Effects of number of winter wheat crops grown successively
on fungal communities on wheat roots
G.L. Bateman
a,*, H. KwasÂna
baIACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
bDepartment of Forest Pathology, Agricultural University, ul. Wojska Polskiego 71c, 60-625 PoznanÂ, Poland
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 graminisvar.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 onlyFusarium culmorumincreased 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 take-all epidemic.#1999 Elsevier Science B.V. All rights reserved.
Keywords:Wheat; Root fungi; Take-all;Gaeumannomyces graminisvar.tritici
1. Introduction
Take-all disease, caused by the root-infecting fun-gusGaeumannomyces graminis(Sacc.) Arx and Oli-vier var. tritici Walker (Ggt), often increases to maximum severity during a sequence of susceptible cereal crops such as wheat. Take-all decline some-times follows severe disease, often in fourth or sub-sequent wheats. A break from the susceptible crop
usually breaks the disease cycle. Although changes in the characteristics of the pathogen itself have been associated with the progress of the epidemic (Cun-ningham, 1975; Bateman et al., 1997), it is usually assumed that changes in populations of other rhizo-sphere microorganisms determine the course of a take-all epidemic by their in¯uence on the take-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 take-all epidemics, or to special cases of suppression other
*Corresponding author.
E-mail address: geoff.bateman@bbsrc.ac.uk (G.L. Bateman)
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 sup-pression. 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 pseudomo-nads, 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,
Trichodermaspp. were implicated in take-all suppres-sion in Western Australian soils as they became acidi®ed (Simon and Sivasithamparam, 1988).
This paper describes comparisons of fungal com-munities 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 experi-ment (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 mÿ2on 25 September 1995. Plots (10 m3 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, suggest-ing 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 cated in the Broadbalk experiment and so four repli-cate subplots (5 m2 m) from the corners of each
main plot (23.2 m6 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 suc-cessive 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
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 (<25% of the root system blackened), moderate (26±75% blackened) or severe (>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) + (2number of plants with moderate
take-all) + (3number of plants with severe
take-all)]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 fun-gal fragments, the remaining fungi mainly represent-ing 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
(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)
patches of plants stunted by take-all suggested that peak take-all was occurring in these plots. There were no fourth wheats or additional longer sequences for comparison in 1997 or 1998.
3.2. Fungi on roots
Sporulating fungi of 50 genera were identi®ed (Table 2). These included some species rarely or never before recorded in Britain or in wheat crops. Fungi of some genera, including Mortierella, Penicillium and
Trichoderma, were identi®ed to species in 1996 only, when it was therefore possible to determine the fre-quency of each species in the fungal communities as well as its incidence on roots. In 1997 and 1998, when not all fungi were identi®ed to species, only incidence on root pieces was determined. Fewer fungi were isolated on PDA than on SNA. Fewer fungi per root piece were isolated in ®rst wheats than in third or continuous wheats in 1996, but with no signi®cant differences (Table 3). In 1997, fewest fungi were again identi®ed on roots of ®rst wheats but there were also more fungi on continuous wheats than on third wheats. The effect of amount of fertiliser N on numbers of fungi was not signi®cant (p= 0.1). In 1998, there were no signi®cant differences between wheat sequences on numbers of fungi. The numbers of different fungi on each root piece were similar for the two ®eld sites.
3.2.1. 1996
On SNA and PDA respectively, 38±51 and 17±30 fungal species were recorded per plot. Individual species were often recorded at different frequencies on the different media. Frequencies of most fungi were not affected by the number of wheat crops. However, depending on the isolation medium,Penicilliumspp. and Fusarium culmorum were more frequent and
Idriella bolleyiless frequent in the ninth wheat than in other crops (Table 4).
3.2.2. 1997
Combining the information from both N treatments,
Acremonium strictumwas more frequent in the ®rst than in the third wheats (Table 5). Chrysosporium pannorumwas more frequent in the ®rst than in other wheats. Cylindrocarpon spp., Fusarium spp. other thanF. culmorum,Penicilliumspp. andPhoma eupyr-enawere more frequent in third than in other wheats.
Epicoccum purpurascens,F. culmorum,Mucor spp.,
Pythium spp. and Trichoderma spp. were more fre-quent in third and continuous wheats than in ®rst wheats. Mucorales were more frequent in third than in ®rst wheats.
3.2.3. 1998
There were fewer signi®cant differences in wheat from Broadbalk ®eld than in 1997 (Table 5). Chry-sosporium pannorum and Cylindrocarpon spp. were more frequent in third than in other wheats. F. cul-morumwas more frequent in continuous than in other wheats. Mucorspp. and Verticilliumspp. were more frequent in third and continuous than in ®rst wheats.
4. Discussion
A large number of constituent fungi of the wheat rhizosphere community were identi®ed:107 species
Table 2
Sporulating fungi identified on wheat roots and the percentage of root pieces from which the fungi were isolated on two agar media
Fungus 1996 1997 1998
SNA PDA SNA or PDAa SNA or PDAa
Absidia cylindrosporaHagem 0.2 0.2 ±e ±
Absidia glaucaHagem 2.0 2.2 ± ±
Absidiaspp. ± 0.6 0.8
Acremonium bacillisporum
(Onions & Barron) W. Gams 1.1 0 ± ±
Acremonium strictumW. Gams 0.8 0 33.6 21.7
Acremoniumsp. 0 0 0.5 1.4
Alternaria alternata(Fr.) Keissler +A. infectoriaSimmons 3.5 0.9 8.4 23.2
Aureobasidium pullulans(de Bary) Arnaud 0 0 42.6 5.7
Botrytis cinereaPers. ex Nocca & Balb. 0 0 0.6 1.0
Broomella acutaSchoem. & E. MuÈll. 0 0 0.1 0
Cephalosporiumsp. 0.2 0 0 0
Chalarastate ofCeratocystis 1.5 0 0 0
Chrysosporium pannorum(Link) Hughes 4.3 0.2 5.5 3.5
Cladosporium cladosporioides(Fres.) de Vreis 3.3 0 9.4 8.2
Cladosporium herbarum(Pers.) Link 50.9 53.7 98.6 94.7
Cladosporium macrocarpumPreuss 0.8 0 0.8 0.5
Cladosporium sphaerospermumPenz. 0.2 0 0 0
Coemansia scorpioideaLinderb 0 0 0.1 0
Coemansia thaxteriLinderb 0 0 0.1 0
Cylindrocarpon destructansPenz. 14.3 2.8 ± ±
Cylindrocarpon macroconidialisBrayford & Samuels 6.5 0.4 ± ±
Cylindrocarponspp. ± ± 2.5 1.0
Dactylaria appendiculataCazau, Aramb. & Cabelloc 2.4 0 0 0
Dactylariasp. 0 0 0.1 0
Dendryophion nanum(Nees ex Gray) Hughes 0.4 0 0 0
cf. Echinostelum elachistonAlexop. 0.9 0 0 0
Epicoccum purpurascensEhrenb. ex Schlecht. 3.0 1.5 18.5 2.5
Exophialasp. 1.5 0 0 0
Exserohilum novae-zelandiaeUpadhyay & Mankauc 1.9 0 0 0
Fusarium avenaceum(Corda) Sacc. 5.1 1.9 2.0 0.6
Fusarium culmorum(W.G. Sm.) Sacc. 33.5 18.0 20.4 11.0
Fusarium dimerumPenz. 0.4 0 0.1 0
Fusarium equiseti(Corda) Sacc. 0.4 0 1.0 0.7
Fusarium flocciferumCorda 2.5 3.3 7.1 0.1
Fusarium graminearumSchwabe 0.2 0 0 0.3
Fusarium merismoidesCorda 3.7 0.2 2.6 6.7
Fusarium oxysporumSchlect. 7.2 3.1 4.0 1.4
Fusarium poae(Peck) Wollenw.c 0 0.2 0 0.3
Fusarium sambucinumFuckel 5.4 1.1 4.1 1.1
Fusarium solani(Mart.) Sacc. 0 1.9 0.2 0.4
Fusarium tricinctum(Corda) Sacc. 0 0.2 26.4 16.0
Non-pathogenicFusariumspp. ± ± 37.8 30.0
Fusariumsp. 0 0.8 0 0
Gliocladium roseumBainier 7.4 7.0 0.5 0.1
Gliocladium virideMatr. 0 0 0.1 0
Gliomastix murorumvar.felina(Marchal) S. Hughes 1.3 0 0 0
Hyalodendronsp. 0.6 0 0.1 0.1
Heteroconium chaetospira(Grove) M.B. Ellis 1.5 0 0.3 0
Humicola griseaTraaen 0.2 0 ± 1.1
Idriella bolleyi 81.1 25.9 55.7 82.6
Table 2 (Continued)
Fungus 1996 1997 1998
SNA PDA SNA or PDAa SNA or PDAa
Metarhizium anisopliaevar.major(Johnston) Tulloch 0 0.6 0 0
Microdochium nivaleSamuels & Hallett 1.7 0.2 0.1 0.6
Monacrosporium psychrophilum(Drechsl.) R. Cook & Dickinsonc 0.2 0 0 0
Monocillium indicumSaksena 2.8 0 ± 0
Monocilliumsp. ± ± 1.6 0
Mortierella alpinaPeyr. 20.8 0.6 ± ±
Mortierella elongataLinn. 31.9 14.5 ± ±
Mortierella exiguaLinn. 1.9 0 ± ±
Mortierella humilisLinn. ex W. Gams 0.4 0 ± ±
Mortierella hygrophilaLinn. 6.7 2.4 ± ±
Mortierella marburgensisLinn. 2.0 0 ± ±
Mortierellasp. 0 0.9 ± ±
Mortierellaspp. ± ± 21.1 25.6
Mucor hiemalisf.hiemalisWehmer 10.5 13.0 ± ±
Mucorspp. ± ± 7.6 7.2
Mucorales ± ± 25.3 30.7
Ochroconis humicola(Barron & Busch) de Hoog & v. Arx 4.1 0.4 0.1 3.1
Paecilomyces farinosus(Holmskiol) A.H.S. Brown & G. Sm. 4.4 0.2 ± ±
Paecilomycesspp. ± ± 0.1 1.0
Papulaspora immersaHotson 0.4 0 1.2 0.4
Papulasporasp. 0 0.2 0 0
Penicillium crustosumThom 0.2 0 ± ±
Penicillium janczewskiiZaleski 3.0 0.9 ± ±
Penicillium miczynskiiZaleski 9.5 4.4 ± ±
Penicillium notatumWestling 0 0.2 ± ±
Penicillium simplicissimum(Oudem.) Thom 0 0.2 ± ±
Penicillium spinulosumThom 2.8 0.6 ± ±
Penicillium viridicatumWestling 19.8 14.6 ± ±
Penicillium vulpinum(Cooke & Massee) Sifert & Sams. 0 0.2 ± ±
Penicilliumspp. ± ± 18.5 21.5
Periconia byssoidesPers. ex Merat 0.6 0 0 0
Periconia macrospinosaLefebvre & Johnson 3.9 0.8 0.1 0
Phialophoraspp.d 0 0 0.4 0.1
Phoma eupyrenaSacc. 31.7 0.9 47.4 41.4
Phoma glomerata(Corda) Wollenw. & Hopchapfel 0.2 0.6 ± ±
Phoma medicaginisvar.pinodella(L.K. Jones) Boerema 0 0.2 ± ±
Phomasp. ± ± 0.1 1.8
Phytophthora cactorum(Lebert & Cohn) Schroeter 0.7 0 0 0
Piptocephalis xenophilaDobbs & English 1.1 0 0 0
Pleurocatena acicularisG. Arnaud ex Aramb.c 20.0 0 0 0
Polyscytalum fecundissimumRiess. 0.2 0 0 0
Pyrenochaetasp. 1.1 0 0 0
Pythium intermediumde Bary 20.5 0.9 ± ±
Pythium irregulareBuisman 0.4 ± ± ±
Pythium ultimumTrow 0.7 ± ± ±
Pythiumspp. ± ± 17.3 1.7
Ramichloridium schulzeri(Sacc.) de Hoog 16.9 0 1.5 0
Ramulispora anguioides(Nirenberg) Crous 0 0 0 0.1
Sporothrix schenckiiHektoen & Perkins 0 0 0.1 0
Tricellula aquaticaJ. Webster 0 0 0 0.4
Trichoderma atrovirideKarsten 0 0.6 ± ±
the fungi recorded are known to be rhizosphere inha-bitants, others may be incidental occupants of the root zone. The latter group is likely to include aquatic fungi such asTricellula aquatica(cf. Bandoni, 1972), found in 1998.
The samples used for identi®cation of fungi were taken in June, when take-all decline might be expected
to be operating in the continuous wheat crops (Hornby, 1992). The experiments were done over three years and, therefore, took some account of differences in fungal communities between years, which are known to occur (MaÈkelaÈ and MaÈki, 1980). Fluctuations within years, and differences between years in timings of such ¯uctuations, were not taken into account.
Table 3
Effects of number of successive wheat crops on the mean number of fungi identified per root piece
No. of wheat crops 1996 1997a 1998a
SNA DPA Low N High N Low or high N
1 4.18 1.80 3.98 4.50 4.24 4.17
3 5.43 2.26 4.57 5.42 4.99 4.02
Continuous 5.05 2.21 5.62 5.78 5.70 4.43
SED 0.734 (4 df) 0.265 (4 df) 0.458 (18 df) 0.324 (18 df) 0.334 (9 df)
p 0.3 0.3 0.6b 0.001 0.5
aSNA or PDA.
bpValue for interaction (amount of NNo. of wheat crops). Table 2 (Continued)
Fungus 1996 1997 1998
SNA PDA SNA or PDAa SNA or PDAa
Trichoderma crassumBissett 1.1 0 ± ±
Trichoderma hamatum(Bon.) Bain. 0.8 0.2 ± ±
Trichoderma harzianumRifai 0 7.4 ± ±
Trichoderma koningiiOudem. 2.6 0 ± ±
Trichoderma longipilisBissett 0.2 0 ± ±
Trichoderma polysporum(Link) Rifai 1.1 5.6 ± ±
Trichoderma viridePers. 4.6 5.9 ± ±
Trichodermaspp. ± ± 14.5 9.9
Ulocladium botrytisPreuss 0 0 0.2 0.6
Verticillium bulbillosumW. Gams & Malla 1.1 0.6 ± ±
Verticillium catenulatum(Kamyschko ex Barron & Onions) W. Gams 1.1 0.2 ± ±
Verticillium fungicola(Preuss) Hassebrauk 0.2 0 ± ±
Verticillium lamellicola(F.E.V. Sm.) W. Gams 5.6 0.2 ± ±
Verticillium lecanii(A.W. Zimmerm.) Viegas 0.2 0 ± ±
Verticillium nigrescensPethybr. 0.9 0 ± ±
Verticilliumsp. 0.6 0 ± ±
Verticilliumspp. ± ± 2.0 1.7
aPercentage occurrence on either SNA or PDA; half-pieces from the same root were identifiable on the two media. (Each percentage is an
average from four subplots of each of two plots treated with different amounts of fertiliser nitrogen for each crop sequence. Totals of 59±60 root pieces per subplot and 476±480 root pieces per crop sequence were examined. Each root piece was halved, the halves being incubated on PDA or SNA; the data presented show occurrence on either or both media.)
bDescribed by KwasÂna et al. (1999). cDescribed by KwasÂna and Bateman (1998).
dProbably includes the take-all pathogen,Gaeumannomyces graminisvar.tritici, which was rarely found sporulating on SNA and whose distinctive hyphal growth was often overgrown by other fungi on PDA.
Differences in species composition between wheat sequences that were found in only one or two years may have occurred in all years but at different times; this could be con®rmed only by repeated sampling and examination of roots from each sequence. LemanÂczyk and Sadowski (1997) found that fewest fungi but greatest diversity occurred in autumn, while most fungi occurred during grain ripening. In a warm climate, fungal counts on wheat roots were found to decline as the plants matured (Ikram-ul-Haq and Iqbal, 1996). Differences in root exudates among cultivars (Gams, 1967) may result in differences in composition of the rhizosphere mycobiota and this, and the differ-ent sampling dates (later in 1996 than in 1997 or 1998), may have contributed to differences between the 1996 crop and the other crops.
The most favoured explanation for take-all decline is an increase in antagonistic microorganisms during
consecutive cropping of the same cereal. Recent information on the complexities and mechanisms of take-all suppression and decline is reviewed in Hornby et al. (1998). Bacteria, especially ¯uorescent pseudo-monads, are often considered as likely agents of take-all suppression (e.g. Cook and Rovira, 1976; Sarniguet et al., 1992). At Rothamsted, evidence of an associa-tion between bacterial populaassocia-tions on plant roots and take-all decline was inconclusive (Brown, 1981). There has been little previous research on associations between take-all epidemics and the fungal community in the rhizosphere in British soils.
Fusarium culmorum was the fungus associated most consistently with the number of wheat crops, being more frequent in continuous wheat than in other wheats. It was therefore associated, more than any of the other fungi identi®ed, with take-all decline. How-ever, populations of F. culmorum, which is a wheat
Table 4
Effects of number of successive wheat crops on frequency of some rhizosphere fungi in mixed populations and on root pieces estimated on low nutrient medium (SNA) or potato dextrose agar (PDA), 1996a
Fungus No. of wheat crops SNA PDA SNA or PDAb
% Logit %c % Logit %c % Logit %c
Frequency as percentage of all fungi identified
Penicilliumspp. 1 5.7 ÿ1.396 7.6 ÿ1.238 6.3 ÿ1.345
3 5.5 ÿ1.420 10.4 ÿ1.093 7.2 ÿ1.274
9 9.9 ÿ1.107 11.4 ÿ1.082 10.4 ÿ1.081
SED (4 df) 1.71 0.1369 4.53 0.2619 1.58 0.1027
p 0.1 0.2 0.7 0.8 0.1 0.1
Fusarium culmorum 1 5.8 ÿ1.402 5.8 ÿ1.377 5.9 ÿ1.402
3 5.8 ÿ1.387 9.6 ÿ1.145 6.8 ÿ1.313 9 8.5 ÿ1.184 9.6 ÿ1.112 8.8 ÿ1.166 SED (4 df) 1.12 0.1024 1.64 0.0932 0.89 0.0865
p 0.1 0.2 0.1 0.09 0.07 0.1
Frequency as percentage of root pieces
Idriella bolleyi 1 82.8 0.775 25.0 ÿ0.587 53.9 0.079
3 87.8 1.013 30.6 ÿ0.478 58.9 0.186
9 72.8 0.483 22.2 ÿ0.795 47.5 ÿ0.051 SED (4 df) 3.69 0.0636 17.58 0.5248 10.70 0.2169
p 0.04 0.08 0.9 0.8 0.6 0.6
Fusarium culmorum 1 25.0 ÿ0.576 11.1 ÿ1.056 18.1 ÿ0.785
3 32.8 ÿ0.366 21.7 ÿ0.672 27.2 ÿ0.508 9 42.8 ÿ0.147 21.1 ÿ0.655 31.9 ÿ0.379 SED (4 df) 8.62 0.1962 2.40 0.0830 4.47 0.1258
p 0.2 0.2 0.02 0.01 0.08 0.07
aOnly those fungi are shown for which treatment effects have small probability values (approaching 0.1) for at least one agar medium. bMean of percentages occurring on SNA and PDA; half-pieces from the same root were not identifiable on the two media.
Table 5
Effects of number of successive wheat crops on frequency of fungi on root pieces
Fungus Number of wheat crops Logit %aroot pieces (back-transformed %)
1997 1998
Acremonium strictum 1 ÿ0.211 (39.1) ÿ0.857 (14.8)
3 ÿ0.527 (25.3) ÿ0.562 (24.0)
Continuous ÿ0.350 (32.7) ÿ0.572 (23.7)
SEDb 0.1260 0.1771
p 0.07 0.2
Chrysosporium pannorum 1 ÿ1.161 (8.4) ÿ1.804 (2.1)
3 ÿ1.772 (2.3) ÿ1.271 (6.8)
Continuous ÿ1.783 (2.3) ÿ2.053 (1.1)
SED 0.2155 0.2483
p 0.01 0.03
Cylindrocarponspp. 1 ÿ2.015 (1.3) ÿ1.804 (2.1)
3 ÿ1.554 (3.8) ÿ1.271 (6.8)
Continuous ÿ2.127 (0.9) ÿ2.053 (1.1)
SED 0.2081 0.2483
p 0.03 0.03
Epicoccum purpurascens 1 ÿ1.069 (10.1) ÿ1.620 (3.3)
3 ÿ0.624 (21.8) ÿ1.914 (1.6)
Continuous ÿ0.688 (19.7) ÿ1.708 (2.7)
SED 0.1635 0.1860
p 0.03 0.3
Fusarium culmorum 1 ÿ1.379 (5.5) ÿ1.338 (5.9)
3 ÿ0.750 (17.8) ÿ1.326 (6.1)
Continuous ÿ0.332 (33.5) ÿ0.732 (18.3)
SED 0.1393 0.2241
p <0.001 0.04
Fusarium tricinctum 1 ÿ0.958 (12.3) ÿ0.794 (16.5)
3 ÿ0.686 (19.7) ÿ1.073 (10.0)
Continuous ÿ0.237 (37.9) ÿ0.678 (20.0)
SED 0.2171 0.1478
p 0.01 0.08
Fusariumspp. (non-pathogens) 1 ÿ0.425 (29.5) ÿ0.432 (29.2)
3 ÿ0.350 (32.7) ÿ0.455 (28.2)
Continuous ÿ0.031 (48.0) ÿ0.410 (30.1)
SED 0.1284 0.1721
p 0.02 1.0
Mucorspp. 1 ÿ1.608 (3.4) ÿ1.642 (3.1)
3 ÿ1.311 (6.3) ÿ1.167 (8.3)
Continuous ÿ1.079 (9.9) ÿ1.187 (8.0)
SED 0.1630 0.1830
p 0.02 0.05
Mucorales 1 ÿ0.785 (16.7) ÿ0.389 (31.0)
3 ÿ0.407 (30.2) ÿ0.489 (26.9)
Continuous ÿ0.527 (25.4) ÿ0.451 (28.4)
SED 0.1382 0.2783
p 0.04 0.9
Penicilliumspp. 1 ÿ1.081 (9.8) ÿ0.686 (19.7)
pathogen, may be expected to increase as wheat cropping is increased. On the other hand, it is rela-tively unimportant as a cause of root disease in the UK, where its main importance is as a cause of brown foot rot and ear blight. Brown foot rot caused byF. culmorumoccurs in warm, dry conditions, usually in summer, when plants are under drought stress (Papen-dick and Cook, 1974). Such conditions did not occur during the period of these experiments when, in other experiments at Rothamsted, brown foot rot was neg-ligible and populations of F. culmorum in the soil remained small (Bateman et al., 1998 and unpublished results). The ability ofF. culmorumto proliferate on roots in the absence of conditions conducive of disease is consistent with its status as a facultative parasite but may be unrelated to its role as a stem-base pathogen. Interactions among pathogenic fungi have been pro-posed as causes of disease suppression (Zogg, 1972) and interactions betweenF. culmorumandG. graminis
var.triticimay occur in nature.F. culmorumis known to show antagonism to the take-all fungus (Lal, 1939). In experiments using inoculum mixtures on roots,F. culmorumsometimes, but not always, decreased the severity of take-all symptoms (Bateman, unpub-lished). It was shown in experiments on inoculated plants that plant damage from root rot was less where both the pathogens F. culmorumand Helminthospor-ium sativum(Bipolaris sorokiniana) were present on the roots than where either was present alone (Leding-ham, 1942). Ledingham (1942) also referred to evi-dence thatF. culmorumcould exacerbate the activity of H. sativum. The effect of pathogen mixtures on disease severity may depend on a balance of environ-mental factors.
No fungus other thanF. culmorumwas associated consistently with number of wheat crops. Cylindro-carponspp., minor pathogens, were sometimes asso-ciated with third wheats; the evidence is insuf®cient to
Table 5 (Continued)
Fungus Number of wheat crops Logit %aroot pieces (back-transformed %)
1997 1998
Continuous ÿ0.476 (27.3) ÿ0.650 (20.9)
SED 0.1398 0.2027
p 0.001 1.0
Phoma eupyrena 1 ÿ0.162 (41.5) ÿ0.168 (41.2)
3 ÿ0.142 (42.5) ÿ0.501 (26.4)
Continuous 0.250 (61.7) 0.169 (57.9)
SED 0.0997 0.5199
p <0.001 0.5
Pythiumspp. 1 ÿ1.190 (8.0) ÿ1.869 (1.8)
3 ÿ0.658 (20.6) ÿ1.948 (1.5)
Continuous ÿ0.778 (16.9) ÿ2.258 (0.6)
SED 0.1948 0.3144
p 0.03 0.5
Trichodermaspp. 1 ÿ1.664 (3.0) ÿ1.068 (10.1)
3 ÿ0.836 (15.3) ÿ1.138 (8.8)
Continuous ÿ0.798 (16.4) ÿ1.164 (8.4)
SED 0.1653 0.2076
p <0.001 0.9
Verticilliumspp. 1 ÿ2.257 (0.6) ÿ2.398 (0.3)
3 ÿ2.024 (1.2) ÿ0.836 (15.3)
Continuous ÿ1.697 (2.8) ÿ0.798 (16.4)
SED 0.2247 0.1460
p 0.07 <0.001
suggest that they too are suppressed by continuous wheat cropping or contribute to the severity of disease in peak take-all years.Trichodermaspp., well known antagonists of fungi, including the take-all fungus (Lal, 1939), have been implicated in take-all suppres-sion in Australia, where suppressuppres-sion was associated also with soil acidi®cation (Simon and Sivasitham-param, 1988). Strains ofIdriella bolleyi can control take-all (Kirk and Deacon, 1987) but, as this fungus always occurs frequently on roots, its effects may depend on its density on individual roots, which was not recorded in the present work.Chrysosporium pannorumis likely to be a coloniser of senescent root cortical tissue and only a weak competitor in the rhizosphere (Widden, 1986). This is consistent with its being most frequent in Broadbalk ®eld in those sequences with fewest fungi per root piece.
Neither total number of fungal species nor the abundance of any single species appears to offer prospects for bioindication of take-all risk or for predicting the stage of a take-all epidemic. Estimates of species richness using diversity indices (Magurran, 1988) were attempted as part of this research (results not shown) but were considered unsatisfactory because of the incompleteness of the fungal commu-nity data. Further development of this might include estimates of species richness based on selected, repre-sentative species. It may also be possible to implement a `biotic effect' method that scores selected species for antagonism to the take-all fungus (Herman, 1985). Any value in these methods might become clear only after regular, frequent monitoring of individual crops, and the implications may be site-speci®c. These approaches would become a practical proposition only with the development of rapid, molecular methods for the detection and quanti®cation of the principal fungi. Such research should be complemented by similar studies on rhizosphere bacteria, including a re-evalua-tion of the role of bacteria in take-all decline on the Rothamsted sites.
Acknowledgements
The research at IACR-Rothamsted was funded by the Biotechnology and Biological Sciences Research Council of the United Kingdom. The Committee of Scienti®c Researches in Poland ®nanced the stay of
the second author at Rothamsted. We thank R.J. Gutteridge for some of the sampling and disease assessments.
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