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Ability of the lumbricid earthworms Aporrectodea rosea and Aporrectodea trapezoides to reduce the severity of take-all under greenhouse and field conditions
Soil Bid. Eiochem. Vol. 26, No. 10, pp. 1291-1297, 1994 Copyright 0 1994 Elsevier Science Ltd
Pergamon
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ACCELERATEDPAPER
ABILITY OF THE LUMBRICID EARTHWORMS APORRECTODEA ROSEA AND APORRECTODEA TRAPEZOIDES TO REDUCE THE SEVERITY OF TAKE-ALL UNDER GREENHOUSE AND FIELD CONDITIONS P. M. STEPHENS,‘* C. W. DAVOREN,’ B. M. DOUBE’.~and M. H. RYDER’.~ ‘Cooperative Research Centre for Soil and Land Management, Private Bag No. 2, Glen Osmond, SA 5064, Australia and %SIRO, Division of Soils, Private Bag No. 2, Glen Osmond, SA 5064, Australia (Accepted 3 July 1994)
Summary-The influence of the earthworms A. rosea and A. trupezoides on wheat plants, grown in soil artificially infested with the take-all fungus Gaeumannomyces graminis var. tritici (Ggt) was examined. In pot trials, using a red-brown earth soil artificially infested with Ggt, the presence of the earthworm A. trapezoides (at a density equivalent to 3 14 or 47 1 m-*) was associated with a significant (P < 0.05) increase in shoot weight and a reduction in the severity of take-all (as measured by a reduction in the percentage length of seminal roots containing take-all lesions). In contrast, in the absence of added Ggt, shoot weight was not significantly (P> 0.05) influenced by the presence of A. trapezoides.Two field trials were conducted, in which A. rosea and A. trupezoides were added, at an equivalent density of 100 or 300 rnm2,to cylinders driven into the soil. In a calcareous sandy loam artificially infested with Ggt, the presence of the earthworms A. rosea or A. trapezoides (at these densities) was associated with a significant (PcO.05) reduction in the severity of take-all disease. Under the same conditions, A. rosea or A. trapezoides (at an equivalent density of 300 m-2) caused a significant increase in shoot weight. In contrast, in the absence of added Ggt. shoot weight was not significantly (P > 0.05) influenced by the presence of A. rosea or A. trapezoides. In a second field trial in a red-brown earth, the presence of the earthworm A. trupezoides (at an equivalent density of 300 rne2) was associated with a significant (P < 0.05) reduction in the severity of take-all disease. Under the same conditions, A. rosea or A. trupezoides did not cause a significant increase in shoot weight. In the absence of Ggt, shoot weight was not significantly (PzO.05) influenced by the presence of A. rosea or A. trapezoides.These results demonstrate the potential of the earthworms A. rosea and A. truperoides, under both greenhouse and field conditions, to reduce the severity of take-all disease on wheat.
INTRODUCTION Gaeumannomyces graminis var. tritici (Ggt), the causative agent of take-all, is the most damaging root disease of wheat (Triticum aestivum L.) worldwide (Huber and McCay-Buis, 1993). In southern Australia, this disease can cause losses in wheat production of Au&100 million per annum (Rovira, 1990). Yield losses as high as 6&75% can occur where there is a high incidence of infected plants (>50%) and high spring rainfall (Roget and Rovira, 1990). The apparent absence of plant genetic resistance and effective chemical controls for take-all, imposes many constraints on farm management practices (Huber and McCay-Buis, 1993). In Australia, control of take-all can be accomplished through the rotation of cereals with a non-host break crop (Kollmorgen et a/., 1983). However, there is concern as to the agronomic sustainability of using extended cropping systems to reduce take-all, due to a decline in cereal grain protein,
*To whom all correspondence
should he addressed.
soil structure and soil fertility. Take-all may also be controlled by the rotation of cereals with a grass-free pasture, in which herbicides are used to remove grasses which host Ggt (MacNish and Nicholas, 1987). However under intense cropping, there is concern about the development of herbicide resistance in grasses (Heap and Knight, 1986). Alternative methods to control take-all need to be developed. Continuous wheat monoculture can lead to a decrease in severity of take-all disease (Gerlagh, 1968; Shipton, 1975). Efforts to isolate the causes of this suppression and use them to control Ggt have focused primarily on isolating microorganisms antagonistic to this fungal pathogen. For example, Ryder and Rovira (1993) isolated a group of non-fluorescent Pseudomonas from the rhizosphere of wheat which gave significant and reproducible disease suppression. Significant control of take-all in the field has been observed using Pseudomonas jfuorescens (Weller and Cook, 1983) and Bacillus species (Capper and Campbell, 1986). The possibility that other larger soil organisms may suppress Ggt has been largely
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overlooked.
P. M. STEPHENS et al. Homma
et al. (1979) and Chakraborty that mycophagous amoebae, isolated from wheat fields, could feed upon G. gvaminis. Addition of mycophagous amoebae to sterilized soil infested with G. graminis var. tritici, resulted in a significant reduction in the severity of take-all (Chakraborty and Warcup, 1985). The disease severity of take-all is increased by a deficiency of essential plant mineral nutrients in soil (Reis et al., 1982; Huber, 1989; Huber and McCay-Buis, 1993). The earthworms Aporrectodea rosea and Aporrectodea trapezoides, which are widespread under wheat in southern Australia (Mele, 1991; Baker et al., 1993a), are able to increase the foliar concentration of a range of macro- and micro-nutrients in wheat (Stephens et al., 1994). These earthworms may therefore help to reduce the severity of take-all disease by increasing the amount of plant available nutrients in soil. Greenhouse studies have also suggested that the earthworm A. trapezoides, at an equivalent density of 1050 mm’, can reduce take-all disease in wheat (M. H. Ryder, B. M. Doube, T. E. Terrace and C. W. Davoren, unpublished results). We have assessed whether the earthworms A. rosea and A. trapezoides can influence the disease severity of take-all on wheat, both in greenhouse trials and at two field sites of contrasting soil types.
et al. (1983) reported
MATERIALS
AND METHODS
Pot experiments Surface soil (&lOcm) was taken from a site at Kapunda, located 75 km north of Adelaide, South Australia. The site contained volunteer pasture on a red-brown earth; classified as a thermic Calcic Natrixeralf (Soil Survey Staff, 1990) and pH 5.0 (H,O). Thesoilwasair-dried,sieved(4mm),adjustedto 75% of field capacity and the equivalent of 400 g airdriedsoilwasaddedto 500mlplasticpots. Soilmoisture was brought back to 75% of field capacity during the experiment by watering pots every 3 days up to a specified weight. Each pot contained either (a) no inoculum, (b) Ggt, (c) A. trapezoides or (d) Ggt + A. trapezoides, with 6 replications of each treatment. Inoculum of Ggt 8 (isolated from infected wheat roots from Avon, South Australia, by H. McDonald in 1981) was produced on dead rye-grass seed (Simon et al., 1987). This inoculum (92 mg) was mixed into the soil in each of the appropriate pots. Pots were placed in a randomized block in a water bath, maintained at 15C with natural light. After 48 h, either 1 (equivalent to 157 me2), 2 (equivalent to 314 m-?) or 3 (equivalent to 471 mm2) adult earthworms of A. trupezoides, each weighing 0.75-1.5 g, were added to the appropriate pots. Aluminium fly wire (mesh size 2 mm) was attached around the circumference of each pot to a height of 150 mm, in order that A. trapezoides did not escape. After a further 5 days, a 10 mm layer of non-infested soil
(adjusted to 75% of field capacity) was added to the surface of each pot and 6 seeds of wheat (Triticum aestiuum cv. Spear) were sown at a depth of 8 mm into each pot. The surface layer of soil protected the seed from early attack by Ggt and thereby increased the uniformity of disease symptoms (Ryder and Rovira. 1993). These were thinned to 3 seedlings pot-‘, 10 days after sowing. At 36 days after sowing, plant roots were rated for disease severity by calculating the percentage length of seminal roots (to 90 mm soil depth) containing Ggt lesions. Plant roots and shoots were then dried at 60’ C and weighed. Earthworms were removed from the pots, washed briefly in distilled water, dried on tissue paper for ca. 10 s and weighed. Field trials Field experiments were conducted in 1993 at: (1) Avon; located 100 km north of Adelaide, South Australia, with a winter dominant rainfall averaging 350 mm year-‘. This soil was classified as a calcareous sandy loam and a Petrocalcic Palexeralf (Soil Survey Staff, 1990) pH 8.2 (H,O), and had been previously sown to wheat. (2) Kapunda; in a field adjacent to the one from which soil was taken for pot experiments. where there was a winter dominant rainfall averaging 520 mm year-‘. This soil was classified as a red-brown earth or thermic Calcic Natrixeralf (Soil Survey Staff, 1990) and had been previously sown to oats. Cylinders of stormwater PVC pipe, 300 mm dia and a height of 210 mm, were inserted into soil to a depth of 150 mm (using an industrial rammer), on 7 June and 26 May at Avon and Kapunda, respectively. Cylinders were constructed and used according to the method of Baker et al. (1993b). On 24 June at Avon and on 15 June at Kapunda, the cylinders still containing soil were removed. Nylon curtain material was attached around the bottom of each cylinder and fastened with 12 mm plastic strapping. The cylinders were subsequently placed back into the soil. Cylinders were established with Ggt and earthworms in all combinations, with 6 replicates treatment-’ in cylinders containing Ggt inoculum. These were all sampled at the end of the experiment. In those cylinders without added Ggt inoculum, there were 12 replicates treatment ‘; of which one half were destructively sampled 42 days after sowing at Avon and 54 days after sowing at Kapunda, in order to assess earthworm survival. Replicates were arranged in a randomized block. On 3 July at both field sites, the top 2 cm of soil was removed from each of the cylinders and 1.05 g of Ggt inoculum (on dead rye-grass seed) was added evenly to the soil surface where appropriate, and the soil was then replaced. Any large fragments of organic matter on the soil surface were removed. Three days later (6 July), A. trupezoides or A. rosea were added to the soil surface of the appropriate cylinders at two different densities: 7 cylinder-’ (equivalent to 100 m-‘) or 21 cylinder- ’
Ability of earthworms to reduce take-all (equivalent to 300 mm2). These earthworms had been collected from a mass-rearing site in a field at the Waite Agricultural Research Institute, Adelaide, and held in pots of the respective field soils for 2 days prior to being added to the cylinders. Twenty wheat seeds (T. aestivum cv. Spear) were sown into each cylinder on 19 July at Avon and on 12 July at Kapunda. Twenty days later at each site seedlings were thinned to 10 cylinder-‘. Twelve additional cylinders (without Ggt inoculum) were also established in the above manner. On 27 July, 21 dead A. rosea or A. trapezoides (killed by exposure to 45°C for 30 min) were placed just under the soil surface in these cylinders. There were 6 replicates of each “dead earthworm” treatment. Plants were harvested on 11 October at Avon (83 days after sowing) and on 6 October at Kapunda (86 days after sowing). Roots were rated for severity of take-all and plant growth was assessed in the manner described previously. At Avon, there was ca. 110 mm of rainfall from the time the earthworms were added through to sampling (6 July-11 October) [measured at Balaklava, 15 km from Avon (Bureau of Meteorology, South Australia)]. An additional 26 mm of rainwater was added to each cylinder by hand. In the years 1990-1992, the average rainfall at Avon from 1 July to 30 September was 129 +47(SD) mm. During this same time period at Avon in 1993, the rainfall was 106 mm. At
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Kapunda, there was ca. 132 mm of rainfall from the time the earthworms were added through to sampling (6 July-6 October). No additional water was added to these cylinders. In the years 199&1992, the average rainfall at Kapunda from 1 July to 30 September was 254*64(SD) mm. During this same period at Kapunda in 1993, the rainfall was 112 mm. The statistical analysis was conducted using Genstat 5 (Anon., 1993). RESULTS
In both pot and field experiments, the variances of the % seminal root length containing Ggt lesions in treatments with added Ggt inoculum, were substantially different from the variances obtained from treatments without added Ggt inoculum. Therefore, the % seminal root length containing Ggt lesions in treatments with and without Ggt inoculum were analysed separately. Pot experiments All earthworms added to pots were recovered at the end of the experiment, except for 4 pots. These were therefore omitted from the statistical analysis. The percentage loss in fresh weight of A. trapezoides in pots containing 1, 2 or 3 individuals was 19.4f 15.7(SD), 18.1f7.2 and 16.4+8.4%, respectively. The introduction of Ggt into soil increased
30
25
20
0 -- Ggt
+ Ggt
Control
(157) t
(314)
(471)
Plus Ggt +
(157) e
(314)
(471)
Minus Ggt _
Fig. 1. Influence of the earthworm A. trupezoides on the severity of take-all disease in wheat, grown in pots in a red-brown earth with or without artificial inoculum of Ggf. Either 0, 1 (equivalent to 157 m-2), 2 (equivalent to 314 m-‘) or 3 (equivalent to 471 m-*) earthworms were added to pots. Columns containing the same letter were not significantly different at the P-co.05 level (using ANOVA).
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P. M. STEPHENS et al.
-- Ggt
+ Ggt
Control
t
Plus Ggt -r
T
MinusGgt
111)
Fig. 2. Influence of the earthworm A. trapezoides on the shoot weight of wheat, grown in pots in a red-brown earth with or without artificial inoculum of Ggt. Either 0, 1 (equivalent to 157 m-‘), 2 (equivalent to 314 m-l) or 3 (equivalent to 471 m-*) earthworms were added to pots. Columns containing the same letter were not significantly different at the P-co.05 level (using ANOVA).
(P-C 0.05) the % seminal root length containing lesions from 0 to 26% (Fig. 1). Under these conditions, the presence of A. trapezoides (at a density of 2 or 3 pot-‘) caused a significant (P~0.05) reduction in root lesions. The addition of Ggt into soil also caused a significant (PC 0.05) reduction of 33% in the shoot dry weight (Fig. 2). The presence of A. trapezoides (at a density of 2 or 3 pot-‘) caused a si~i~cant (PcO.05) increase in the shoot dry weight in the presence of Ggt, although the plant top growth was still significantly lower than that of wheat grown in the absence of Ggt. In the absence of Ggt, A. trape~aides did not influence shoot dry weight or the amount of take-all disease. Field trials
In field trials in the calcareous sandy loam at Avon, one cylinder (out of 72) contributed 11.6 times more to the residual mean square (error) of disease rating compared to the mean residual square of the other observations and prevented any significant differences between treatments. This one value was omitted from the statistical analysis. In the calcareous sandy loam, 88 + B(SE)% of A. trapezoides and 69+ 19(SD)% of A. rosea were recovered from cylinders 42 days after sowing. At the termination of the experiment (83 days after sowing), 25.8~23.5(SD)%ofA.trapezoidesand5.0~10.2%0f A. rosea were recovered from the cylinders. There was a reduction in earthworm number in each cylinder,
making it impossible to calculate the relative loss in earthworm weight. The addition of Ggt into the calcareous sandy loam caused a substantial increase in the incidence of take-all disease on wheat (Table I ). The introduction of the equivalent of 7 or 21 A. rosea or A. tmpezoides cylinder-‘, resulted in a significant (P~0.05) reduction in root lesions; with an average reduction of 34*5(SD)% in take-all lesions. There was no significant difference in the reduction of take-all lesions with either earthworm species or density. In the presence of earthwo~s and Ggr, the amount of take-all disease was still significantly (P < 0.05) higher than that of wheat grown in the absence of added Ggt. The addition of Ggt caused a 27% reduction (P-cO.05) in shoot weight (Table 1). In the presence of Ggt inoculum, the addition of 21 A. rosea or A. trapezoides cylinder-’ (but not 7 cylinder -I), was associated with a significant (P~0.05) increase in shoot weight and resulted in shoot weights not significantly (P > 0.05) different to that of the control (minus Ggt inoculum). In contrast, in the absence of Ggt, the addition of A. rosea or A. trapezoides, at either 7 or 2 1 cylinder- ‘, had no significant (P> 0.05) effect upon shoot weight. In the red-brown earth at Kapunda, 83 + 12(SD)% of A. trapezoides and 8 1+ 21% of A. rosea were recovered from the cylinders 54 days after sowing. At the end of the experiment (86 days after sowing), two
Ability of earthworms to reduce take-all cylinders had holes in the nylon material with < 24% of earthworms present. These 2 cylinders were omitted from the statistical analysis. At the end of the experiment, 83.5 + 16.7(SD)% of A. trapezoides and 85.3 f21.5% of A. rosea were recovered from the remaining cylinders. In those cylinders where the same number of earthworms were recovered at the end of the experiment as were added at the start, A. trapezoides had apparently lost 37.0 &-8.0% of their fresh weight, while A. rosea had lost 19.8 f 11.O%. The addition of Ggt inoculum into the red-brown earth, caused the percentage length of seminal roots containing Ggt lesions to increase from 2 to 48%. Under these conditions, only the introduction of A. trapezoides, at a density of 21 cylinder-‘, caused a significant (PcO.05) reduction (reduced to 28%) in the length of seminal roots containing take-all lesions. However, the amount of take-all disease was still significantly (PC 0.05) higher than that of wheat grown in the absence of Ggt. The addition of 7 or 21 A. rosea cylinder-’ or 7 A. trapezoides cylinder-’ did not cause a significant (P>O.O5) reduction in Ggt lesions. The addition of Ggt caused a 43% reduction (P < 0.05) in shoot weight. Under these conditions, the addition of A. rosea and A. trapezoides (at either 7 or 21 cylinder-‘) did not cause a significant (P>O.O5) increase in shoot weight. However, shoot weight in the presence of 7 or 21 A. rosea cylinder-i and 21 A. trapezoides cylinder-’ was not significantly different from the control (minus Ggt). In the absence of added Ggt inoculum, the addition of A. rosea or A. trapezoides (at either 7 or 21 cylinder’) did not have a significant (P> 0.05) effect upon shoot weight.
DISCUSSION
Our study showed that under field conditions Table 1. Influence of the earthworms A. mea weight of wheat, grown under field conditions Treatment
+ Ggt Control + A. mea (7 cylinder- I) + A. rmea (21 cylinder-‘) + A. trapezoides (7 cylinderr’) + A. trapezoides(21 cylinder-‘) - Ggt Control + A. mea (7 cylinder- ‘) +A. rmea (21 cylinder-‘) + A. trapezoides (7 cylinder-‘) + A. trapezoides (21 cylinderr’) + A. rmea (21 cylinder-’ ; added dead) + A. trapemides (21 cylinder-’ ; added dead)
the
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earthworms A. rosea and A. trapezoides have the potential to reduce the severity of take-all on wheat. The mechanism by which A. rosea and A. trapezoides reduced take-all disease is not known. Previous studies have shown that direct drilling, compared to conventional cultivation, may increase the incidence of take-all disease (Moore and Cook, 1984). One possible explanation for this result is that soil disturbance reduced take-all disease. As movement of A. rosea and A. trapezoides causes disruption of the soil profile, soil disturbance may be one mechanism by which A. rosea and A. trapezoides reduced the severity of take-all. In addition, earthworms have been shown to reduce the microbial biomass (Ruz-Jerez et al., 1992) and suppress the development of various fungi in soil (Keogh and Christensen, 1976; Striganova et al., 1989). Earthworms may have therefore also suppressed the saprophytic activity of Ggt in soil, by ingesting fungal mycelium or by producing unfavourable conditions for Ggt in their casts or tunnel linings. Wheat is more susceptible to take-all if nutritionally deficient in any of several macro- or micro-nutrients, including P, Mg, K, Mn, Cu, Zn or Fe (Reis et al., 1982). The report that A. rosea can, under certain conditions, increase the foliar concentration in wheat of Ca, Cu, K, Mn, N, Na and P and that A. trapezoides can increase the foliar concentration of Al, Ca, Fe, K, Mn, N and Na (Stephens et al., 1994) would suggest that these earthworms may also have reduced take-all disease by increasing the amounts of plant available nutrients in soil. Earthworms may increase plant nutrient availability in soil by enhancing organic matter breakdown and the release of plant nutrients (Lavelle et al., 1989). Plant nutrient availability may also be influenced by the higher enzymatic activity in earthworm casts compared to uningested soil (Businelli et al., 1984; Mulongoy and Bedoret, 1989) disease severity of take-all and shoot dry sandy loam soil with or without artificial inoculum
and A. trapezoides on the in a calcareous of Ggr
% Seminal root length containing Gat lesions* 22.2 * 4. I ta 14.8f8.5b 16.0+5.4b 13.5k5.0b 14.2+4.6b
0.1+0.2 0.0 + 0.0 0.0 5 0.0 0.0 * 0.0 0.0 + 0.0 0.0 + 0.0 0.0 * 0.0
Shoot dry weight
1.67kO.52~ 1.98 f 0.29bc 2.26 + 0.49ab 2.10~0.31bc 2.19k0.4ab
2.29k0.2ab 2.23 f 0.53a 2.62 f 0.29a 2.58k0.30a 2.57+0.33a 2.36+0.53ab 2.03 f0.44bc
Either 0, 7 (equivalent to 100 mm2) or 21 (equivalent to 300 m-2) A. traperoides or A. rosea were added to the appropriate cylinders. Columns containing the same letter were not significantly different at the PiO.05 level (usina ANOVA). *As the variances of the % seminal root length containing Ggt lesions in cylinders without added Ggr inoculum were substantially different from the variances obtained from cylinders with added Ggf inoculum, treatments with and without added Ggt inoculum were analysed separately. tstandard deviation.
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P. M. STEPHENS et al.
or by the secretion of calcium carbonate influencing the solubility and availability of plant nutrients (Edwards and Lofty, 1977). Increased nutrient availability in soil may also be derived from the loss of nutrients from both live and dead earthworms. In our study, earthworms which were not recovered at the end of the experiment in the field and which had died rather than escaped, may have helped reduce the incidence of take-all by increasing the soil nutrient pool. However in pot trials with no earthworm mortality, A. trapezoides reduced the disease severity of take-all. This would suggest that earthworm death was not the main mechanism by which take-all disease was reduced. It will be important to determine the mechanisms by which A. rosea and A. trapezoides reduced take-all in order to ascertain the feasibility of using these earthworms to reduce this disease in the field. In red-brown earth soils, numbers as high as 138 A. trapezoides m-’ and 675 A. rosea mm2 have been recorded under a wheat-pasture rotation in South Australia (Buckerfield, 1993). Results both in pots and in the field, suggest that A. trapezoides is unlikely to affect take-all disease at a density of 138 m-* and may therefore be unlikely to affect this disease in most fields cropped to wheat in southern Australia. Further field trials are required to test this. In calcareous sand soils in southern Australia, earthworms are usually only found in relatively low numbers (< 50 m-‘), with A. rosea being the dominant earthworm present (Mele, 1991). Our study demonstrated that under field conditions, A. rosea at a number equivalent to 100 m-*, can reduce take-all disease in this soil type. Increasing the density of A. rosea to 300 m-*, did not cause a further reduction in the percentage length of seminal roots containing take-all lesions. Further field trials are required in calcareous sand soils, to assess whether A. rosea at numbers < 50 m-* can also reduce take-all. Methods, such as direct drilling (Buckerfield, 1993), which further increase and sustain earthworm numbers in these soil types, may facilitate the ability of A. rosea and A. trapezoides to reduce this disease. Climate, as well as soil type, is likely to influence the ability of earthworms to reduce take-all disease. At Kapunda, only 44% of the rainfall that was recorded in July-September in 1990-1992, was observed during the same period in 1993. This would surely have resulted in a lower average soil matric potential, which in turn may have reduced earthworm activity (Kretzschmar, 1991). If the reduction in take-all was correlated with earthworm activity, in years with an average rainfall the inhibition of disease by earthworms may have been even greater. In addition, as the amount of spring rain is a major influence on the development of take-all (Roget and Rovira, 1990), the environmental conditions conducive to earthworms reducing take-all disease may also correlate with those environmental conditions conducive to take-all. In conclusion, our results demonstrate that under field conditions the earthworms A. rosea and A.
trapezoides have the potential to reduce the incidence of take-all disease on wheat. This study, together with the report that A. trapezoides can reduce the disease severity of Rhizoctonia solani Kuhn on wheat (Stephens et al., 1993) would suggest that earthworms, under certain conditions, may influence the disease severity of soil-borne plant fungal pathogens on wheat. Further field trials, using natural rather than artificial inoculum of Ggt and in which earthworms are not restricted to cylinders, are required to confirm this. Acknowledgements-This work was supported in part by the Grains Research and Development Corporation, Australia. Thanks are due to Dr Ray-Correll for help in statistical analysis, MS Teri Terrace for technical advice and Dr Clive Pankhurst and David Roget for critical reading of this manuscript. REFERENCES
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Kollmorgen J. F., Griffiths J. B. and Walsgott D. N. (1983) The effects of various crops on the survival and carry-over of the wheat take-all fungus Gaeumannomyces graminis var. tritici. Plant Pathology 32, 73-77. Kretzschmar A. (1991) Burrowing ability of the earthworm Aporrecfodea longa limited by soil compaction and water ootential. Biology and Fertility of Soils 11. 48-51. Lavelle P., Barois?., Martin A:, Zaidi Z. and Schaefer R. (1989) Management of earthworm populations in agro-ecosystems: a possible way to maintain soil quality? In Ecology of Arable Land, Perspectives and Challenges (M. Clarholm, L. Bergstrom, Eds), pp. 1099122. Kluwer, London. MacNish G. C. and Nicholas D. A. (1987) Some effects of field history on the relationship between grass production in subterranean clover pasture, grain yield and take-all (Gaeumannomyees graminis var. tritici) in a subsequent crop of wheat at Bannister, Western Australia. Australian Journal of Agricultural Research 38, 101l-1018. Mele P. (199 1) What species and how many on local farms. Earthworms, improving soilfor agriculture. Proceedings of a Conference held at TAFE College Wangaratta, Victoria. Australian Institute of Agricultural Science Occasional Publication No. 62. Moore K. J. and Cook R. J. (1984) Increased take-all of wheat with direct drilling in the Pacific Northwest. Phytopathology 74, 10441~9.
Mulongoy K. and Bedoret A. (1989) Properties ofworm casts and surface soils under various plant covers in the humid tropics. Soil Biology & Biochemistry 21, 197-203. Reis E. M.. Cook R. J. and McNeil B. L. (1982) Effect of mineral nutrition on take-all of wheat. Phytopathology 72, 224229.
Roget D. K. and Rovira A. D. (1990) The relationships
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between incidence of infection by the take-all fungus (Gaeumannomyces graminis var. tritict] rainfall, and yield of wheat in South Australia. Australian Journal Experimental Agriculture 31, 509-S 13. Rovira A. D. (1990) The impact of soil and crop management practices on soil-borne root diseases and wheat yields. Soil Use and Management 6, 195-200.
Ruz-Jerez B. E., Ball P. R. and Tillman R. W. (1992) Laboratory assessment of nutrient release from a pasture soil receiving grass or clover residues, in the presence or absence of Lumbricus rubellus or Eisenia fetida. Soil Biology & Biochemistry 24, 1529-1534. Rvder M. H. and Rovira A. D. (1993) Biological control of ‘take-all of glasshouse-grown wheat using strains of Pseudomonas corrugata isolated from wheat field soil. Soil Biology & Biochemistry 25, 3 1l-320. Shipton P. J. (1975) Take-all decline during cereal monoculture. In Biology and Control of Soil-borne Plant Pathogens (G. W. Bruehl, Ed.), pp. 137-144. American Phytopathological Society, St Paul, Minn. Simon A., Rovira A. D. and Foster R. C. (1987) Inocula of Gaeumannomyces graminis var. tritici for field and glasshouse studies. Soil Biology & Biochemistry 19, 363-370.
Soil Survey Staff (1990) Keys to Soil Taxonomy, 4th edn. Soil Management Support Services Technical Monograph No. 19, VirginiaPolytechnic Institute and State University, Blacksburg, Va. Stephens P. M., Davoren C. W., Doube B. M., Ryder M. H., Benger A. M., Neate S. M. (1993) Reduced severity of Rhizoctonia solani disease on wheat seedlings associated with the presence of the earthworm Aporrectodea trapezoides (Lumbricidae). Soil Biology & Biochemistry 25, 1477-1484.
Stephens P. M., Davoren C. W., Doube B. M. and Ryder M. H. (1994) Ability of theearthworms Aporrectodea rosea and Aporrectodea trapezoides to increase plant growth and the foliar concentration of elements in wheat (Triticum aestivum cv. Spear) in a sandy loam soil. Biology and Fertility of Soils. In press. Striganova B. R., Marfenina 0. E. and Ponomarenko V. A. (1989) Some aspects of the effect of earthworms on soil fungi. Biology Bulletin Academy of Science, USSR 15, 460-463.
Weller D. M. and Cook R. J. (1983) Suppression of take-all of wheat by seed treatments with fluorescent pseudomonads. Phytopathology 73, 463469.
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ACCELERATEDPAPER
ABILITY OF THE LUMBRICID EARTHWORMS APORRECTODEA ROSEA AND APORRECTODEA TRAPEZOIDES TO REDUCE THE SEVERITY OF TAKE-ALL UNDER GREENHOUSE AND FIELD CONDITIONS P. M. STEPHENS,‘* C. W. DAVOREN,’ B. M. DOUBE’.~and M. H. RYDER’.~ ‘Cooperative Research Centre for Soil and Land Management, Private Bag No. 2, Glen Osmond, SA 5064, Australia and %SIRO, Division of Soils, Private Bag No. 2, Glen Osmond, SA 5064, Australia (Accepted 3 July 1994)
Summary-The influence of the earthworms A. rosea and A. trupezoides on wheat plants, grown in soil artificially infested with the take-all fungus Gaeumannomyces graminis var. tritici (Ggt) was examined. In pot trials, using a red-brown earth soil artificially infested with Ggt, the presence of the earthworm A. trapezoides (at a density equivalent to 3 14 or 47 1 m-*) was associated with a significant (P < 0.05) increase in shoot weight and a reduction in the severity of take-all (as measured by a reduction in the percentage length of seminal roots containing take-all lesions). In contrast, in the absence of added Ggt, shoot weight was not significantly (P> 0.05) influenced by the presence of A. trapezoides.Two field trials were conducted, in which A. rosea and A. trupezoides were added, at an equivalent density of 100 or 300 rnm2,to cylinders driven into the soil. In a calcareous sandy loam artificially infested with Ggt, the presence of the earthworms A. rosea or A. trapezoides (at these densities) was associated with a significant (PcO.05) reduction in the severity of take-all disease. Under the same conditions, A. rosea or A. trapezoides (at an equivalent density of 300 m-2) caused a significant increase in shoot weight. In contrast, in the absence of added Ggt. shoot weight was not significantly (P > 0.05) influenced by the presence of A. rosea or A. trapezoides. In a second field trial in a red-brown earth, the presence of the earthworm A. trupezoides (at an equivalent density of 300 rne2) was associated with a significant (P < 0.05) reduction in the severity of take-all disease. Under the same conditions, A. rosea or A. trupezoides did not cause a significant increase in shoot weight. In the absence of Ggt, shoot weight was not significantly (PzO.05) influenced by the presence of A. rosea or A. trapezoides.These results demonstrate the potential of the earthworms A. rosea and A. truperoides, under both greenhouse and field conditions, to reduce the severity of take-all disease on wheat.
INTRODUCTION Gaeumannomyces graminis var. tritici (Ggt), the causative agent of take-all, is the most damaging root disease of wheat (Triticum aestivum L.) worldwide (Huber and McCay-Buis, 1993). In southern Australia, this disease can cause losses in wheat production of Au&100 million per annum (Rovira, 1990). Yield losses as high as 6&75% can occur where there is a high incidence of infected plants (>50%) and high spring rainfall (Roget and Rovira, 1990). The apparent absence of plant genetic resistance and effective chemical controls for take-all, imposes many constraints on farm management practices (Huber and McCay-Buis, 1993). In Australia, control of take-all can be accomplished through the rotation of cereals with a non-host break crop (Kollmorgen et a/., 1983). However, there is concern as to the agronomic sustainability of using extended cropping systems to reduce take-all, due to a decline in cereal grain protein,
*To whom all correspondence
should he addressed.
soil structure and soil fertility. Take-all may also be controlled by the rotation of cereals with a grass-free pasture, in which herbicides are used to remove grasses which host Ggt (MacNish and Nicholas, 1987). However under intense cropping, there is concern about the development of herbicide resistance in grasses (Heap and Knight, 1986). Alternative methods to control take-all need to be developed. Continuous wheat monoculture can lead to a decrease in severity of take-all disease (Gerlagh, 1968; Shipton, 1975). Efforts to isolate the causes of this suppression and use them to control Ggt have focused primarily on isolating microorganisms antagonistic to this fungal pathogen. For example, Ryder and Rovira (1993) isolated a group of non-fluorescent Pseudomonas from the rhizosphere of wheat which gave significant and reproducible disease suppression. Significant control of take-all in the field has been observed using Pseudomonas jfuorescens (Weller and Cook, 1983) and Bacillus species (Capper and Campbell, 1986). The possibility that other larger soil organisms may suppress Ggt has been largely
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overlooked.
P. M. STEPHENS et al. Homma
et al. (1979) and Chakraborty that mycophagous amoebae, isolated from wheat fields, could feed upon G. gvaminis. Addition of mycophagous amoebae to sterilized soil infested with G. graminis var. tritici, resulted in a significant reduction in the severity of take-all (Chakraborty and Warcup, 1985). The disease severity of take-all is increased by a deficiency of essential plant mineral nutrients in soil (Reis et al., 1982; Huber, 1989; Huber and McCay-Buis, 1993). The earthworms Aporrectodea rosea and Aporrectodea trapezoides, which are widespread under wheat in southern Australia (Mele, 1991; Baker et al., 1993a), are able to increase the foliar concentration of a range of macro- and micro-nutrients in wheat (Stephens et al., 1994). These earthworms may therefore help to reduce the severity of take-all disease by increasing the amount of plant available nutrients in soil. Greenhouse studies have also suggested that the earthworm A. trapezoides, at an equivalent density of 1050 mm’, can reduce take-all disease in wheat (M. H. Ryder, B. M. Doube, T. E. Terrace and C. W. Davoren, unpublished results). We have assessed whether the earthworms A. rosea and A. trapezoides can influence the disease severity of take-all on wheat, both in greenhouse trials and at two field sites of contrasting soil types.
et al. (1983) reported
MATERIALS
AND METHODS
Pot experiments Surface soil (&lOcm) was taken from a site at Kapunda, located 75 km north of Adelaide, South Australia. The site contained volunteer pasture on a red-brown earth; classified as a thermic Calcic Natrixeralf (Soil Survey Staff, 1990) and pH 5.0 (H,O). Thesoilwasair-dried,sieved(4mm),adjustedto 75% of field capacity and the equivalent of 400 g airdriedsoilwasaddedto 500mlplasticpots. Soilmoisture was brought back to 75% of field capacity during the experiment by watering pots every 3 days up to a specified weight. Each pot contained either (a) no inoculum, (b) Ggt, (c) A. trapezoides or (d) Ggt + A. trapezoides, with 6 replications of each treatment. Inoculum of Ggt 8 (isolated from infected wheat roots from Avon, South Australia, by H. McDonald in 1981) was produced on dead rye-grass seed (Simon et al., 1987). This inoculum (92 mg) was mixed into the soil in each of the appropriate pots. Pots were placed in a randomized block in a water bath, maintained at 15C with natural light. After 48 h, either 1 (equivalent to 157 me2), 2 (equivalent to 314 m-?) or 3 (equivalent to 471 mm2) adult earthworms of A. trupezoides, each weighing 0.75-1.5 g, were added to the appropriate pots. Aluminium fly wire (mesh size 2 mm) was attached around the circumference of each pot to a height of 150 mm, in order that A. trapezoides did not escape. After a further 5 days, a 10 mm layer of non-infested soil
(adjusted to 75% of field capacity) was added to the surface of each pot and 6 seeds of wheat (Triticum aestiuum cv. Spear) were sown at a depth of 8 mm into each pot. The surface layer of soil protected the seed from early attack by Ggt and thereby increased the uniformity of disease symptoms (Ryder and Rovira. 1993). These were thinned to 3 seedlings pot-‘, 10 days after sowing. At 36 days after sowing, plant roots were rated for disease severity by calculating the percentage length of seminal roots (to 90 mm soil depth) containing Ggt lesions. Plant roots and shoots were then dried at 60’ C and weighed. Earthworms were removed from the pots, washed briefly in distilled water, dried on tissue paper for ca. 10 s and weighed. Field trials Field experiments were conducted in 1993 at: (1) Avon; located 100 km north of Adelaide, South Australia, with a winter dominant rainfall averaging 350 mm year-‘. This soil was classified as a calcareous sandy loam and a Petrocalcic Palexeralf (Soil Survey Staff, 1990) pH 8.2 (H,O), and had been previously sown to wheat. (2) Kapunda; in a field adjacent to the one from which soil was taken for pot experiments. where there was a winter dominant rainfall averaging 520 mm year-‘. This soil was classified as a red-brown earth or thermic Calcic Natrixeralf (Soil Survey Staff, 1990) and had been previously sown to oats. Cylinders of stormwater PVC pipe, 300 mm dia and a height of 210 mm, were inserted into soil to a depth of 150 mm (using an industrial rammer), on 7 June and 26 May at Avon and Kapunda, respectively. Cylinders were constructed and used according to the method of Baker et al. (1993b). On 24 June at Avon and on 15 June at Kapunda, the cylinders still containing soil were removed. Nylon curtain material was attached around the bottom of each cylinder and fastened with 12 mm plastic strapping. The cylinders were subsequently placed back into the soil. Cylinders were established with Ggt and earthworms in all combinations, with 6 replicates treatment-’ in cylinders containing Ggt inoculum. These were all sampled at the end of the experiment. In those cylinders without added Ggt inoculum, there were 12 replicates treatment ‘; of which one half were destructively sampled 42 days after sowing at Avon and 54 days after sowing at Kapunda, in order to assess earthworm survival. Replicates were arranged in a randomized block. On 3 July at both field sites, the top 2 cm of soil was removed from each of the cylinders and 1.05 g of Ggt inoculum (on dead rye-grass seed) was added evenly to the soil surface where appropriate, and the soil was then replaced. Any large fragments of organic matter on the soil surface were removed. Three days later (6 July), A. trupezoides or A. rosea were added to the soil surface of the appropriate cylinders at two different densities: 7 cylinder-’ (equivalent to 100 m-‘) or 21 cylinder- ’
Ability of earthworms to reduce take-all (equivalent to 300 mm2). These earthworms had been collected from a mass-rearing site in a field at the Waite Agricultural Research Institute, Adelaide, and held in pots of the respective field soils for 2 days prior to being added to the cylinders. Twenty wheat seeds (T. aestivum cv. Spear) were sown into each cylinder on 19 July at Avon and on 12 July at Kapunda. Twenty days later at each site seedlings were thinned to 10 cylinder-‘. Twelve additional cylinders (without Ggt inoculum) were also established in the above manner. On 27 July, 21 dead A. rosea or A. trapezoides (killed by exposure to 45°C for 30 min) were placed just under the soil surface in these cylinders. There were 6 replicates of each “dead earthworm” treatment. Plants were harvested on 11 October at Avon (83 days after sowing) and on 6 October at Kapunda (86 days after sowing). Roots were rated for severity of take-all and plant growth was assessed in the manner described previously. At Avon, there was ca. 110 mm of rainfall from the time the earthworms were added through to sampling (6 July-11 October) [measured at Balaklava, 15 km from Avon (Bureau of Meteorology, South Australia)]. An additional 26 mm of rainwater was added to each cylinder by hand. In the years 1990-1992, the average rainfall at Avon from 1 July to 30 September was 129 +47(SD) mm. During this same time period at Avon in 1993, the rainfall was 106 mm. At
1293
Kapunda, there was ca. 132 mm of rainfall from the time the earthworms were added through to sampling (6 July-6 October). No additional water was added to these cylinders. In the years 199&1992, the average rainfall at Kapunda from 1 July to 30 September was 254*64(SD) mm. During this same period at Kapunda in 1993, the rainfall was 112 mm. The statistical analysis was conducted using Genstat 5 (Anon., 1993). RESULTS
In both pot and field experiments, the variances of the % seminal root length containing Ggt lesions in treatments with added Ggt inoculum, were substantially different from the variances obtained from treatments without added Ggt inoculum. Therefore, the % seminal root length containing Ggt lesions in treatments with and without Ggt inoculum were analysed separately. Pot experiments All earthworms added to pots were recovered at the end of the experiment, except for 4 pots. These were therefore omitted from the statistical analysis. The percentage loss in fresh weight of A. trapezoides in pots containing 1, 2 or 3 individuals was 19.4f 15.7(SD), 18.1f7.2 and 16.4+8.4%, respectively. The introduction of Ggt into soil increased
30
25
20
0 -- Ggt
+ Ggt
Control
(157) t
(314)
(471)
Plus Ggt +
(157) e
(314)
(471)
Minus Ggt _
Fig. 1. Influence of the earthworm A. trupezoides on the severity of take-all disease in wheat, grown in pots in a red-brown earth with or without artificial inoculum of Ggf. Either 0, 1 (equivalent to 157 m-2), 2 (equivalent to 314 m-‘) or 3 (equivalent to 471 m-*) earthworms were added to pots. Columns containing the same letter were not significantly different at the P-co.05 level (using ANOVA).
1294
P. M. STEPHENS et al.
-- Ggt
+ Ggt
Control
t
Plus Ggt -r
T
MinusGgt
111)
Fig. 2. Influence of the earthworm A. trapezoides on the shoot weight of wheat, grown in pots in a red-brown earth with or without artificial inoculum of Ggt. Either 0, 1 (equivalent to 157 m-‘), 2 (equivalent to 314 m-l) or 3 (equivalent to 471 m-*) earthworms were added to pots. Columns containing the same letter were not significantly different at the P-co.05 level (using ANOVA).
(P-C 0.05) the % seminal root length containing lesions from 0 to 26% (Fig. 1). Under these conditions, the presence of A. trapezoides (at a density of 2 or 3 pot-‘) caused a significant (P~0.05) reduction in root lesions. The addition of Ggt into soil also caused a significant (PC 0.05) reduction of 33% in the shoot dry weight (Fig. 2). The presence of A. trapezoides (at a density of 2 or 3 pot-‘) caused a si~i~cant (PcO.05) increase in the shoot dry weight in the presence of Ggt, although the plant top growth was still significantly lower than that of wheat grown in the absence of Ggt. In the absence of Ggt, A. trape~aides did not influence shoot dry weight or the amount of take-all disease. Field trials
In field trials in the calcareous sandy loam at Avon, one cylinder (out of 72) contributed 11.6 times more to the residual mean square (error) of disease rating compared to the mean residual square of the other observations and prevented any significant differences between treatments. This one value was omitted from the statistical analysis. In the calcareous sandy loam, 88 + B(SE)% of A. trapezoides and 69+ 19(SD)% of A. rosea were recovered from cylinders 42 days after sowing. At the termination of the experiment (83 days after sowing), 25.8~23.5(SD)%ofA.trapezoidesand5.0~10.2%0f A. rosea were recovered from the cylinders. There was a reduction in earthworm number in each cylinder,
making it impossible to calculate the relative loss in earthworm weight. The addition of Ggt into the calcareous sandy loam caused a substantial increase in the incidence of take-all disease on wheat (Table I ). The introduction of the equivalent of 7 or 21 A. rosea or A. tmpezoides cylinder-‘, resulted in a significant (P~0.05) reduction in root lesions; with an average reduction of 34*5(SD)% in take-all lesions. There was no significant difference in the reduction of take-all lesions with either earthworm species or density. In the presence of earthwo~s and Ggr, the amount of take-all disease was still significantly (P < 0.05) higher than that of wheat grown in the absence of added Ggt. The addition of Ggt caused a 27% reduction (P-cO.05) in shoot weight (Table 1). In the presence of Ggt inoculum, the addition of 21 A. rosea or A. trapezoides cylinder-’ (but not 7 cylinder -I), was associated with a significant (P~0.05) increase in shoot weight and resulted in shoot weights not significantly (P > 0.05) different to that of the control (minus Ggt inoculum). In contrast, in the absence of Ggt, the addition of A. rosea or A. trapezoides, at either 7 or 2 1 cylinder- ‘, had no significant (P> 0.05) effect upon shoot weight. In the red-brown earth at Kapunda, 83 + 12(SD)% of A. trapezoides and 8 1+ 21% of A. rosea were recovered from the cylinders 54 days after sowing. At the end of the experiment (86 days after sowing), two
Ability of earthworms to reduce take-all cylinders had holes in the nylon material with < 24% of earthworms present. These 2 cylinders were omitted from the statistical analysis. At the end of the experiment, 83.5 + 16.7(SD)% of A. trapezoides and 85.3 f21.5% of A. rosea were recovered from the remaining cylinders. In those cylinders where the same number of earthworms were recovered at the end of the experiment as were added at the start, A. trapezoides had apparently lost 37.0 &-8.0% of their fresh weight, while A. rosea had lost 19.8 f 11.O%. The addition of Ggt inoculum into the red-brown earth, caused the percentage length of seminal roots containing Ggt lesions to increase from 2 to 48%. Under these conditions, only the introduction of A. trapezoides, at a density of 21 cylinder-‘, caused a significant (PcO.05) reduction (reduced to 28%) in the length of seminal roots containing take-all lesions. However, the amount of take-all disease was still significantly (PC 0.05) higher than that of wheat grown in the absence of Ggt. The addition of 7 or 21 A. rosea cylinder-’ or 7 A. trapezoides cylinder-’ did not cause a significant (P>O.O5) reduction in Ggt lesions. The addition of Ggt caused a 43% reduction (P < 0.05) in shoot weight. Under these conditions, the addition of A. rosea and A. trapezoides (at either 7 or 21 cylinder-‘) did not cause a significant (P>O.O5) increase in shoot weight. However, shoot weight in the presence of 7 or 21 A. rosea cylinder-i and 21 A. trapezoides cylinder-’ was not significantly different from the control (minus Ggt). In the absence of added Ggt inoculum, the addition of A. rosea or A. trapezoides (at either 7 or 21 cylinder’) did not have a significant (P> 0.05) effect upon shoot weight.
DISCUSSION
Our study showed that under field conditions Table 1. Influence of the earthworms A. mea weight of wheat, grown under field conditions Treatment
+ Ggt Control + A. mea (7 cylinder- I) + A. rmea (21 cylinder-‘) + A. trapezoides (7 cylinderr’) + A. trapezoides(21 cylinder-‘) - Ggt Control + A. mea (7 cylinder- ‘) +A. rmea (21 cylinder-‘) + A. trapezoides (7 cylinder-‘) + A. trapezoides (21 cylinderr’) + A. rmea (21 cylinder-’ ; added dead) + A. trapemides (21 cylinder-’ ; added dead)
the
1295
earthworms A. rosea and A. trapezoides have the potential to reduce the severity of take-all on wheat. The mechanism by which A. rosea and A. trapezoides reduced take-all disease is not known. Previous studies have shown that direct drilling, compared to conventional cultivation, may increase the incidence of take-all disease (Moore and Cook, 1984). One possible explanation for this result is that soil disturbance reduced take-all disease. As movement of A. rosea and A. trapezoides causes disruption of the soil profile, soil disturbance may be one mechanism by which A. rosea and A. trapezoides reduced the severity of take-all. In addition, earthworms have been shown to reduce the microbial biomass (Ruz-Jerez et al., 1992) and suppress the development of various fungi in soil (Keogh and Christensen, 1976; Striganova et al., 1989). Earthworms may have therefore also suppressed the saprophytic activity of Ggt in soil, by ingesting fungal mycelium or by producing unfavourable conditions for Ggt in their casts or tunnel linings. Wheat is more susceptible to take-all if nutritionally deficient in any of several macro- or micro-nutrients, including P, Mg, K, Mn, Cu, Zn or Fe (Reis et al., 1982). The report that A. rosea can, under certain conditions, increase the foliar concentration in wheat of Ca, Cu, K, Mn, N, Na and P and that A. trapezoides can increase the foliar concentration of Al, Ca, Fe, K, Mn, N and Na (Stephens et al., 1994) would suggest that these earthworms may also have reduced take-all disease by increasing the amounts of plant available nutrients in soil. Earthworms may increase plant nutrient availability in soil by enhancing organic matter breakdown and the release of plant nutrients (Lavelle et al., 1989). Plant nutrient availability may also be influenced by the higher enzymatic activity in earthworm casts compared to uningested soil (Businelli et al., 1984; Mulongoy and Bedoret, 1989) disease severity of take-all and shoot dry sandy loam soil with or without artificial inoculum
and A. trapezoides on the in a calcareous of Ggr
% Seminal root length containing Gat lesions* 22.2 * 4. I ta 14.8f8.5b 16.0+5.4b 13.5k5.0b 14.2+4.6b
0.1+0.2 0.0 + 0.0 0.0 5 0.0 0.0 * 0.0 0.0 + 0.0 0.0 + 0.0 0.0 * 0.0
Shoot dry weight
1.67kO.52~ 1.98 f 0.29bc 2.26 + 0.49ab 2.10~0.31bc 2.19k0.4ab
2.29k0.2ab 2.23 f 0.53a 2.62 f 0.29a 2.58k0.30a 2.57+0.33a 2.36+0.53ab 2.03 f0.44bc
Either 0, 7 (equivalent to 100 mm2) or 21 (equivalent to 300 m-2) A. traperoides or A. rosea were added to the appropriate cylinders. Columns containing the same letter were not significantly different at the PiO.05 level (usina ANOVA). *As the variances of the % seminal root length containing Ggt lesions in cylinders without added Ggr inoculum were substantially different from the variances obtained from cylinders with added Ggf inoculum, treatments with and without added Ggt inoculum were analysed separately. tstandard deviation.
1296
P. M. STEPHENS et al.
or by the secretion of calcium carbonate influencing the solubility and availability of plant nutrients (Edwards and Lofty, 1977). Increased nutrient availability in soil may also be derived from the loss of nutrients from both live and dead earthworms. In our study, earthworms which were not recovered at the end of the experiment in the field and which had died rather than escaped, may have helped reduce the incidence of take-all by increasing the soil nutrient pool. However in pot trials with no earthworm mortality, A. trapezoides reduced the disease severity of take-all. This would suggest that earthworm death was not the main mechanism by which take-all disease was reduced. It will be important to determine the mechanisms by which A. rosea and A. trapezoides reduced take-all in order to ascertain the feasibility of using these earthworms to reduce this disease in the field. In red-brown earth soils, numbers as high as 138 A. trapezoides m-’ and 675 A. rosea mm2 have been recorded under a wheat-pasture rotation in South Australia (Buckerfield, 1993). Results both in pots and in the field, suggest that A. trapezoides is unlikely to affect take-all disease at a density of 138 m-* and may therefore be unlikely to affect this disease in most fields cropped to wheat in southern Australia. Further field trials are required to test this. In calcareous sand soils in southern Australia, earthworms are usually only found in relatively low numbers (< 50 m-‘), with A. rosea being the dominant earthworm present (Mele, 1991). Our study demonstrated that under field conditions, A. rosea at a number equivalent to 100 m-*, can reduce take-all disease in this soil type. Increasing the density of A. rosea to 300 m-*, did not cause a further reduction in the percentage length of seminal roots containing take-all lesions. Further field trials are required in calcareous sand soils, to assess whether A. rosea at numbers < 50 m-* can also reduce take-all. Methods, such as direct drilling (Buckerfield, 1993), which further increase and sustain earthworm numbers in these soil types, may facilitate the ability of A. rosea and A. trapezoides to reduce this disease. Climate, as well as soil type, is likely to influence the ability of earthworms to reduce take-all disease. At Kapunda, only 44% of the rainfall that was recorded in July-September in 1990-1992, was observed during the same period in 1993. This would surely have resulted in a lower average soil matric potential, which in turn may have reduced earthworm activity (Kretzschmar, 1991). If the reduction in take-all was correlated with earthworm activity, in years with an average rainfall the inhibition of disease by earthworms may have been even greater. In addition, as the amount of spring rain is a major influence on the development of take-all (Roget and Rovira, 1990), the environmental conditions conducive to earthworms reducing take-all disease may also correlate with those environmental conditions conducive to take-all. In conclusion, our results demonstrate that under field conditions the earthworms A. rosea and A.
trapezoides have the potential to reduce the incidence of take-all disease on wheat. This study, together with the report that A. trapezoides can reduce the disease severity of Rhizoctonia solani Kuhn on wheat (Stephens et al., 1993) would suggest that earthworms, under certain conditions, may influence the disease severity of soil-borne plant fungal pathogens on wheat. Further field trials, using natural rather than artificial inoculum of Ggt and in which earthworms are not restricted to cylinders, are required to confirm this. Acknowledgements-This work was supported in part by the Grains Research and Development Corporation, Australia. Thanks are due to Dr Ray-Correll for help in statistical analysis, MS Teri Terrace for technical advice and Dr Clive Pankhurst and David Roget for critical reading of this manuscript. REFERENCES
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