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Effects of soil temperature and moisture on survival of Coniothyrium minitans conidia in central China
Biological Control 55 (2010) 27–33
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Effects of soil temperature and moisture on survival of Coniothyrium minitans conidia in central China Long Yang a, Guo Qing Li a,*, Ya Qin Long a, Guo Ping Hong b, Dao Hong Jiang a, Hung-Chang Huang c,1 a
The State Key Laboratory of Agricultural Microbiology, The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China Hubei Province Meteorological Science and Technology Service Center, Wuhan 430074, China c Agriculture and Agri-Food Canada,Research Centre, Lethbridge, Alberta, Canada b
a r t i c l e
i n f o
Article history: Received 24 February 2010 Accepted 21 June 2010 Available online 1 July 2010 Keywords: Coniothyrium minitans Survival Temperature Soil moisture Biological control
a b s t r a c t A study was conducted to investigate effects of soil temperature and moisture on survival of the mycoparasite Coniothyrium minitans in soils free of its host fungus Sclerotinia sclerotiorum in central China. Survival of C. minitans conidia was monitored in non-irrigated soil (yellow-brown clay soil) and in submerged soil under the influence of seasonal fluctuations of soil temperature from 0.1 to 37 °C and precipitation from 1.2 to 10.4 mm d1. Results showed that in non-irrigated soil, C. minitans survived for 750 days with the concentration decreasing from 7.2 105 to 3.9 104 cfu g1 soil. In submerged soil, C. minitans survived for 150 days over the summer-to-autumn period and the winter-to-spring period with the concentration decreasing more rapidly than that in non-irrigated soil. Studies on the effects of constant temperature and moisture on survival of C. minitans in soil showed that at 4, 10, 20 and 28 °C, C. minitans survived for 360 days in soil containing 6.3%, 18.5% or 45.4% of water (w w1). At 30, 35, 37 and 40 °C, survival of C. minitans became moisture dependent. Good survival in soil containing 6.3% of water and poor survival in soil containing 18.5% or 45.4% of water were observed. C. minitans survived very poorly (61 day) at 45 and 50 °C irrespective of soil moisture. These results suggest that C. minitans can adapt to soil temperature and moisture conditions in central China and can be a promising agent used to treat soil for control of S. sclerotiorum. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Coniothyrium minitans Campbell is a mycoparasite of Sclerotinia sclerotiorum (Lib.) de Bary (Campbell, 1947). It is an effective agent for control of crop diseases caused by S. sclerotiorum (Huang, 1980; de Vrije et al., 2001; Whipps et al., 2008). Examples of commercial products of C. minitans registered for control of sclerotinia diseases are ContansÒ WG (Prophyta Biologischer Pflanzenschutz GmbH, Malchow, Germany) in Germany, and KONIÒ (Biovéd Biological Plant Protection Product Produicing Co., Kemestaródfa, Hungry) in Hungary. C. minitans is an obligate mycoparasite of Sclerotinia spp. and no detectable saprophytic growth by C. minitans in natural soils has ever been observed (Adams, 1990; Whipps and Gerlagh, 1992; Bennett et al., 2003). Thus, infection of sclerotia of S. sclerotiorum by C. minitans has been regarded as an important mechanism for survival of this mycoparasite in nature (Whipps and Gerlagh, 1992). Investigators in UK reported that C. minitans survived in soil
* Corresponding author. E-mail address: [email protected] (G.Q. Li). 1 Present address: Plant Pathology Division, Taiwan Agricultural Research Institute, Wufeng, Taichung 41301, Taiwan. 1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.06.010
contaminated with sclerotia of S. sclerotiorum for two years or longer (Tribe, 1957; Budge and Whipps, 1991; McQuilken and Whipps, 1995; McQuilken et al., 1995, 1997a). Bennett et al. (2006) indicated that C. minitans-infected sclerotia of S. sclerotiorum could serve as reservoirs for survival of C. minitans in soil. However, information remains unavailable on survival of C. minitans in natural soil in the absence of sclerotia of S. sclerotiorum. Several reports indicated that temperature is an important factor affecting survival of C. minitans. McQuilken and Whipps (1995) and McQuilken et al. (1997a) indicated that the shelf-life of C. minitans conidia grown on solid substrates was longer at 5 and 15 °C than at 30 °C. Huang and Erickson (2002) reported that C. minitans could over-winter in soil in western Canada, where the soil temperature was subfreezing in winter. Several previous studies suggest that soil type might not be as important as temperature in affecting survival of C. minitans in soil, as this organism has been detected in various types of soil distributing in temperate regions (Campbell, 1947; Huang, 1981; Sandys-Winsch et al., 1993; Li et al., 1995; Gao et al., 2002). Information on the effect of soil moisture or water content on survival of C. minitans remains scant, except one report by Bennett et al. (2006), who demostrated that, under dry conditions, most conidia of C. minitans exuded from pycnidia lost viability after 10 months.
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Hubei Province is located in the middle range of Yangtze River in central China and is one of the most important regions for cultivation of paddy rice (Oryza sativa L.) and oilseed rape (Brassica napus L. and B. campestris L.) in China. The rotation of oilseed rape with paddy rice has become a routine agronomic practice in many counties in Hubei Province. The climate of Hubei Province is humid and hot in summer, dry and temperate in autumn, dry and cold in winter, and humid and temperate in spring. Under these climatic conditions, S. sclerotiorum can survive as sclerotia in summer, autumn and winter, and germinate carpogenically to produce ascospores in spring, thereby causing sclerotinia diseases (leaf blight, stem rot, pod rot and seed rot) on winter oilseed rape, as well as on other crops. Li et al. (1995, 2006) showed that C. minitans is an effective mycoparasite for destroying sclerotia of S. sclerotiorum in soil and for suppression of sclerotinia diseases of oilseed rape caused by ascospores of S. sclerotiorum. However, data on survival of C. minitans conidia under summer and winter conditions in nonirrigated soil and in paddy soil in central China is lacking. The objectives of this study are: (i) to determine survival of C. minitans conidia in non-irrigated soil and in water-submerged soil under field conditions in Wuhan, a representative area in central China; and (ii) to assess the effects of soil temperature and moisture on survival of C. minitans conidia in soil. 2. Materials and methods 2.1. Strain SV-5-2 of C. minitans and inoculum preparation A mutant strain SV-5-2 of C. minitans was used in this study. It was derived from the wild-type strain Chy-1 of C. minitans isolated from a soil sample collected from Chang Yang County, Hubei Province, China (Li et al., 1995). Strain SV-5-2 was highly resistant to the fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione], and was similar to its parental stain Chy-1 in mycelial growth rate, percentage of conidial germination on agar media, infection of sclerotia of S. sclerotiorum and in suppression of colonization of flower petals of oilseed rape by S. sclerotiorum (Miao and Li, 2006). The fungicide-resistance trait in strain of SV-5-2 was genetically stable (Miao and Li, 2006). Yang et al. (2007) showed that the fungicide-resistance trait in strain SV-5-2 is a useful marker for monitoring survival of C. minitans on flower petals of oilseed rape. It might also be useful for monitoring survival of C. minitans in soil. Stock cultures of strain SV-5-2 of C. minitans were maintained at 4 °C on potato dextrose agar (PDA) made of fresh potato. Working cultures were established by transferring agar plugs of stock cultures containing conidia and mycelia onto PDA plates (9 cm diam.), which were then incubated at 20 °C in the dark for 4 weeks. To prepare conidial inoculum for each experiment, conidial suspensions of C. minitans strain SV-5-2 (1 107 conidia ml1) were pipetted on PDA in Petri dishes (9 cm diam.), 100 ll per dish, and the conidial suspension drops were spread over the agar surface using a sterilized glass rod. The dishes were sealed with PARAFILMÒ M (Chicago, IL, USA) and incubated at 20 °C in the dark for four weeks. Sterile distilled water (SDW) was added to the C. minitans culture in each dish and the colony surface of C. minitans was gently rubbed with a sterilized spatula to suspend conidia. The suspensions were filtered through four layers of sterilized cheesecloth to remove mycelial fragments. The conidial concentration in the suspensions was determined using a hemocytometer. 2.2. Soil preparation The soil used for all the experiments in this study was collected from the top 5 cm of the soil profile of a field near the campus of
Huazhong Agricultural University (HAU), Wuhan, China. The field had been planted for oilseed rape for many years. The soil is a yellow-brown clay with a pH value of 6.0, the carbon content of 1.1% (w w1) and the nitrogen content of 0.1% (w w1) and is widely distributed in central China (Xi, 1994). The water holding capacity for this soil was 45.4% (w w1). Soil samples were air-dried, crushed to a fine powder and sieved through a 2-mm-mesh screen to remove sclerotia of S. sclerotiorum, sand particles and plant debris (Hoes and Huang, 1975). 2.3. Survival of C. minitans in a non-irrigated field This experiment was aimed at assessing the long-term survival of C. minitans in non-irrigated soils under natural field conditions in central China. The field for this experiment was located in the campus of Huazhong Agricultural University (HAU), Wuhan (E114°210 and N30°370 ) and oilseed rape had been planted for many years in this field. The experiment was begun in Oct. 01 of 2004 and ended in Nov. 01 of 2006. This period covered three autumn seasons (2004, 2005 and 2006), two spring seasons (2005 and 2006), two winter seasons (2004 and 2005) and two summer seasons (2005 and 2006) (Fig. 1). Under the influence of the subtropical climate, rain precipitation and soil temperature (5 cm depth) in Wuhan fluctuated in these seasons. The average rain precipitation was high in the summer seasons (4.2 mm day1), followed by the spring seasons (3.4 mm day1), but was low in the autumn seasons (2.1 mm day1) and in the winter seasons (1.8 mm day1). The average daily soil temperature was also high in the summer seasons (30 °C), followed by the autumn seasons (21 °C) and the spring seasons (19 °C), but was low in the winter seasons (6 °C). Conidial suspensions of C. minitans (5 107 conidia ml1) were incorporated into the prepared fine soil at the ratio of 1:20 (v w1). Soil mixed with sterile distilled water was used as a control. C. minitans-infested soil or the control soil were added to plastic pots (9 11 cm, diameter height), 150 g soil per pot. The pots were buried in the field at the depth of 8–9 cm and were arranged in rows at 20 cm row spacing and 15 cm pot spacing within each row. Treatments were arranged in a randomized complete block design. To simulate conditions of commercial fields for cultivation of oilseed rape, seeds of oilseed rape (B. napus cultivar Zhong You Za No. 3) were sown between rows of the plastic pots in this field for two seasons (2004.10–2005.05 and 2005.10–2006.05). Weeds in the field were manually removed in each season. For detection of viable C. minitans in soil, three plastic pots (replicates) of each treatment were randomly retrieved immediately after burial (Oct. 01, 2004) and at half-month intervals thereafter till Nov. 01 of 2006. Four 10-g sub-samples of soil were collected from each sampled pot (1–5 cm in depth). One of the sub-samples from each sampled pot was added to 100 ml SDW in a 250-ml Erlenmeyer flask, which was then shaken on a rotary shaker at 200 rpm for 30 min. The resulting suspension was serially diluted to 102, 103 and 104 with SDW and 0.1 ml of each suspension was spread on a plate of PDA containing 500 lg a.i. ml1 vinclozolin (BASF Trading Co. Ltd. Shanghai, China), 500 lg a.i. ml1 of streptomycin sulfate (North China Pharmaceutical Company Ltd., Shi Jia Zhuang, China) and 500 lg a.i. ml1 of benzylpenicillin sodium (North China Pharmaceutical Company Ltd, Shi Jia Zhuang, China) (Yang et al., 2007). There were five dishes for each dilution level. After incubation at 20 °C in the dark for 6 days, colonies of C. minitans developed on each agar dish were identified and recorded (Yang et al., 2007). The other three 10-g sub-samples of soil from each sampled pot were used for determination of the soil water content by drying at 105 °C for 48 h (Bao, 2000). The number of viable C. minitans in each sub-sample of soil was expressed as colony-forming units per gram of dry soil (cfu g1 dry soil).
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Fig. 1. Dynamics of average daily rain precipitation (A), average soil temperature (B) and log10 values of viable Coniothyrium minitans (C) in the assay from October 1, 2004 to November 1, 2006 in a non-irrigated field in Wuhan, Hubei, China. Vertical bars in the graph C represent standard errors of means (n = 3).
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2.4. Survival of C. minitans in submerged soil To assess survival of C. minitans under simulated rice-growing conditions, two experiments were conducted in submerged soils over two seasonal intervals. The first experiment was done in summer and autumn (the summer-to-autumn assay) from May 17 to Oct. 29 of 2005 and was repeated from May 21 to Oct. 22 of 2006. The second experiment was done in winter and spring (the winter-to-spring assay) from Nov. 27 of 2005 to Apr. 30 of 2006 and was repeated from Nov. 20 of 2006 to Apr. 23 of 2007. There were two treatments in each experiment: C. minitans and the untreated control (without C. minitans). The experiments were conducted in the same field used to study survival of C. minitans in the non-irrigated soils. The experimental design and the procedures to bury the soil-containing plastic pots to the field, to retrieve soil-containing pots and to quantify viable C. minitans in soil were the same as described above. In these assays, soil in each plastic pot was constantly maintained submerged under a 2 cm depth of sterile distilled water. 2.5. Weather data Soil temperature (at a depth of 5 cm) and daily rainfall data for the period of each experiment were provided by Wuhan Weather Bureau (WWB), Wuhan, China from a WWB weather station located near the experimental field (about 1.5 km) for this study. The daily temperature was based on the average of data collected at 2 am, 8 am, 2 pm and 8 pm for each day. 2.6. Effect of temperature and soil moisture on survival of C. minitans To determine effects of temperature and soil moisture on survival of C. minitans, an experiment was conducted under controlled conditions. The conidial suspension of C. minitans strain SV-5-2 (1 107 conidia ml1) was mixed with finely sieved soil to adjust the conidial concentration to about 1 106 conidia g1 soil. The C. minitans-infested soil was placed in sterile Petri dishes (6 cm diam.) at 30 g soil per dish. Sterile distilled water was added to each dish to adjust the water content to 6.3% (w w1) as the lowmoisture treatment (about 1500 kpa), to 18.5% as the moderate-moisture treatment (about 100 kpa) or to 45.4% as the high-moisture treatment (about 0 kpa). The dishes were individually sealed with PARAFILMÒ M, incubated in the dark at 4, 10, 20, 28, 30, 35, 37, 40, 45 or 50 °C. Soil in dishes with the water moisture content of 6.3%, 18.5% or 45.4%, but without inoculation with C. minitans, was used as controls. For temperatures at 4–28 °C, three dishes for each moisture treatment were sampled immediately after incubation (0 d) and then at 30-day intervals for 360 days. For temperatures at 30–40 °C, three dishes for each moisture treatment were sampled at 0, 1, 3 and 7 days after incubation, and then at 7-, 14- or 28-day intervals for 180–210 days. For temperatures at 45 and 50 °C, three dishes of soil for each moisture treatment were sampled at daily intervals for 7 days. Four 5-g sub-samples of soil were removed from the soil in each sampled dish, one for determination of viable C. minitans and the other three for determination of the soil water content (Bao, 2000). Concentrations of viable C. minitans were calculated and expressed as cfu g1 dry soil. 2.7. Data analyses Data on colony-forming units of C. minitans per gram of dry soil were individually log10-transformed before each analysis. Linear regression analysis was used to analyze the relationship between the concentration of viable C. minitans in soil (log10 values) and days after soil incorporation with C. minitans using the GLM proce-
dure in SAS software (SAS Institute, Cary, NC, USA, Version 8.0, 1999). The significance of the relative coefficient value (r) for each linear regression equation was determined by Student’s T test at P = 0.05 level. Data on log10 values of viable C. minitans for the three-moisture treatments at each temperature and at each sampling date were analyzed using analysis of variance (ANOVA) in the SAS software. Treatment means of different treatments in each test were separated using the Least Significance Difference (LSD) Test at the P = 0.05 level.
3. Results 3.1. Survival of C. minitans in non-irrigated soil Results showed that C. minitans strain SV-5-2 survived in the nonirrigated soil for 750 days (Oct. 01 of 2004–Nov. 01 of 2006) (Fig. 1). The concentration of viable C. minitans decreased with time from the initial of 7.2 105 cfu g1 soil (log10 value 5.9) to the final of 3.9 104 cfu g1 soil (log10 value 4.6). Regression analysis showed that log10 values (Y) of the concentration of viable C. minitans in soil were inversely proportional to the days (X) after incorporation of C. minitans into soil: Y = 6.3161 0.0023X (r = 0.8379, P < 0.01). 3.2. Survival of C. minitans in submerged soil C. minitans strain SV-5-2 could be retrieved from the submerged soil throughout the burial period for 150 days which covered a summer-autumn period (May 17–Oct. 29 of 2005 and May 21– Oct. 22 of 2006) in the summer-to-autumn assay and a winter– spring period (Nov. 27 of 2005–Apr. 30 of 2006 and Nov. 20 of 2006–Apr. 23 of 2007) in the winter-to-spring assay (Fig. 2). The concentration of viable C. minitans decreased with time. The relationship between log10 values (Y) of the concentration of viable C. minitans in soil and days (X) after incorporation of C. minitans in soil closely fitted the linear equation: Y = aX + b, where the slope constant a varied from 0.0047 to 0.0151, the intercept constant b varied from 4.6264 to 6.5017 and the relative coefficient r varied from 0.8646 to 0.9741 (P < 0.01). The absolute value of a in the equations for the summer-to-autumn assay in 2005 (a = 0.0151) and in 2006 (a = 0.0082) was greater than that in the equations for the winter-to-spring assay in 2005–2006 (a = 0.0065) and 2006–2007 (a = 0.0047) (Fig. 2). This result indicates that under submerged conditions, the concentration of viable C. minitans in soil decreased more rapidly in the summer-autumn period than in the winter–spring period. 3.3. Effect of soil temperature and moisture on survival of C. minitans At 4, 10, 20 and 28 °C, log10 values of viable C. minitans per gram of soil decreased slightly over the 360-day period (Fig. 2). Statistical analyses showed that under each temperature at each sampling date, log10 values of C. minitans were not significantly different (P > 0.05) among the three-moisture treatments (6.3%, 18.5% and 45.4% of water contents). At 30, 35, 37 and 40 °C, viable C. minitans experienced little or no loss of viability in low-moisture soils over the incubation period of 180 or 210 days (Fig. 3). However, under each of these temperatures, survival of C. minitans was greatly reduced for both the moderate-moisture treatment and the high-moisture treatment. In moderate-moisture soil incubated at 30, 35, 37 and 40 °C, viable C. minitans was retrieved for periods shorter than 120, 35, 14 and 3 days, respectively. In high-moisture soil incubated at these four temperatures, viable C. minitans was retrieved for periods shorter than 120, 14, 3 and 3 days, respectively.
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Fig. 2. Dynamics of log10 values of viable Coniothyrium minitans in the water-logged field in Wuhan, Hubei, China, in the summer-autumn test (2005 and 2006) and in the winter–spring test (2005–2006 and 2006–2007). Vertical bars in each graph represent standard errors of means (n = 3).
At 45 and 50 °C, survival of C. minitans was very poor for all the three-moisture treatments. After incubation for 1 day, viable C. minitans was retrieved only from the low-moisture soils with the average log10 values of viable C. minitans decreasing from 4.7 to 2.7 (Fig. 3). However, viable C. minitans was not retrieved from any soil samples of the other two moisture treatments at 45 °C and from any soil samples of all the three-moisture treatments at 50 °C. After incubation for 3 to 7 days, no viable C. minitans was retrieved from any of the three-moisture soils incubated at 45 and 50 °C.
4. Discussion Previous reports showed that occurrence of C. minitans in nature is frequently in association with sclerotia of Sclerotinia species (Campbell, 1947; Tribe, 1957; Hoes and Huang, 1975; Huang, 1977, 1981; Whipps and Gerlagh, 1992). Tribe (1957) proposed that sclerotia of Sclerotinia species might play a protective role in the longterm survival of C. minitans in soils and this hypothesis was confirmed by Bennett et al. (2006) using scanning electron microscopy to study the in situ survival of conidia of C. minitans on the surface and in the medulla of sclerotia of S. sclerotiorum in soils. Our study revealed that C. minitans could survive for 750 days in soil free of sclerotia of S. sclerotiorum under field conditions. Results from the controlled experiment demonstrated that C. minitans could survive for 360 days at 4–28 °C in soil also free of sclerotia of S. sclerotiorum. These findings provide direct evidence that the presence of sclerotia of S. sclerotiorum might not be essential for the long-term survival of C. minitans in soil. Previous studies showed that C. minitans cannot conduct any saprophytic growth in natural soils (Tribe, 1957). Dormancy of conidia of C. minitans in soil free of sclerotia of S. sclerotiroum might be the mechanism responsible for the long-term survival of this fungus observed in this study. The optimum temperature range for C. minitans to conduct mycelial growth and infection of S. sclerotiorum sclerotia is 15– 25 °C and the maximum temperature is 30 °C (Campbell, 1947; Huang and Erickson, 2008; McQuilken et al., 1997b). In central China, the climate is hot in summer, whereas temperate in spring. Soil temperature can reach up to 30 °C or higher in summer, or as low
as zero or more in winter. Our finding reveals that C. minitans can survive for >750 days in a non-irrigated soil under field conditions. This result suggests that C. minitans can over-summer and overwinter in central China. It further suggests that C. minitans might have a great potential to be used as a biocontrol agent to treat soil for elimination of sclerotia of S. sclerotiorum. Our previous studies involving a pot experiment in Wuhan (Wang et al., 2006) and a small-scale field experiment in Er Zhou, an area close to Wuhan (Yang et al., 2009), showed that application of C. minitans to S. sclerotiorum-contaminated soil in the early summer after harvest of oilseed rape, or in the middle of autumn at seed sowing or seedling transplanting of oilseed rape, could reduce the number of apothecia produced from sclerotia of S. sclerotiorum. Previous studies demonstrated that efficacy of C. minitans in suppression of apothecium production by S. sclerotiorum and in suppression of sclerotinia diseases on greenhouse lettuce is greatly affected by the rate of C. minitans conidia inoculated in soil (Jones and Whipps, 2002; Gerlagh et al., 2003; Jones et al., 2003; Yang et al., 2009). The minimum conidial concentration of C. minitans for effective suppression of carpogenic germination of S. sclerotiorum was 1 103 conidia g1 soil according to our previous study (Yang et al., 2009). In the present study, the concentration of C. minitans in non-irrigated soils after two years remained higher than this level. Therefore, soil treatment of C. minitans appears a promising measure for suppression of S. sclerotiorum in central China. Field studies on a larger scale to evaluate the efficacy of soil application of C. minitans in suppression of sclerotinia diseases of oilseed rape and other crops are warranted. Previous studies reported that sclerotia of S. sclerotiorum or S. minor survived less than 7–8 weeks in flooded soils, especially in summer (Moore, 1949; Abawi et al., 1985). In the present study C. minitans survived for 150 days in submerged soil over both summer–autumn and winter–spring periods, longer than sclerotia of Sclerotinia spp., under the same conditions. Long-term survival of C. minitans in paddy soil suggests that it might be possible to integrate soil treatment with C. minitans with the oilseed rape–rice rotation system to achieve effective control of sclerotinia stem rot of oilseed rape. Effects of constant temperature and soil moisture on survival of C. minitans were evaluated in the indoor experiment of this
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Fig. 3. Effects of soil temperature and soil moisture on survival of Coniothyrium minitans. The concentration of viable C. minitans in soil (cfu g1 dry soil) was log10transformed and vertical bars in each graph represent standard errors of means (n = 3).
study. Similar to the outdoor experiments, soil used in our indoor experiment was not sterilized. Long-term survival of C. minitans in non-irrigated soil under field conditions and in indoor soils incubated at 4–28 °C suggests that a close correlation in survival of C. minitans might exist between the outdoor and indoor experiments. Results about better survival of C. minitans at 4–28 °C (irrespective of soil moisture) than at 30 °C or higher (moisturedependent) in the indoor experiment, provide experimental evidence that soil temperature and moisture can affect survival of C. minitans in soil. As observed in this study, soil temperatures (5 cm depth) fluctuate yearly within the range of 1 to 37 °C in Wuhan, a representative area in central China. Although soil temperatures exceeding 30 °C can frequently occur in this area, the durations are usually brief. Thus, soil temperature in central China might not be highly adverse to survival of C. minitans, especially in non-irrigated soils.
Acknowledgments This study was funded by the Grants of the Natural Science Foundation of China (Grant Nos. 30471165 and 30971953), the Special Fund for Public Welfare Projects (Agriculture) of China (Grant No. nyhyzx07-054) and the ‘‘863’’ High-Tech Program of China (Grant No. 2006AA10A211). We thank Miss Qing-Lin Fu, College of Resources and Environment, Huazhong Agricultural University, for testing properties of the soil used in this study.
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Li, G.Q., Wang, D.B., Zhang, S.H., Dan, H.H., 1995. Studies on the mycoparasite Coniothyrium minitans I: characterization of biological properties and natural distribution in Hubei Province. Journal of Huazhong Agricultural University 14, 125–129. Li, G.Q., Huang, H.C., Miao, H.J., Erickson, R.S., Jiang, D.H., Xiao, Y.N., 2006. Biological control of sclerotinia diseases of rapeseed by aerial applications of the mycoparasite Coniothyrium minitans. European Journal of Plant Pathology 114, 345–355. McQuilken, M.P., Mitchell, S.J., Budge, S.P., Whipps, J.M., Fenlon, J.S., Archer, S.A., 1995. Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape. Plant Pathology 44, 883–896. McQuilken, M.P., Whipps, J.M., 1995. Production, survival and evaluation of solidsubstrate inocula of Coniothyrium minitans against Sclerotinia sclerotiorum. European Journal of Plant Pathology 101, 101–110. McQuilken, M.P., Budge, S.P., Whipps, J.M., 1997a. Production, survival and evaluation of liquid culture-produced inocula of Coniothyrium minitans against Sclerotinia sclerotiorum. Biocontrol Science and Technology 7, 23–36. McQuilken, M.P., Budge, S.P., Whipps, J.M., 1997b. Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of C. minitans. Mycological Research 101, 11–17. Miao, H.J., Li, G.Q., 2006. Mutation of Coniothyrium minitans tolerant to the fungicide vinclozolin and evaluation of the biocontrol potential of the selected mutants. Chinese Journal of Biological Control 22, 75–79. Moore, W.D., 1949. Flooding as a means of destroying sclerotia of Sclerotinia sclerotiorum. Phytopathology 39, 920–927. Sandys-Winsch, C., Whipps, J.M., Gerlagh, M., Kruse, M., 1993. World distribution of the sclerotial mycoparasite Coniothyrium minitans. Mycological Research 97, 1175–1178. Tribe, H.T., 1957. On the parasitism of Sclerotinia trifoliorum by Coniothyrium minitans. Transactions of the British Mycological Society 64, 489–499. Wang, Y.C., Li, G.Q., Fang, Y., Jiang, D.H., 2006. Production of conidia by the mycoparasite Coniothyrium minitans on stubbles of oilseed rape. Chinese Journal of Biological Control 22, 308–312. Whipps, J.M., Gerlagh, M., 1992. Biology of Coniothyrium minitans and its potential for use in disease biocontrol. Mycological Research 96, 897–907. Whipps, J.M., Sreenivasaprasad, S., Muthumeenakshi, S., Rogers, C.W., Challen, M.P., 2008. Use of Coniothyrium minitans as a biocontrol agent and some molecular aspects of sclerotial mycoparasitism. European Journal of Plant Pathology 121, 323–330. Xi, C.F., 1994. Soil Taxonomy. China Agriculture Press, Beijing, China. pp. 279. Yang, L., Miao, H.J., Li, G.Q., Yin, L.M., Huang, H.C., 2007. Survival of the mycoparasite Coniothyrium minitans on flower petals of oilseed rape under field conditions in central China. Biological Control 40, 179–186. Yang, L., Li, G.Q., Jiang, D.H., Huang, H.C., 2009. Water-assisted dissemination of conidia of the mycoparasite Coniothyrium minitans in soil. Biocontrol Science and Technology 19, 779–796.
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Biological Control journal homepage: www.elsevier.com/locate/ybcon
Effects of soil temperature and moisture on survival of Coniothyrium minitans conidia in central China Long Yang a, Guo Qing Li a,*, Ya Qin Long a, Guo Ping Hong b, Dao Hong Jiang a, Hung-Chang Huang c,1 a
The State Key Laboratory of Agricultural Microbiology, The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China Hubei Province Meteorological Science and Technology Service Center, Wuhan 430074, China c Agriculture and Agri-Food Canada,Research Centre, Lethbridge, Alberta, Canada b
a r t i c l e
i n f o
Article history: Received 24 February 2010 Accepted 21 June 2010 Available online 1 July 2010 Keywords: Coniothyrium minitans Survival Temperature Soil moisture Biological control
a b s t r a c t A study was conducted to investigate effects of soil temperature and moisture on survival of the mycoparasite Coniothyrium minitans in soils free of its host fungus Sclerotinia sclerotiorum in central China. Survival of C. minitans conidia was monitored in non-irrigated soil (yellow-brown clay soil) and in submerged soil under the influence of seasonal fluctuations of soil temperature from 0.1 to 37 °C and precipitation from 1.2 to 10.4 mm d1. Results showed that in non-irrigated soil, C. minitans survived for 750 days with the concentration decreasing from 7.2 105 to 3.9 104 cfu g1 soil. In submerged soil, C. minitans survived for 150 days over the summer-to-autumn period and the winter-to-spring period with the concentration decreasing more rapidly than that in non-irrigated soil. Studies on the effects of constant temperature and moisture on survival of C. minitans in soil showed that at 4, 10, 20 and 28 °C, C. minitans survived for 360 days in soil containing 6.3%, 18.5% or 45.4% of water (w w1). At 30, 35, 37 and 40 °C, survival of C. minitans became moisture dependent. Good survival in soil containing 6.3% of water and poor survival in soil containing 18.5% or 45.4% of water were observed. C. minitans survived very poorly (61 day) at 45 and 50 °C irrespective of soil moisture. These results suggest that C. minitans can adapt to soil temperature and moisture conditions in central China and can be a promising agent used to treat soil for control of S. sclerotiorum. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Coniothyrium minitans Campbell is a mycoparasite of Sclerotinia sclerotiorum (Lib.) de Bary (Campbell, 1947). It is an effective agent for control of crop diseases caused by S. sclerotiorum (Huang, 1980; de Vrije et al., 2001; Whipps et al., 2008). Examples of commercial products of C. minitans registered for control of sclerotinia diseases are ContansÒ WG (Prophyta Biologischer Pflanzenschutz GmbH, Malchow, Germany) in Germany, and KONIÒ (Biovéd Biological Plant Protection Product Produicing Co., Kemestaródfa, Hungry) in Hungary. C. minitans is an obligate mycoparasite of Sclerotinia spp. and no detectable saprophytic growth by C. minitans in natural soils has ever been observed (Adams, 1990; Whipps and Gerlagh, 1992; Bennett et al., 2003). Thus, infection of sclerotia of S. sclerotiorum by C. minitans has been regarded as an important mechanism for survival of this mycoparasite in nature (Whipps and Gerlagh, 1992). Investigators in UK reported that C. minitans survived in soil
* Corresponding author. E-mail address: [email protected] (G.Q. Li). 1 Present address: Plant Pathology Division, Taiwan Agricultural Research Institute, Wufeng, Taichung 41301, Taiwan. 1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.06.010
contaminated with sclerotia of S. sclerotiorum for two years or longer (Tribe, 1957; Budge and Whipps, 1991; McQuilken and Whipps, 1995; McQuilken et al., 1995, 1997a). Bennett et al. (2006) indicated that C. minitans-infected sclerotia of S. sclerotiorum could serve as reservoirs for survival of C. minitans in soil. However, information remains unavailable on survival of C. minitans in natural soil in the absence of sclerotia of S. sclerotiorum. Several reports indicated that temperature is an important factor affecting survival of C. minitans. McQuilken and Whipps (1995) and McQuilken et al. (1997a) indicated that the shelf-life of C. minitans conidia grown on solid substrates was longer at 5 and 15 °C than at 30 °C. Huang and Erickson (2002) reported that C. minitans could over-winter in soil in western Canada, where the soil temperature was subfreezing in winter. Several previous studies suggest that soil type might not be as important as temperature in affecting survival of C. minitans in soil, as this organism has been detected in various types of soil distributing in temperate regions (Campbell, 1947; Huang, 1981; Sandys-Winsch et al., 1993; Li et al., 1995; Gao et al., 2002). Information on the effect of soil moisture or water content on survival of C. minitans remains scant, except one report by Bennett et al. (2006), who demostrated that, under dry conditions, most conidia of C. minitans exuded from pycnidia lost viability after 10 months.
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Hubei Province is located in the middle range of Yangtze River in central China and is one of the most important regions for cultivation of paddy rice (Oryza sativa L.) and oilseed rape (Brassica napus L. and B. campestris L.) in China. The rotation of oilseed rape with paddy rice has become a routine agronomic practice in many counties in Hubei Province. The climate of Hubei Province is humid and hot in summer, dry and temperate in autumn, dry and cold in winter, and humid and temperate in spring. Under these climatic conditions, S. sclerotiorum can survive as sclerotia in summer, autumn and winter, and germinate carpogenically to produce ascospores in spring, thereby causing sclerotinia diseases (leaf blight, stem rot, pod rot and seed rot) on winter oilseed rape, as well as on other crops. Li et al. (1995, 2006) showed that C. minitans is an effective mycoparasite for destroying sclerotia of S. sclerotiorum in soil and for suppression of sclerotinia diseases of oilseed rape caused by ascospores of S. sclerotiorum. However, data on survival of C. minitans conidia under summer and winter conditions in nonirrigated soil and in paddy soil in central China is lacking. The objectives of this study are: (i) to determine survival of C. minitans conidia in non-irrigated soil and in water-submerged soil under field conditions in Wuhan, a representative area in central China; and (ii) to assess the effects of soil temperature and moisture on survival of C. minitans conidia in soil. 2. Materials and methods 2.1. Strain SV-5-2 of C. minitans and inoculum preparation A mutant strain SV-5-2 of C. minitans was used in this study. It was derived from the wild-type strain Chy-1 of C. minitans isolated from a soil sample collected from Chang Yang County, Hubei Province, China (Li et al., 1995). Strain SV-5-2 was highly resistant to the fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione], and was similar to its parental stain Chy-1 in mycelial growth rate, percentage of conidial germination on agar media, infection of sclerotia of S. sclerotiorum and in suppression of colonization of flower petals of oilseed rape by S. sclerotiorum (Miao and Li, 2006). The fungicide-resistance trait in strain of SV-5-2 was genetically stable (Miao and Li, 2006). Yang et al. (2007) showed that the fungicide-resistance trait in strain SV-5-2 is a useful marker for monitoring survival of C. minitans on flower petals of oilseed rape. It might also be useful for monitoring survival of C. minitans in soil. Stock cultures of strain SV-5-2 of C. minitans were maintained at 4 °C on potato dextrose agar (PDA) made of fresh potato. Working cultures were established by transferring agar plugs of stock cultures containing conidia and mycelia onto PDA plates (9 cm diam.), which were then incubated at 20 °C in the dark for 4 weeks. To prepare conidial inoculum for each experiment, conidial suspensions of C. minitans strain SV-5-2 (1 107 conidia ml1) were pipetted on PDA in Petri dishes (9 cm diam.), 100 ll per dish, and the conidial suspension drops were spread over the agar surface using a sterilized glass rod. The dishes were sealed with PARAFILMÒ M (Chicago, IL, USA) and incubated at 20 °C in the dark for four weeks. Sterile distilled water (SDW) was added to the C. minitans culture in each dish and the colony surface of C. minitans was gently rubbed with a sterilized spatula to suspend conidia. The suspensions were filtered through four layers of sterilized cheesecloth to remove mycelial fragments. The conidial concentration in the suspensions was determined using a hemocytometer. 2.2. Soil preparation The soil used for all the experiments in this study was collected from the top 5 cm of the soil profile of a field near the campus of
Huazhong Agricultural University (HAU), Wuhan, China. The field had been planted for oilseed rape for many years. The soil is a yellow-brown clay with a pH value of 6.0, the carbon content of 1.1% (w w1) and the nitrogen content of 0.1% (w w1) and is widely distributed in central China (Xi, 1994). The water holding capacity for this soil was 45.4% (w w1). Soil samples were air-dried, crushed to a fine powder and sieved through a 2-mm-mesh screen to remove sclerotia of S. sclerotiorum, sand particles and plant debris (Hoes and Huang, 1975). 2.3. Survival of C. minitans in a non-irrigated field This experiment was aimed at assessing the long-term survival of C. minitans in non-irrigated soils under natural field conditions in central China. The field for this experiment was located in the campus of Huazhong Agricultural University (HAU), Wuhan (E114°210 and N30°370 ) and oilseed rape had been planted for many years in this field. The experiment was begun in Oct. 01 of 2004 and ended in Nov. 01 of 2006. This period covered three autumn seasons (2004, 2005 and 2006), two spring seasons (2005 and 2006), two winter seasons (2004 and 2005) and two summer seasons (2005 and 2006) (Fig. 1). Under the influence of the subtropical climate, rain precipitation and soil temperature (5 cm depth) in Wuhan fluctuated in these seasons. The average rain precipitation was high in the summer seasons (4.2 mm day1), followed by the spring seasons (3.4 mm day1), but was low in the autumn seasons (2.1 mm day1) and in the winter seasons (1.8 mm day1). The average daily soil temperature was also high in the summer seasons (30 °C), followed by the autumn seasons (21 °C) and the spring seasons (19 °C), but was low in the winter seasons (6 °C). Conidial suspensions of C. minitans (5 107 conidia ml1) were incorporated into the prepared fine soil at the ratio of 1:20 (v w1). Soil mixed with sterile distilled water was used as a control. C. minitans-infested soil or the control soil were added to plastic pots (9 11 cm, diameter height), 150 g soil per pot. The pots were buried in the field at the depth of 8–9 cm and were arranged in rows at 20 cm row spacing and 15 cm pot spacing within each row. Treatments were arranged in a randomized complete block design. To simulate conditions of commercial fields for cultivation of oilseed rape, seeds of oilseed rape (B. napus cultivar Zhong You Za No. 3) were sown between rows of the plastic pots in this field for two seasons (2004.10–2005.05 and 2005.10–2006.05). Weeds in the field were manually removed in each season. For detection of viable C. minitans in soil, three plastic pots (replicates) of each treatment were randomly retrieved immediately after burial (Oct. 01, 2004) and at half-month intervals thereafter till Nov. 01 of 2006. Four 10-g sub-samples of soil were collected from each sampled pot (1–5 cm in depth). One of the sub-samples from each sampled pot was added to 100 ml SDW in a 250-ml Erlenmeyer flask, which was then shaken on a rotary shaker at 200 rpm for 30 min. The resulting suspension was serially diluted to 102, 103 and 104 with SDW and 0.1 ml of each suspension was spread on a plate of PDA containing 500 lg a.i. ml1 vinclozolin (BASF Trading Co. Ltd. Shanghai, China), 500 lg a.i. ml1 of streptomycin sulfate (North China Pharmaceutical Company Ltd., Shi Jia Zhuang, China) and 500 lg a.i. ml1 of benzylpenicillin sodium (North China Pharmaceutical Company Ltd, Shi Jia Zhuang, China) (Yang et al., 2007). There were five dishes for each dilution level. After incubation at 20 °C in the dark for 6 days, colonies of C. minitans developed on each agar dish were identified and recorded (Yang et al., 2007). The other three 10-g sub-samples of soil from each sampled pot were used for determination of the soil water content by drying at 105 °C for 48 h (Bao, 2000). The number of viable C. minitans in each sub-sample of soil was expressed as colony-forming units per gram of dry soil (cfu g1 dry soil).
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Fig. 1. Dynamics of average daily rain precipitation (A), average soil temperature (B) and log10 values of viable Coniothyrium minitans (C) in the assay from October 1, 2004 to November 1, 2006 in a non-irrigated field in Wuhan, Hubei, China. Vertical bars in the graph C represent standard errors of means (n = 3).
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2.4. Survival of C. minitans in submerged soil To assess survival of C. minitans under simulated rice-growing conditions, two experiments were conducted in submerged soils over two seasonal intervals. The first experiment was done in summer and autumn (the summer-to-autumn assay) from May 17 to Oct. 29 of 2005 and was repeated from May 21 to Oct. 22 of 2006. The second experiment was done in winter and spring (the winter-to-spring assay) from Nov. 27 of 2005 to Apr. 30 of 2006 and was repeated from Nov. 20 of 2006 to Apr. 23 of 2007. There were two treatments in each experiment: C. minitans and the untreated control (without C. minitans). The experiments were conducted in the same field used to study survival of C. minitans in the non-irrigated soils. The experimental design and the procedures to bury the soil-containing plastic pots to the field, to retrieve soil-containing pots and to quantify viable C. minitans in soil were the same as described above. In these assays, soil in each plastic pot was constantly maintained submerged under a 2 cm depth of sterile distilled water. 2.5. Weather data Soil temperature (at a depth of 5 cm) and daily rainfall data for the period of each experiment were provided by Wuhan Weather Bureau (WWB), Wuhan, China from a WWB weather station located near the experimental field (about 1.5 km) for this study. The daily temperature was based on the average of data collected at 2 am, 8 am, 2 pm and 8 pm for each day. 2.6. Effect of temperature and soil moisture on survival of C. minitans To determine effects of temperature and soil moisture on survival of C. minitans, an experiment was conducted under controlled conditions. The conidial suspension of C. minitans strain SV-5-2 (1 107 conidia ml1) was mixed with finely sieved soil to adjust the conidial concentration to about 1 106 conidia g1 soil. The C. minitans-infested soil was placed in sterile Petri dishes (6 cm diam.) at 30 g soil per dish. Sterile distilled water was added to each dish to adjust the water content to 6.3% (w w1) as the lowmoisture treatment (about 1500 kpa), to 18.5% as the moderate-moisture treatment (about 100 kpa) or to 45.4% as the high-moisture treatment (about 0 kpa). The dishes were individually sealed with PARAFILMÒ M, incubated in the dark at 4, 10, 20, 28, 30, 35, 37, 40, 45 or 50 °C. Soil in dishes with the water moisture content of 6.3%, 18.5% or 45.4%, but without inoculation with C. minitans, was used as controls. For temperatures at 4–28 °C, three dishes for each moisture treatment were sampled immediately after incubation (0 d) and then at 30-day intervals for 360 days. For temperatures at 30–40 °C, three dishes for each moisture treatment were sampled at 0, 1, 3 and 7 days after incubation, and then at 7-, 14- or 28-day intervals for 180–210 days. For temperatures at 45 and 50 °C, three dishes of soil for each moisture treatment were sampled at daily intervals for 7 days. Four 5-g sub-samples of soil were removed from the soil in each sampled dish, one for determination of viable C. minitans and the other three for determination of the soil water content (Bao, 2000). Concentrations of viable C. minitans were calculated and expressed as cfu g1 dry soil. 2.7. Data analyses Data on colony-forming units of C. minitans per gram of dry soil were individually log10-transformed before each analysis. Linear regression analysis was used to analyze the relationship between the concentration of viable C. minitans in soil (log10 values) and days after soil incorporation with C. minitans using the GLM proce-
dure in SAS software (SAS Institute, Cary, NC, USA, Version 8.0, 1999). The significance of the relative coefficient value (r) for each linear regression equation was determined by Student’s T test at P = 0.05 level. Data on log10 values of viable C. minitans for the three-moisture treatments at each temperature and at each sampling date were analyzed using analysis of variance (ANOVA) in the SAS software. Treatment means of different treatments in each test were separated using the Least Significance Difference (LSD) Test at the P = 0.05 level.
3. Results 3.1. Survival of C. minitans in non-irrigated soil Results showed that C. minitans strain SV-5-2 survived in the nonirrigated soil for 750 days (Oct. 01 of 2004–Nov. 01 of 2006) (Fig. 1). The concentration of viable C. minitans decreased with time from the initial of 7.2 105 cfu g1 soil (log10 value 5.9) to the final of 3.9 104 cfu g1 soil (log10 value 4.6). Regression analysis showed that log10 values (Y) of the concentration of viable C. minitans in soil were inversely proportional to the days (X) after incorporation of C. minitans into soil: Y = 6.3161 0.0023X (r = 0.8379, P < 0.01). 3.2. Survival of C. minitans in submerged soil C. minitans strain SV-5-2 could be retrieved from the submerged soil throughout the burial period for 150 days which covered a summer-autumn period (May 17–Oct. 29 of 2005 and May 21– Oct. 22 of 2006) in the summer-to-autumn assay and a winter– spring period (Nov. 27 of 2005–Apr. 30 of 2006 and Nov. 20 of 2006–Apr. 23 of 2007) in the winter-to-spring assay (Fig. 2). The concentration of viable C. minitans decreased with time. The relationship between log10 values (Y) of the concentration of viable C. minitans in soil and days (X) after incorporation of C. minitans in soil closely fitted the linear equation: Y = aX + b, where the slope constant a varied from 0.0047 to 0.0151, the intercept constant b varied from 4.6264 to 6.5017 and the relative coefficient r varied from 0.8646 to 0.9741 (P < 0.01). The absolute value of a in the equations for the summer-to-autumn assay in 2005 (a = 0.0151) and in 2006 (a = 0.0082) was greater than that in the equations for the winter-to-spring assay in 2005–2006 (a = 0.0065) and 2006–2007 (a = 0.0047) (Fig. 2). This result indicates that under submerged conditions, the concentration of viable C. minitans in soil decreased more rapidly in the summer-autumn period than in the winter–spring period. 3.3. Effect of soil temperature and moisture on survival of C. minitans At 4, 10, 20 and 28 °C, log10 values of viable C. minitans per gram of soil decreased slightly over the 360-day period (Fig. 2). Statistical analyses showed that under each temperature at each sampling date, log10 values of C. minitans were not significantly different (P > 0.05) among the three-moisture treatments (6.3%, 18.5% and 45.4% of water contents). At 30, 35, 37 and 40 °C, viable C. minitans experienced little or no loss of viability in low-moisture soils over the incubation period of 180 or 210 days (Fig. 3). However, under each of these temperatures, survival of C. minitans was greatly reduced for both the moderate-moisture treatment and the high-moisture treatment. In moderate-moisture soil incubated at 30, 35, 37 and 40 °C, viable C. minitans was retrieved for periods shorter than 120, 35, 14 and 3 days, respectively. In high-moisture soil incubated at these four temperatures, viable C. minitans was retrieved for periods shorter than 120, 14, 3 and 3 days, respectively.
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Fig. 2. Dynamics of log10 values of viable Coniothyrium minitans in the water-logged field in Wuhan, Hubei, China, in the summer-autumn test (2005 and 2006) and in the winter–spring test (2005–2006 and 2006–2007). Vertical bars in each graph represent standard errors of means (n = 3).
At 45 and 50 °C, survival of C. minitans was very poor for all the three-moisture treatments. After incubation for 1 day, viable C. minitans was retrieved only from the low-moisture soils with the average log10 values of viable C. minitans decreasing from 4.7 to 2.7 (Fig. 3). However, viable C. minitans was not retrieved from any soil samples of the other two moisture treatments at 45 °C and from any soil samples of all the three-moisture treatments at 50 °C. After incubation for 3 to 7 days, no viable C. minitans was retrieved from any of the three-moisture soils incubated at 45 and 50 °C.
4. Discussion Previous reports showed that occurrence of C. minitans in nature is frequently in association with sclerotia of Sclerotinia species (Campbell, 1947; Tribe, 1957; Hoes and Huang, 1975; Huang, 1977, 1981; Whipps and Gerlagh, 1992). Tribe (1957) proposed that sclerotia of Sclerotinia species might play a protective role in the longterm survival of C. minitans in soils and this hypothesis was confirmed by Bennett et al. (2006) using scanning electron microscopy to study the in situ survival of conidia of C. minitans on the surface and in the medulla of sclerotia of S. sclerotiorum in soils. Our study revealed that C. minitans could survive for 750 days in soil free of sclerotia of S. sclerotiorum under field conditions. Results from the controlled experiment demonstrated that C. minitans could survive for 360 days at 4–28 °C in soil also free of sclerotia of S. sclerotiorum. These findings provide direct evidence that the presence of sclerotia of S. sclerotiorum might not be essential for the long-term survival of C. minitans in soil. Previous studies showed that C. minitans cannot conduct any saprophytic growth in natural soils (Tribe, 1957). Dormancy of conidia of C. minitans in soil free of sclerotia of S. sclerotiroum might be the mechanism responsible for the long-term survival of this fungus observed in this study. The optimum temperature range for C. minitans to conduct mycelial growth and infection of S. sclerotiorum sclerotia is 15– 25 °C and the maximum temperature is 30 °C (Campbell, 1947; Huang and Erickson, 2008; McQuilken et al., 1997b). In central China, the climate is hot in summer, whereas temperate in spring. Soil temperature can reach up to 30 °C or higher in summer, or as low
as zero or more in winter. Our finding reveals that C. minitans can survive for >750 days in a non-irrigated soil under field conditions. This result suggests that C. minitans can over-summer and overwinter in central China. It further suggests that C. minitans might have a great potential to be used as a biocontrol agent to treat soil for elimination of sclerotia of S. sclerotiorum. Our previous studies involving a pot experiment in Wuhan (Wang et al., 2006) and a small-scale field experiment in Er Zhou, an area close to Wuhan (Yang et al., 2009), showed that application of C. minitans to S. sclerotiorum-contaminated soil in the early summer after harvest of oilseed rape, or in the middle of autumn at seed sowing or seedling transplanting of oilseed rape, could reduce the number of apothecia produced from sclerotia of S. sclerotiorum. Previous studies demonstrated that efficacy of C. minitans in suppression of apothecium production by S. sclerotiorum and in suppression of sclerotinia diseases on greenhouse lettuce is greatly affected by the rate of C. minitans conidia inoculated in soil (Jones and Whipps, 2002; Gerlagh et al., 2003; Jones et al., 2003; Yang et al., 2009). The minimum conidial concentration of C. minitans for effective suppression of carpogenic germination of S. sclerotiorum was 1 103 conidia g1 soil according to our previous study (Yang et al., 2009). In the present study, the concentration of C. minitans in non-irrigated soils after two years remained higher than this level. Therefore, soil treatment of C. minitans appears a promising measure for suppression of S. sclerotiorum in central China. Field studies on a larger scale to evaluate the efficacy of soil application of C. minitans in suppression of sclerotinia diseases of oilseed rape and other crops are warranted. Previous studies reported that sclerotia of S. sclerotiorum or S. minor survived less than 7–8 weeks in flooded soils, especially in summer (Moore, 1949; Abawi et al., 1985). In the present study C. minitans survived for 150 days in submerged soil over both summer–autumn and winter–spring periods, longer than sclerotia of Sclerotinia spp., under the same conditions. Long-term survival of C. minitans in paddy soil suggests that it might be possible to integrate soil treatment with C. minitans with the oilseed rape–rice rotation system to achieve effective control of sclerotinia stem rot of oilseed rape. Effects of constant temperature and soil moisture on survival of C. minitans were evaluated in the indoor experiment of this
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Fig. 3. Effects of soil temperature and soil moisture on survival of Coniothyrium minitans. The concentration of viable C. minitans in soil (cfu g1 dry soil) was log10transformed and vertical bars in each graph represent standard errors of means (n = 3).
study. Similar to the outdoor experiments, soil used in our indoor experiment was not sterilized. Long-term survival of C. minitans in non-irrigated soil under field conditions and in indoor soils incubated at 4–28 °C suggests that a close correlation in survival of C. minitans might exist between the outdoor and indoor experiments. Results about better survival of C. minitans at 4–28 °C (irrespective of soil moisture) than at 30 °C or higher (moisturedependent) in the indoor experiment, provide experimental evidence that soil temperature and moisture can affect survival of C. minitans in soil. As observed in this study, soil temperatures (5 cm depth) fluctuate yearly within the range of 1 to 37 °C in Wuhan, a representative area in central China. Although soil temperatures exceeding 30 °C can frequently occur in this area, the durations are usually brief. Thus, soil temperature in central China might not be highly adverse to survival of C. minitans, especially in non-irrigated soils.
Acknowledgments This study was funded by the Grants of the Natural Science Foundation of China (Grant Nos. 30471165 and 30971953), the Special Fund for Public Welfare Projects (Agriculture) of China (Grant No. nyhyzx07-054) and the ‘‘863’’ High-Tech Program of China (Grant No. 2006AA10A211). We thank Miss Qing-Lin Fu, College of Resources and Environment, Huazhong Agricultural University, for testing properties of the soil used in this study.
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