®eld
site at Rothamsted, found little evidence of consistent
associations between amounts of take-all in sequences
of wheat crops and rhizoplane populations of bacterial
groups suspected of being involved in take-all suppression. Fluorescent pseudomonads were thought to
be involved in take-all suppression in northwestern
USA (Cook and Rovira, 1976). In Montana, USA,
different causes of suppression, including pseudomonads, mycoparasitism and antagonistic fungi, were
thought to be involved, in different combinations,
on different sites (Andrade et al., 1994). In France,
antagonistic ¯uorescent pseudomonads were found to
be associated with the use of NH4+ fertilisers, the use
of which can lessen take-all in some circumstances
(Sarniguet et al., 1992). Although there have been
reports describing the fungal ¯ora of wheat roots (e.g.
MaÈkelaÈ and MaÈki, 1980; LemanÂczyk and Sadowski,
1997), studies on the occurrence of rhizosphere fungi
in relation to take-all epidemics and suppression have
usually concerned individual species. For example,
Trichoderma spp. were implicated in take-all suppression in Western Australian soils as they became
acidi®ed (Simon and Sivasithamparam, 1988).
This paper describes comparisons of fungal communities in the rhizosphere of wheat in crops in
different stages of take-all epidemics, including some
presumed to be in take-all decline. The times of
sampling were just before or immediately after
anthesis; early summer is likely to be a time when
suppression is operating during take-all decline
(Hornby, 1992). The fungal populations assessed
were principally those of the rhizoplane of the upper
part of the root system, which is usually the site of
the most damaging phase of take-all development
in late spring and early summer in the locality of
the experiments.
2. Materials and methods
2.1. Field sites
Two wheat ®elds on Rothamsted Farm, on silty
clay-loam with ¯ints, were sampled in 1996±1998.
Soil pH is maintained at ca. 6.5±7.0 by applications of
lime every sixth year. Fertiliser nitrogen was applied
as ammonium nitrate (34.5% N).
2.1.1. 1996
Samples were taken from a crop sequence experiment (coded CS/323) on West Barn®eld, described by
Hornby and Gutteridge (1995) and Hornby (1998). In
the 1995±1996 growing season, all plots were in
winter wheat after the completion of the main part
of the crop sequence experiment. Wheat cv. Mercia,
seed-treated with bitertanol + fuberidazole (Sibutol),
was sown at 380 seeds mÿ2 on 25 September 1995.
Plots (10 m 3 m) of three treatments from each of
three randomised blocks were used. The treatments
sampled were ®rst wheat after oats, third wheat after
oats, and ninth (continuous) wheat. Third wheat crops
were sampled because of visible evidence (moderate
root disease and little stunting of plants) that take-all
was increasing but had not reached its peak. Stunting
was more apparent in plots of fourth wheats, suggesting that peak take-all had been reached. Standard
inputs included fertiliser nitrogen at 30 kg haÿ1 on
7 March and 170 kg haÿ1 on 15 April.
2.1.2. 1997
Samples were taken from the long-term `Classical'
experiment on Broadbalk ®eld. Wheat cv. Hereward,
seed-treated with ¯udioxonil (Beret Gold) and fonofos
(Fonofos Seed Treatment), was sown at 380 seeds mÿ2
on 15 October 1996. Individual treatments (crop
sequences, fertilisers and other inputs) are not replicated in the Broadbalk experiment and so four replicate subplots (5 m 2 m) from the corners of each
main plot (23.2 m 6 m) were sampled for fungal
isolations. First, third and continuous (39th) wheats,
from sections 5, 7 and 9, respectively, were sampled.
Two plots with different amounts of fertiliser nitrogen,
applied on 11 April, were sampled from each section:
plot 7 (96 kg N haÿ1) and plot 9 (192 kg N haÿ1). The
®ve-year rotation in sections 5 and 7 was three successive wheat crops following oats and potatoes.
2.1.3. 1998
The Broadbalk experiment was sampled as in 1997.
Seed of wheat cv. Hereward was treated and sown as in
the previous year, but on 21 October 1997. In this year
the ®rst wheat was in section 3, the third wheat in
section 4 and the continuous (32nd) wheat in section 1.
Rotations in sections 3 and 4 were the same as in
sections 5 and 7. Only plot 9 (192 kg N haÿ1, applied
on 24 March) in each section was sampled.
273
G.L. Bateman, H. KwasÂna / Applied Soil Ecology 13 (1999) 271±282
2.2. Sampling
Plants for disease assessment were sampled on 1
July 1996 at growth stage (GS) 73 (Zadoks et al.,
1974), 9 July 1997 at GS 77 and 9 July 1998 at GS 75
by digging up ten 20-cm lengths of row (®ve for ®rst
wheats on Broadbalk), located in a W-pattern, from
each whole plot.
For assessment of fungal communities, a minimum
of 10 plants was dug from ®ve random positions in
each plot or subplot on 25 June 1996, when the plants
were at GS 69 (anthesis complete), on 2 June 1997, at
GS 53 (ears partly emerged), and on 1 June 1998, at
GS 51 (ear emergence just beginning). Most of the
length of the shoots was cut off and the remainder of
the plants stored in open plastic bags at 5oC for 10 days
(1996) or 1 day (1997 and 1998) before processing
was resumed.
2.3. Disease assessments
Take-all severity was assessed on the washed root
system of each plant as slight (75% blackened). A take-all rating
(TAR; 0±300) was calculated for each plot by the
equation TAR = 100 [(number of plants with slight
take-all) + (2 number of plants with moderate takeall) + (3 number of plants with severe takeall)] total number of plants (Dyke and Slope, 1978).
2.4. Identification of fungi on roots
After soaking and washing in running water, six to
eight randomly chosen 1-cm root pieces were cut from
the upper parts of the root system of each plant,
1.5 cm from their points of attachment. Each set
of root pieces was washed 20 times, for 3 min each
time, by shaking vigorously in 10 ml sterile distilled
water. Fresh sterile water, cooled to 5oC, was used for
each wash. This method is similar to that described by
Holdenrieder and Sieber (1992), who used ultrasonic
agitation rather than shaking. Twenty washes were
considered suf®cient to remove most detachable fungal fragments, the remaining fungi mainly representing those that have a vegetative existence on the root
surface (Harley and Waid, 1955). The root pieces were
dried in a sterile air ¯ow on sterile ®lter paper and each
was cut into two 0.5-cm pieces. One piece was put
onto potato dextrose agar (PDA; Oxoid) and one onto
low nutrient agar (SNA) containing KH2PO4 at
1 g lÿ1, KNO3 at 1 g lÿ1, MgSO47H2O at 0.5 g lÿ1,
KCl at 0.5 g lÿ1, glucose at 0.2 g lÿ1, sucrose at
0.2 g lÿ1 and Agar Technical (Oxoid) at 15 g lÿ1
(Nirenberg, 1976). Both media contained penicillin,
streptomycin sulphate and chloramphenicol. In 1997
and 1998, the positions of the two halves of each root
piece on the agar plates were recorded for subsequent
re-identi®cation. There were 60 root pieces per plot or
subplot on each medium. They were incubated for
three days at 20oC followed by two days or more at
5oC. The plates were examined microscopically at
intervals from three days to two months. Sporulating
fungi were identi®ed. Further subcultures onto PDA
(slants) and SNA were made as necessary. Sporulation
of some subcultures was encouraged by incubation
under near-ultraviolet light at 15o or 25oC, or in
daylight.
3. Results
3.1. Take-all severity
Take-all was absent from ®rst wheat crops and
slight to moderate in third and continuous wheat crops
(Table 1). TARs suggest that take-all had not begun to
build up in ®rst wheats, had begun building up but had
not reached peak severity in third wheats, and had
declined below peak severity in continuous wheats.
The mean TAR for fourth wheats in the experiment on
West Barn®eld in 1996 was only 109 (replicate plots
had TARs 64, 135 and 128), but the presence of
Table 1
Effects of number of successive wheat crops on take-all severity
No. of wheat crops Take-all rating (0±300)
1996
1
3
Continuous
SED (50 df)
p
0
47
91
26.5