| Coniothyrium minitans is a promising biocontrol agent(BCA) of Sclerotinia sclerotiorum,the causal agent of sclerotinia stem rot(SSR) of oilseed rape.C.minitans can be used in two approaches for control SSR of oilseed rape:spray application on floral parts for suppression of infection by ascospores of S.sclerotiorum on flower petals and soil application for destruction of sclerotia of S.sclerotiorum.In order to evaluate the potential of C.minitans as a BCA for SSR of oilseed rape,to improve the efficacy of C. minitans and to establish convenient methods for application of C.minitans,we investigated survival of C.minitans on flower petals of oilseed rape and in natural soil under field condition in central China and factors affecting survival of C.minitans. Meanwhile,water-assisted dissemination of C.minitans conidia in soil was studied.On the basis of resistance against the fungicide vinclozolin and to the two bactericides benzylpencillin sodium and streptomycin sulfate,a selective medium containing vinclozolin(500μg a.i./ml),benzylpencillin sodium(500μg a.i./ml) and streptomycin sulfate(500μg a.i./ml) was formulated.C.minitans strain SV-5-2 could conduct normal conidial germination and mycelial growth on this medium.Therefore,the medium could be used for monitoring C.minitans released to environment.Meanwhile,a PCR-based assay was developed to specifically detect C.minitans.This molecular detection assay was successfully used in studying the interaction between C.minitans and S.sclerotiorum,parasitism of sclerotia S.sclerotiorum by C.minitans and in detection of C.minitans in sterile soil.Using this medium,survival of C.minitans on flower petals of oilseed rape was tracked.Results of the five field experiments(A-E) conducted in 2004 and 2005 showed that C.minitans strain SV-5-2 applied to flower racemes of oilseed rape could be detected for five days in four experiments and three days in one experiment.The rate of survival of C.minitans decreased with time and at the last sampling date,the survival rate was 17.3%, 14.3%,35.0%,62.6%and 1.9%for experiments A,B,C,D,and E,respectively. Moreover,survival of C.minitans was affected by weather conditions.Relatively,high rates of survival of C.minitans were observed under sunny weather in the experiments C and D,implying that conidia of C.minitans are tolerant to sunlight irradiation for a period of at least 5 days in the experiment C and 3 days in the experiment D.Low survival rates of C.minitans were observed under light rain weather in the experiment A,and heavy rain weather in the experiments B and E.The low recovery rate of C.minitans under rainy conditions suggests a possibility of rain wash of the inoculum of C.minitans applied to the florets of oilseed rape.Field experiments in two seasons were conducted to evaluate the efficacy of applying the mycoparasite C.minitans alone or mixed with the fungicide dimethachlon to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases.Results showed that compared to the control treatment,a significant reduction of SSR by aerial application of C.minitans alone or mixed with dimethachlon was observed.The recommended conidial concentration of C.minitans aerial applied alone at the flowering stage to control sclerotinia diseases was 1×106 conidia/ml.In order to evaluate the potential of the mycoparasite C.minitans to control S. sclerotiorum in central China,a study was conducted to investigate survival of C. minitans in soil under field conditions in Wuhan,China,using the most probable number technique on potato dextrose agar(PDA).Results showed that in non-irrigated soil,C. minitans strain SV-5-2 survived for 10 and 25 months in the first and the second assay, respectively.The concentration of viable C.minitans had a decreased from 5.1×105 to 8.0×104 cfu g-1 soil in the first assay and from 7.2×105 to 3.9×104 cfu g-1 soil in the second assay.In water-submerged soil,survival of C.minitans lasted for 5 months both in summer-autumn seasons and in winter-spring seasons.The population of C.minitans declined quickly,especially in the summer-autumn seasons.In addition,effects of temperature and soil water content on survival of C.minitans were characterized by incubating C.minitans-incorporated soil of different water treatments at 4,10,20 or 28℃for 30-360 days,at 30 or 35℃for 1-210 days,at 37 or 40℃for 1-180 days,and at 45 or 50℃for 1-7 days,and periodically detecting viable C.minitans on PDA.Results showed that at 4-28℃,C.minitans survived one year in soil containing 6.3%,18.5%or 45.4%of water and no significant difference(P＞0.05) in the concentration of viable C.minitans was detected among the three water treatments at each temperature.At 30,35,37 and 40℃,survival of C.minitans depended on soil water content,lasting for 210 days at 30 and 35℃,and for 180 days at 37 and 40℃,in soil containing 6.3%of water,whereas for 120,21,14 and 3 days in soil containing 18.5%of water and for 120,14,3 and 3 days in soil containing 45.4%of water for these temperatures,respectively.At 45 and 50℃,C. minitans survived one day or shorter in soil of the three water treatments.These results suggest that C.minitans can survive for a long time in soil under climatic conditions in central China and can be used as soil treatment for suppression of S.sclerotiorum.Results of potting experiment indicated that the conidial concentration of C.minitans greatly affected the number of apothecia produced by S.sclerotiorum in three consecutive seasons.There was a tendency of decrease in the cumulative number of apothecia with the increase in the concentration of C.minitans conidia from 0(control) to 1×107 conidia/g soil.The minimum conidial concentration of C.minitans for significant(P＜0.05) suppression of apothecium production by S.sclerotiorum was 10-1000 conidia/g soil.Results of field experiments in two seasons showed that soil treated with C.minitans in the conidial concentration of 1×105 to 1×107 conidia/ml,greatly suppressed apothecia produced and controlled sclerotinia diseases caused by S.sclerotiorum.Water-assisted dispersal of conidia of C.minitans in four soils(yellow-brown soil, red-clay soil,fluvo-aquic soil and black soil) and one sand was studied.Resuks showed that conidia of C.minitans could be disseminated with water and spread in soil or sand for 16-20 cm vertically and for 5-10 cm horizontally.The conidial concentration of C. minitans was logarithmically reduced with the increase in the depth or distance.These results suggest that soil application of C.minitans in plowed fields might be integrated in irrigations.Results of potting experiments in two seasons showed that sclerotial burial depth in soil greatly affected carpogenic germination by sclerotia of S.sclerotiorum in soil treated with C.minitans or water alone(control).In treatments of C.minitans and the control,sclerotia buried in soil at 0-0.5 cm in depth produced much more apothecia than those buried at 5 cm in depth and no apothecia formation was observed for sclerotia buried at 10 cm in depth in the entire season.Suppressive effect of C.minitans on carpogenic germination of S.sclerotiorum was also observed in comparison with the control treatment.Effect of a compound fertilizer on mycelial growth,conidial germination of C. minitans and on infection of sclerotia of S.sclerotiorum by this mycoparasite was studied. Results showed that the compound fertilizer could significantly affect the mycelial growth and conidial germination of C.minitans and the degree of inhibition depends on the concentration of the fertilizer,however,without any lethal effect on C.minitans.The compound fertilizer did not affect the infection of sclerotia of S.sclerotiorum by C. minitans.This study suggests that the compound fertilizer was compatible with C. minitans.Results of potting experiment indicated that C.minitans mixed with the compound fertilizer greatly reduced the number of apothecia produced by S.sclerotiorum sclerotia in three consecutive seasons.Results of field experiments in two seasons showed that soil treated with C.minitans mixed with the compound fertilizer greatly suppressed apothecia produced and controlled sclerotinia diseases caused by S.sclerotiorum.Three potting experiments were conducted to determine the effects of soil treatment with three mycoparasites on production of apothecia of S.sclerotiorum.All the three species of fungi assessed,C.minitans,Talaromyces flavus and Trichoderma atroviride were the effective agents for reducing carpogenic germination of S.sclerotiorum sclerotia. The efficacy of C.minitans mixed with T.flavus or T.atroviride reduced formation of apothecia of S.sclerotiorum were better than all the three species of fungi treated alone. Effects were greatest when inoculum of the three mycoparasites were applied in October, at the time of sowing,rather than when they were applied in May.Based on the results of this study,four convenient techniques for application of C. minitans to SSR of oilseed rape were established,including aerial application of C. minitans alone or mixed with low-dose of the fungicide dimethachlon,water-assisted delivery of C.minitans to soil,integrated use of compound fertilizer and C.minitans and combination of C.minitans with Talaromyces flavus or Trichoderma atroviride.
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