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Abstract Bacillus subtilis HAS is a biocontrol strain with better inhibitory effect on Sporisorium scitaminea Sydow. In order to further understand its action range， with fungal pathogens in sugarcane production as targets， confrontation tests and tests of inhibitory effect of secondary metabolites on growth of pathogens were carried out. The results showed that B. subtilis HAS not only had better inhibitory effect on S. Scitaminea， but also inhibited the growth of various fungal pathogens in sugarcane production. The inhibitory effect was the best on Ceratocystis adiposum， Thielaviopsis paradoxa， Fusarium moniliforme and Colletotrichum fuleatum， which was reflected by the radius of inhibitory zone over 10 mm. The inhibitory rates of the secondary metabolites were about 70%. It indicates that B. subtilis HAS has wide antifungal property， providing reference for further application of this strain.
Key words Bacillus subtilis HAS； Confrontation training； Secondary metabolites； Inhibitory effect
Bacillus subtilis is a kind of biological bacterium of plant disease with the widest application having broad application prospect. It has the characteristics of endogenous spores and strong tolerance to heat and good resistance. B. subtilis not only widely exists in external environments including soil and plant root surface， but also serves as a kind of common endophytic bacterium in plant， especially in plant root and stem parts. Studies both at home and abroad show that B. Subtilis has good field control efficiency against various diseases as well as very remarkbaly yield increasing effect.
Sugarcane smut is widely distributed in various sugarcane-growing areas in the world[1-2]. This disease was found in KwaZulu-Natal as early as 1877， and then reported in most countries in Asia and Africa in the Eastern Hemisphere[2-7]. Sugracane smut was found in Guangzhou of China in 1932， and then appeared in various areas of China. At present， surgarcane smut occurs at every place where sugarcane is planted[3-7]. This disease is mainly controlled by selecting resistant varieties， but due to the lack of resistant resources and differentiation of physilogical races of sugarcane smut， there are many difficulties in the breeding of sugarcane varieties resistant to sugarcane smut[2].
B. subtilis HAS is a biocntrol strain with better inhibitory effect on sugarcane smut[8-9]， which has been preserved in Common Microorgainsm Center of China Committee for Culture Collection of Microorganisms， with a preservation number CGMCC No. 6590. In this study， with various funfal pathogens in sugarcane production as targets， the inhibitory effect of B. subtilis HAS on them was studied， so as to provide reference for further extenstion and application of the biocontrol strain B. subtilis HAS. Materials and methods
Materials
Tested strains included biocontrol strain HAS， and pathogenic fungi， Sporisorium scitaminea Sydow， Ceratocystis adiposum， Thielaviopsis paradoxa， Fusarium moniliforme， Colletotrichum fuleatum， Cercospora taiwanensis， Dimeriella sacchari Hansford， Cercospora longipes Butler， Phyllostita sorghina Sacc， Rhizoctonia solani Kuhn and Bipolaris sacchari Shoemaker. All the strains were isolated and preserved in laboratory.
Methods
Confrontation culture of strain HAS and fungal pathogens
Various strains were activated with PDA medium. Fugal cakes of each strain were made with a puncher with a diameter of 6 mm， and inoculated in the middle of PDA plates， and the biocontrol strain HAS was inoculated 2 cm far from each fungal cake. The plate free of inoculation of HAS was set as control. Each fungal strain was inoculated into three plates and cultured in a constant-temperature incubator at 28 ℃ under inverted state. The radius of the inhibitory zone of biocontrol strain HAS was measured.
Inhibitory effect of secondary metabolites of strain HAS on fungal pathogens
The preserved strain was transferred to LB plate， and cultured at 37 ℃ for 24 h. Then， the activated strain was inoculated into a 500 ml triangular flask containing 200 ml of LB liquid medium， and cultured at 37 ℃ under 170 r/min for 24 h. After the culture， centrifugation was performed at 10 000 r/min for 10 min to remove the bacteria， and the supernatant was divided into two parts， one of which was extracted with ethyl acetate， obtaining the organic phase which was placed in a ventilation hood to allow volatilization of ethyl acetate until the liquid was about 10 ml； and the other part was precipitated after the addition of ammonium sulfate until the saturability of ammonium sulfate reached 90%， and then centrifuged， and the precipitate was added with 10 ml of 0.01 M phosphate buffer to dissolve it for later use.
With above fungal pathogens as target， the antifungal activity of the extract was determined indoor by growth rate determination method. Under sterile condition， Fugal cakes of each tested strain were made with a puncher with a diameter of 6 mm for later use. After sterilization of medium， the medium was added into plates， and after cooling， coating was performed with 200 μl of the prepared solution. The fungal cakes were placed on prepared medium with the mycelium downward according to one fungal cake per plate. The fungi were cultured at 28 ℃ for 5 d. The diameter of each colony was measured by crossing method for two times. Each test had three replicates， and the inhibitory rate was calculated using the average value of three replicates： Inhibitory rate=[（Colonial diameter of control-Diameter of fungal cake）-（Colonial diameter of treatment-Diameter of fungal cake）]/（Colonial diameter of control-Diameter of fungal cake）×100%. Results and Analysis
Inhibitory effect of HAS on fungal pathogens
It could be seen from the inhibitory effect on the 11 fungal pathogens including S. scitaminea （Table 1） that strain HAS had wide inhibitory effect. B. subtilis HAS had the best inhibitory effect on S. scitaminea and T. paradoxa causing root rot of sugarcane， F. Moniliforme causing top rot of sugarcane and C. Fuleatum causing leaf rot of sugarcane， second best inbitory effect on C. adiposum， C. taiwanensis， C. Longipes and R. solani， and certain inhibitory effect on D. sacchari， P. Sorghina and B. Sacchari， indicating that HAS has broad application prosepect.
Inhibitory effect of secondary metabolites of strain HAS on 11 fungal pathogens
The secondary metabolites of strain HAS extracted with organic solvent had no inhibitory effect against fungal pathogens， while the precipitate obtained with ammonium sulfate had certain inhibitory effect against various fungal pathogens （Table 2）. It indicates that proteins in the secondary metabolites of strain HAS might be the main components having antifungal activity. Also， the secondary metabolites of strain HAS differed in the inhibitory effect on various fungal pathogens. The inhibitory rates against C. adiposum， T. paradoxa， F. moniliforme and C. fuleatum were 70.80%， 70.09%， 69.46% and 70.58%， respectively. The inhibitory rates on S. scitaminea， C. taiwanensis， C. longipes and R. solani were the second highest， and those on D. sacchari， P. sorghina and B. sacchari were only around 50%， 50.57%， 53.11% and 49.19%， respectively.
Discussion
In this study， the inhibitory effect of strain HAS was detected with fungal pathogens of sugarcane in production as targets. The results showed that HAS had very good inhibitory effect on most fungal pathogens， especially T. paradoxa， F. moniliforme， C. fuleatum and C. adiposum. However， whether the action mechanisms of strain HAS against these pathogens are the same or close to that against S. scitaminea still need further demonstration.
The antimicrobial substances of microorganisms are metabolites， which often have specificity and strong disease prevention effect and are attracting more and more attention[10]. In this study， the results showed that the antimicrobial substances in the secondary metabolites of biocontrol strain B. subtilis HAS had remarkable inhibitory effect on growth of various fungal pathogens of sugarcane， and especially， the inhibitory rates against C. adiposum， T. paradoxa， F. moniliforme and C. fuleatum reached 70%. The inhibitory rates against S. scitaminea， C. taiwanensis， C. longipes and R. solani were about 60%， while those against D. sacchari， P. sorghina and B. sacchari were only about 50%. The inhibitory effect is remarkably divided into three different ranges， and whether the antimicrobial substances in the secondary metabolites of biocontrol strain B. subtilis HAS are several kinds or several types of different components still needs further study. References
[1] SCHENCK S， MPEAR H， LIU Z， et al. Genetic variation of Ustilago scitaminea pathotypes in Hawaii evaluated by host range and AFLP marker[J]. Sugar Cane International， 2005， 23： 15-19.
[2] BYTHER RS， STEINER GW， AWISMER C. New sugarcane diseases reported in Hawaii[J]. Sugarcane Pathologists Newsletter， 1971， 7： 18-21.
[3] RILEY LT， JUBB TF， CROFT B. First outbreak of sugarcane smut in Australia[J]. Proc. XXIII ISSCT Conger， New Delhi， India， 1999：333-336.
[4] QUE YX， XU LP， LIN JW， et al. Molecular variation of Sporisorium scitamineum in Mainland China revealed by RAPD and SRAP markers[J]. Plant Disease， 2012， 96（10）： 1519-1525.
[5] HOY JW. Incidence of sugarcane smut in Louisiana and its effects on yield[J]. Plant Dis.， 1986， 70（1）：59-60.
[6] XU LP， CHEN RK. Current status and prospects of smut and smut resistance breeding in sug arcane[J]. Fujian Journal of Agricultural Sciences， 2000，15（2）： 26-31. （in Chinese）
[7] XIONG GR， ZHANG SZ. Study on sugarcane smut[J]. Journal of Agricultural Catastrophology， 2012， 2（4）： 8-10. （in Chinese）
[8] XIONG GR， ZHAO GF， WU SR， et al. Screening and identification of a biocontrol strain， HAS antagonistic to Sporisorium scitaminea Sydow[J]. Chinese Journal of Tropical Crops， 2013， 34（6）： 1-7. （in Chinese）
[9] XIONG GR， WU SR， ZHANG SZ， et al. Bacillus subtilis HAS and its application in control of Sporisorium scitaminea Sydow： China， ZL201210452707.7[P]. 2013-01-30. （in Chinese）
[10] LI L， QIAO YS， GU BK. Advances in the study of plant endophytic bacteria and their effects on biological control of plant diseases[J]. Jiangsu Journal of Agricultural Sciences， 2008， 24（6）： 969- 974. （in Chinese）
Key words Bacillus subtilis HAS； Confrontation training； Secondary metabolites； Inhibitory effect
Bacillus subtilis is a kind of biological bacterium of plant disease with the widest application having broad application prospect. It has the characteristics of endogenous spores and strong tolerance to heat and good resistance. B. subtilis not only widely exists in external environments including soil and plant root surface， but also serves as a kind of common endophytic bacterium in plant， especially in plant root and stem parts. Studies both at home and abroad show that B. Subtilis has good field control efficiency against various diseases as well as very remarkbaly yield increasing effect.
Sugarcane smut is widely distributed in various sugarcane-growing areas in the world[1-2]. This disease was found in KwaZulu-Natal as early as 1877， and then reported in most countries in Asia and Africa in the Eastern Hemisphere[2-7]. Sugracane smut was found in Guangzhou of China in 1932， and then appeared in various areas of China. At present， surgarcane smut occurs at every place where sugarcane is planted[3-7]. This disease is mainly controlled by selecting resistant varieties， but due to the lack of resistant resources and differentiation of physilogical races of sugarcane smut， there are many difficulties in the breeding of sugarcane varieties resistant to sugarcane smut[2].
B. subtilis HAS is a biocntrol strain with better inhibitory effect on sugarcane smut[8-9]， which has been preserved in Common Microorgainsm Center of China Committee for Culture Collection of Microorganisms， with a preservation number CGMCC No. 6590. In this study， with various funfal pathogens in sugarcane production as targets， the inhibitory effect of B. subtilis HAS on them was studied， so as to provide reference for further extenstion and application of the biocontrol strain B. subtilis HAS. Materials and methods
Materials
Tested strains included biocontrol strain HAS， and pathogenic fungi， Sporisorium scitaminea Sydow， Ceratocystis adiposum， Thielaviopsis paradoxa， Fusarium moniliforme， Colletotrichum fuleatum， Cercospora taiwanensis， Dimeriella sacchari Hansford， Cercospora longipes Butler， Phyllostita sorghina Sacc， Rhizoctonia solani Kuhn and Bipolaris sacchari Shoemaker. All the strains were isolated and preserved in laboratory.
Methods
Confrontation culture of strain HAS and fungal pathogens
Various strains were activated with PDA medium. Fugal cakes of each strain were made with a puncher with a diameter of 6 mm， and inoculated in the middle of PDA plates， and the biocontrol strain HAS was inoculated 2 cm far from each fungal cake. The plate free of inoculation of HAS was set as control. Each fungal strain was inoculated into three plates and cultured in a constant-temperature incubator at 28 ℃ under inverted state. The radius of the inhibitory zone of biocontrol strain HAS was measured.
Inhibitory effect of secondary metabolites of strain HAS on fungal pathogens
The preserved strain was transferred to LB plate， and cultured at 37 ℃ for 24 h. Then， the activated strain was inoculated into a 500 ml triangular flask containing 200 ml of LB liquid medium， and cultured at 37 ℃ under 170 r/min for 24 h. After the culture， centrifugation was performed at 10 000 r/min for 10 min to remove the bacteria， and the supernatant was divided into two parts， one of which was extracted with ethyl acetate， obtaining the organic phase which was placed in a ventilation hood to allow volatilization of ethyl acetate until the liquid was about 10 ml； and the other part was precipitated after the addition of ammonium sulfate until the saturability of ammonium sulfate reached 90%， and then centrifuged， and the precipitate was added with 10 ml of 0.01 M phosphate buffer to dissolve it for later use.
With above fungal pathogens as target， the antifungal activity of the extract was determined indoor by growth rate determination method. Under sterile condition， Fugal cakes of each tested strain were made with a puncher with a diameter of 6 mm for later use. After sterilization of medium， the medium was added into plates， and after cooling， coating was performed with 200 μl of the prepared solution. The fungal cakes were placed on prepared medium with the mycelium downward according to one fungal cake per plate. The fungi were cultured at 28 ℃ for 5 d. The diameter of each colony was measured by crossing method for two times. Each test had three replicates， and the inhibitory rate was calculated using the average value of three replicates： Inhibitory rate=[（Colonial diameter of control-Diameter of fungal cake）-（Colonial diameter of treatment-Diameter of fungal cake）]/（Colonial diameter of control-Diameter of fungal cake）×100%. Results and Analysis
Inhibitory effect of HAS on fungal pathogens
It could be seen from the inhibitory effect on the 11 fungal pathogens including S. scitaminea （Table 1） that strain HAS had wide inhibitory effect. B. subtilis HAS had the best inhibitory effect on S. scitaminea and T. paradoxa causing root rot of sugarcane， F. Moniliforme causing top rot of sugarcane and C. Fuleatum causing leaf rot of sugarcane， second best inbitory effect on C. adiposum， C. taiwanensis， C. Longipes and R. solani， and certain inhibitory effect on D. sacchari， P. Sorghina and B. Sacchari， indicating that HAS has broad application prosepect.
Inhibitory effect of secondary metabolites of strain HAS on 11 fungal pathogens
The secondary metabolites of strain HAS extracted with organic solvent had no inhibitory effect against fungal pathogens， while the precipitate obtained with ammonium sulfate had certain inhibitory effect against various fungal pathogens （Table 2）. It indicates that proteins in the secondary metabolites of strain HAS might be the main components having antifungal activity. Also， the secondary metabolites of strain HAS differed in the inhibitory effect on various fungal pathogens. The inhibitory rates against C. adiposum， T. paradoxa， F. moniliforme and C. fuleatum were 70.80%， 70.09%， 69.46% and 70.58%， respectively. The inhibitory rates on S. scitaminea， C. taiwanensis， C. longipes and R. solani were the second highest， and those on D. sacchari， P. sorghina and B. sacchari were only around 50%， 50.57%， 53.11% and 49.19%， respectively.
Discussion
In this study， the inhibitory effect of strain HAS was detected with fungal pathogens of sugarcane in production as targets. The results showed that HAS had very good inhibitory effect on most fungal pathogens， especially T. paradoxa， F. moniliforme， C. fuleatum and C. adiposum. However， whether the action mechanisms of strain HAS against these pathogens are the same or close to that against S. scitaminea still need further demonstration.
The antimicrobial substances of microorganisms are metabolites， which often have specificity and strong disease prevention effect and are attracting more and more attention[10]. In this study， the results showed that the antimicrobial substances in the secondary metabolites of biocontrol strain B. subtilis HAS had remarkable inhibitory effect on growth of various fungal pathogens of sugarcane， and especially， the inhibitory rates against C. adiposum， T. paradoxa， F. moniliforme and C. fuleatum reached 70%. The inhibitory rates against S. scitaminea， C. taiwanensis， C. longipes and R. solani were about 60%， while those against D. sacchari， P. sorghina and B. sacchari were only about 50%. The inhibitory effect is remarkably divided into three different ranges， and whether the antimicrobial substances in the secondary metabolites of biocontrol strain B. subtilis HAS are several kinds or several types of different components still needs further study. References
[1] SCHENCK S， MPEAR H， LIU Z， et al. Genetic variation of Ustilago scitaminea pathotypes in Hawaii evaluated by host range and AFLP marker[J]. Sugar Cane International， 2005， 23： 15-19.
[2] BYTHER RS， STEINER GW， AWISMER C. New sugarcane diseases reported in Hawaii[J]. Sugarcane Pathologists Newsletter， 1971， 7： 18-21.
[3] RILEY LT， JUBB TF， CROFT B. First outbreak of sugarcane smut in Australia[J]. Proc. XXIII ISSCT Conger， New Delhi， India， 1999：333-336.
[4] QUE YX， XU LP， LIN JW， et al. Molecular variation of Sporisorium scitamineum in Mainland China revealed by RAPD and SRAP markers[J]. Plant Disease， 2012， 96（10）： 1519-1525.
[5] HOY JW. Incidence of sugarcane smut in Louisiana and its effects on yield[J]. Plant Dis.， 1986， 70（1）：59-60.
[6] XU LP， CHEN RK. Current status and prospects of smut and smut resistance breeding in sug arcane[J]. Fujian Journal of Agricultural Sciences， 2000，15（2）： 26-31. （in Chinese）
[7] XIONG GR， ZHANG SZ. Study on sugarcane smut[J]. Journal of Agricultural Catastrophology， 2012， 2（4）： 8-10. （in Chinese）
[8] XIONG GR， ZHAO GF， WU SR， et al. Screening and identification of a biocontrol strain， HAS antagonistic to Sporisorium scitaminea Sydow[J]. Chinese Journal of Tropical Crops， 2013， 34（6）： 1-7. （in Chinese）
[9] XIONG GR， WU SR， ZHANG SZ， et al. Bacillus subtilis HAS and its application in control of Sporisorium scitaminea Sydow： China， ZL201210452707.7[P]. 2013-01-30. （in Chinese）
[10] LI L， QIAO YS， GU BK. Advances in the study of plant endophytic bacteria and their effects on biological control of plant diseases[J]. Jiangsu Journal of Agricultural Sciences， 2008， 24（6）： 969- 974. （in Chinese）