Survival of the biocontrol agents Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 on the spikes of barley in the field

Survival of the biocontrol agents Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 on the spikes of barley in the field 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:bxzhang@zju.edu.cn †E-mail:bxzhang@zju.edu.cn bxzhang@zju.edu.cn 2 2 3 4 5 Department of Plant Protection, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Department of Plant Protection, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Academy of Agriculture Science, Hangzhou 310021, China Academy of Agriculture Science, Hangzhou 310021, China Department of Biological Sciences, University of Wisconsin-Milwaukee, WI 53211, USA Department of Biological Sciences, University of Wisconsin-Milwaukee, WI 53211, USA Department of Horticulture, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China Department of Horticulture, School of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China School of Life Science, Zhejiang University, Hangzhou 310029, China School of Life Science, Zhejiang University, Hangzhou 310029, China Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease that results in extensive yield losses to wheat and barley. A green fluorescent protein (GFP) expressing plasmid pRP22-GFP was constructed for monitoring the colonization of two biocontrol agents, Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116, on the spikes of barley and their effect on suppression of FHB. Survival and colonization of the Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 strains on spikes of barley were observed by tracking the bacterial transformants with GFP expression. Our field study revealed that plasmid pRP22-GFP was stably maintained in the bacterial strains without selective pressure. The retrieved GFP-tagged strains showed that the bacterial population fluctuation accorded with that of the rain events. Furthermore, both biocontrol strains gave significant protection against FHB on spikes of barley in fields. The greater suppression of barley FHB disease was resulted from the treatment of barley spikes with biocontrol agents before inoculation with F. graminearum . GFP Survival Brevibacillus brevis and Bacillus subtilis Spikes Barley Biocontrol INTRODUCTION 1 Soil-borne pathogens, including Pythium spp. and Fusarium spp., cause significant yield losses in horticulture and agriculture crops (Mao et al., 1997 1997 ). Current practices for controlling plant diseases are based largely on disease resistant crops, cultivation management in fields and application of synthetic pesticides (Elizabeth and Emmert, 1999 1999 ). Biological control using antagonistic microbes to reduce the use of chemical pesticides in a system of integrated plant disease management, offers a powerful alternative to control plant diseases. Biocontrol of soil-borne pathogens depends on the successful establishment and survival of microbial inocula. While biocontrol agents are often effective in the laboratory, the level of control achieved in the field is sometimes unpredictable. Some of these failures can be attributed to inadequate establishment and survival of microbial inocula (O’Callaghan et al., 2001 2001 ). To assess survival of biocontrol agents in fields, methods to detect the released strains and distinguish them from indigenous bacteria are needed. Classical studies of population dynamics of beneficial bacteria introduced into the environment have depended upon detection of culturable cells (Araujo et al., 1994 1994 ; Boelens et al., 1994 1994 ). To distinguish the introduced cells from the indigenous population, the former has generally been tagged with a traceable marker (Kragelund et al., 1995 1995 ). Antibiotic resistance markers have so far almost always been relied on (Chao et al., 1986 1986 ; Wilson, 1995 1995 ; Nautiyal et al., 2002 2002 ), but the value of this approach is limited by the fact that selective plating provides little information on the localization and distribution of the marked strains in the environmental samples (Scott et al., 1998 1998 ). The green fluorescent protein (GFP) reporter has recently been found to be a potentially valuable tool for tracking the survival (Ramos et al., 2002 2002 ) and localization of microbial cells (Xi et al., 1999 1999 ) and for monitoring gene expression in micro-niches (Lewis and Marston, 1999 1999 ). The gfp gene can be identified by noninvasive methods. Neither substrates, other enzymes nor co-factors are required for detection of GFP (Douka et al., 2001 2001 ). In addition, mature GFP is extremely pH (pH 6~10) and temperature tolerant (up to 60 °C). Fusarum head blight (FHB), primarily caused by F. graminearum , is a devastating disease that results in extensive yield and quality losses to wheat and barley throughout the world (Urrea et al., 2002 2002 ; Capettini et al., 2003 2003 ). After infection, Fusarium mycotoxins are accumulated and the products of these crops are harmful to both people and animal stock (Ingalls, 1996 1996 ; Onji et al., 1998 1998 ; Placinta et al., 1999 1999 ). Among the pathogens, F. graminearum is the most common Fusarium species producing deoxynivalenol (DON) (Olsson et al., 2002 2002 ). Two biocontrol agents (BCAs) which were isolated in our laboratory inhibited F. graminearum growth significantly in laboratory assays. The two strains were identified as Bre vibacillus brevis and Bacillus subtilis respectively by the methods of Klement et al.( 1990 1990 ), combined with 16 srDNA sequence analysis (GenBank accession number is AY897210 and AY897211, respectively). In order to track the biocontrol strains on the spikes of barley, a plasmid pRP22-GFP based on the pGFPuv replicon constitutively expressing the gfp gene was constructed. This paper described the construction of a broad-host-range plasmid expressing the gfp gene. Its use in survival and colonization studies of two biocontrol agents, B. brevis ZJY-1 and B. subtilis ZJY-116 on spikes of barley was demonstrated. In addition, the effect of disease suppression of two FHB biocontrol agents of barley in fields was also investigated. MATERIALS AND METHODS 1 Plasmid construction 2 The vector pRP22, an E. coli-B. subtilis shuttle plasmid was kindly provided by Professor Chen, Jiangsu Academy of Agriculture Science. The plasmid (pRP22-GFP) carrying the gfp gene was constructed by the following strategy. The xylR gene and the xylA promoter were amplified as a single product from B. subtilis 168 chromosomal DNA using the primers Pr1 and Pr2. Gene GFPuv was amplified from the plasmid pGFPuv using the primers Pr3 and Pr4. The above PCR products were digested with Hin d III and ligated together. The resulting ligation product was reamplified with primers Pr1 and Pr4 and inserted into the Sph I -Kpn I site of plasmid pRP22 (Table 1 1 ). Underlining indicates Sph I (GCATGC), Kpn I (GGTACC), Hin d III (AAGCTT) restriction sites Strains Relevant characteristics Antibiotic resistance Reference/source B. subtilis 168 Contains xy1R gene and xy1A promoter This study E. coli TG1 This study Vectors pGFPuv gfp gene Amp r Clontech pRP22 Shuttle vector Cm r Amp r Zhi-Yi Cheng Oligo-nucleotide 2 Sequence Restriction site(s) Pr1 2 5′-CACAT GCATGC CATGTCACTGTTGCTTCAG-3′ Sph I Pr2 2 5′-C GGTACC CAT AAGCTT GTGATTTCCCCCTTA-3′ Kpn I/ Hin d III Pr3 2 5′-GCGC AAGCTT ATGAGTAAAGGAGAAGAA-3′ Hin d III Pr4 2 5′-GG GGTACC ATTATTTTTGACACCAGA-3′ Kpn I Plasmid transformation 2 The plasmid pRP22-GFP was transformed into the two biocontrol agents, B. brevis ZJY-1 and B. subtilis ZJY-116, by the methods described in Chang and Cohen ( 1979 1979 ) with slight modifications. Media and solutions compositions were as follows: SMM buffer consisting of 0.5 mol/L sucrose, 0.02 mol/L maleate and 0.02 mol/L MgCl 2, pH 6.5 adjusted with NaOH. SMMP medium was prepared by mixing equal volumes of fourfold strength Penassay broth (Difco) and double strength SMM. PEG solution (40%, w / V ) containing 40 g PEG (Sigma, approx. MW 6000) and 50 ml double strength SMM buffer in 100 ml. Luria-Bertain (LB) broth containing Tryptone 10 g, yeast extract 5 g, NaCl 10 g in 1 L, adjusted pH 7.5 with NaOH. The DM3 regeneration medium consisted of the following sterile solutions (per liter: 200 ml 4% agar, 500 ml 1 mol/L sodium succinate (pH 7.3), 100 ml 5% Difco Casamino acids, 50 ml 10% Difco yeast extract, 100 ml 3.5% K 2 HPO 4 and 1.5% KH 2 PO 4 , 25 ml 20% glucose, 20 ml 1 mol/L MgCl 2 , and 5 ml filter-sterilized 2% bovine serum albumin (added to the mixture when the temperature was ~55 °C). Mid-log phase cell cultures, freshly grown in LB at 37 °C to A 600 nm 0.6, were harvested by centrifugation (2000 r/min) for 10 min with a 10 ml Eppendorf tube and re-suspended in a 500 µl volume of SMMP solution. Four µl lysozyme (50 mg/ml) was added into the suspension and the suspension was incubated at 37 °C with gentle shaking (100 r/min) for about 2 h. Cells were then harvested by centrifugating the suspension at 2000 r/min for 10 min, washed once by resuspending them gently in SMMP and pelleted a second time. The washed protoplast was brought to 500 µl volume by adding SMMP. After mixing completely, 1×10 −6 ~5 µg plasmid DNA in 50 µl TE buffer (10 mmol/L Tris-HCl, 1 mmol/l EDTA, pH 8.0) was mixed immediately with an equal volume of double strength SMM, followed by 1.5 ml 40% ( w / V ) PEG. After 2 min, 5 ml SMMP medium was added to the mixture to dilute the PEG, and protoplasts were recovered by centrifugation for 10 min at 2000 r/min. The treated protoplasts were re-suspended in 1 ml SMMP and incubated for 2 h at 30 °C on shaker (100 r/min) to enable phenotypic expression of genetic determinants carried by the plasmid before plating them onto chloramphenicol (C m ) amended (3×10 −6 g/L) DM3 media for direct selection of transformants. The final fluorescent colonies were obtained by incubation of protoplasts on the DM3 regeneration plates at 37 °C for 2 d. Plasmid stability 2 To determine the stability of the pRP22-GFP, transconjugants of two BCA were grown in liquid LB medium for several generations. Plasmid loss under these conditions was determined by plating onto solid LB medium with and without selective pressure (C m ) and assaying for the GFP phenotypes. The number of colony forming units (CFU) was determined by the Miles and Misra drop count method on solid agar medium (Somasegaran and Hoben, 1994 1994 ). The number of generations was determined by using the equation (Miller, 1972 1972 ): n =[ln( N t )−ln( N 0 )]/(ln2) where n is the number of generations, N t is CFU of the culture at final time (before each successive dilution) and N 0 is the CFU at zero time of the culture (after each successive dilution). Pathogen culture and inoculation 2 The pathogen F. graminearum was isolated from diseased spikes of barley on the farm of Zhejiang University, Hangzhou, China. For experiment use, the pathogen was grown on potato dextrose agar (PDA) at 28 °C for 5 d and the resulting mycelial plugs were inoculated into 500 ml flasks containing 250 ml of sterilized Charles culture medium with the following compositions (per liter: 2 g KNO 3 , 0.5 g KCl, 1 g K 2 HPO 4 , 0.5 g MgSO 4 , 0.1 g FeSO 4 , 30 g Sucrose). F. graminearum spores were formed by incubation in flasks at 25 °C on a rotary shaker (220 r/min) for 7 d. The spore filtrate was obtained by filtration of the culture medium with double layer sterile gauzes. The spores were counted using hemacytometer under a light microscope and then diluted them to an ultimate sporule of 1×10 6 CFU/ml. The resulting suspension was ready for spraying on spikes in the field during mid-anthesis (50% of the flowering barley). Population dynamics of the biocontrol agents in the barley spikes 2 The two transformed strains, of B. brevis ZJY-1 and B. subtilis ZJY-116, were streaked onto LB plate containing 3×10 −6 g/L C m (LB+C m ). After one day incubation at 37 °C, a single colony was used to inoculate sterile LB (250 ml) into a 500 ml flask which was then incubated overnight on a shaker (200 r/min) at 30 °C. Samples of cultures were centrifuged at 3000 r/min for 15 min at 4 °C, the cells were then resuspended in sterile distilled water (SDW). Bacterial suspensions of B. brevis ZJY-1 and B. subtilis ZJY-116 were adjusted to 6.8×10 7 CFU/ml and 7.4×10 7 CFU/ml respectively before inoculation onto barley spikes. The experiments were conducted at the farm of Zhejiang University in Hangzhou, China. In order to study whether the existence of the pathogen affects the population dynamics of B. brevis ZJY-1 and B. subtilis ZJY-116 in barley spikes, during the mid-anthesis of barley, the suspension of the two GFP-tagged biocontrol agents was sprayed onto spikes that has been inoculated with F. graminearum or SDW the day before application of these biocontrol agents. Two groups of treatments (barley spikes inoculated with either F. graminearum or SDW), three replicates with a total of 6 plots (1 m 2 per plot) for each bacteria. Sampling and statistical analysis 2 After inoculation, sampling was carried out at intervals of 1, 4, 7, 10 d… until the barley could be harvested. Ten grains were harvested each time at random from the spikes of each replicate, a total of 30 grains were removed from each treatment aseptically and placed in SDW (30 ml) in a 150 ml flask, shaken by hand for 5 min and left to stand for 10 min. An SDW dilution series was set up (0.1 ml to 0.9 ml), and 0.1 ml serial dilutions were plated onto LB+C m plates incubated at 37 °C for about 24 h. Then the colony forming units (CFU) with obvious fluorescence were counted under ultraviolet (UV) light. To understand the final distribution of GFP-tagged strains on spikes when the spikes matured, the husks, awns and kernels from 30 harvested grains of each treatment were aseptically detached with sterile nipper and placed in SDW (30 ml) respectively. The introduced strains from every part were obtained and counted using the previously described methods. Biocontrol effects of two biocontrol agents 2 The experiment was conducted in 2 m×1 m plots at the same farm. Survival test was conducted in November 2003. The barley seeds (variety: MOREX ) were planted in the field with conventional patten. During the mid-anthesis (April, 2004), 5 L spore concentration of F. graminearum was sprayed onto the spikes until runoff. To study the optimal time to apply B. brevis ZJY-1 and B. subtilis ZJY-116 in fields, 1.5 L biocontrol agents of the same concentration as that used in the survival test were inoculated the day before onto the pathogen-sprayed spikes, and after being inoculated respectively. There were three controls designed for this study: (1) healthy control–the spikes treated with SDW only; (2) infected control–the spikes sprayed with pathogen F. graminearum ; and (3) chemical control–the spikes previously sprayed with F. graminearum and treated with 0.75 L carbendazim (1 mg/ml) a day later. The experiments were randomly designed with two treatment groups (the biocontrol agents inoculated before and after the spikes were sprayed with F. graminearum ), three replicates of each group, three replicates of each control, resulting in a total of 21 plots. Sampling and statistical analysis 2 When the spikes ripened, 20 spikes from each plot were randomly collected. Infection percentage of the spikes infected by fungi was calculated as follows: Infection (%)=(number of grains infected by a fungus/total number of grains)×100%. The mean infection percentage and disease suppression were respectively calculated. Statistical analyses were conducted using the general linear models procedures of the Statistical Analysis System version 6.08 (SAS Institute, Inc., Cary, NC). Experiments results were analyzed using standard analysis of variance (ANOVA) and results were separated by the LSD test at P =0.05. RESULTS 1 Plasmid construction and strains transformation 2 The vector pRP22-GFP constructed in this work was an E. coli-B. subtilis shuttle plasmid carrying gfp , chloramphenicol and ampicillin resistance genes. The transformants of B. brevis ZJY-1 and B. subtilis ZJY-116, designated as ZJY-1G and ZJY-116G, were successfully obtained using bacterial protoplasts as transformants. Sixty percent of the transformants of each strains regenerated cell walls. The GFP fluorescence from all transformants was easily observed under UV radiation by fluorescence microscopy (Fig. 1a 1a and Fig. 1b 1b ). The high levels of expression of fluorescence in the present constructs make them suitable for ecological studies even under normal field environments. Stability of pRP22-GFP plasmid in BCAs 2 Studies showed that after 50 generations in the absence of antibiotic pressure, 55.3% strain ZJY-1G cells and 47.6% strain ZJY-116G cells were C m resistant and showed GFP fluorescence. Survival of GFP-tagged strains on spikes 2 The behaviour of the marked strain on both infected and healthy spikes was broadly similar. Although the number of ZJY-1G cells on healthy spikes showed a small increase a day after inoculation, the number steadily decreased in the first 13 d in both infected and healthy spikes, from 6.8×10 7 CFU/ml initially to 4×10 5 CFU/grain on infected spikes and 1×10 5 CFU/grain on healthy spikes, then the population began to recover gradually and the dynamics of introduced strains was closely related to the rain events in the field. Fig. 2 2 showed that each period of rain inevitably reduced the number of marked agents and that heavier rain led to fewer surviving strains. For example, a heaviest rainfall (25 mm) corresponded with a lowest population on both infected and healthy spikes 25 d after inoculation. Compared with ZJY-1G, smaller population fluctuation was observed in ZJY-116G on both infected and healthy spikes. In addition, a higher number of ZJY-116G cells was observed on healthy spikes than on Fusarium infected spikes throughout the experiments (Fig. 3 3 ). Applied at suspension of 7.4×10 7 CFU/ml, strain ZJY-116G decreased steadily to 1×10 5 CFU/grain on Fusarium infected spikes and 2.9×10 5 CFU/grain on healthy spikes 13 d later, followed by a gradual increase, then the trend resembled the survival seen in the strain ZJY-1G. Comparison of the distribution of these two GFP-tagged strains on the barley spikes showed that the inoculated strains were mostly distributed on the husks with the least amount distributed on the kernels (Table 2 2 ). G: The number of GFP-tagged bacterial strains (CFU/grain) T: The number of total bacteria (CFU/grain) 3 Strains 6 Part of spike 2 Kernel 2 Crust 2 Awn G a a T b b G T G T ZJY-1 1.01×10 4 1.00×10 8 2.16×10 5 6.72×10 7 5.60×10 4 3.80×10 7 ZJY-116 1.21×10 4 7.98×10 7 4.32×10 5 1.33×10 8 5.40×10 4 3.80×10 7 The biocontrol effects of two biocontrol agents 2 Typical symptom of FHB developed about 10 d after inoculation with the suspension of F. graminearum . Some spikes took on a light brown colour in initial stages and brown grains resulted when the spikes matured, however, no visual infection was observed on the untreated control, the spikes treated with SDW only. All biocontrol agents treatments showed effective suppression of FHB as compared with the no-bacteria control treatment (Table 3 3 ). Greater disease suppression resulted from treatments when the biocontrol agents were sprayed before F. graminearum was inoculated. Biocontrol agents were sprayed a day before the spikes were inoculated with F. graminearum Biocontrol agents were sprayed a day after the spikes were inoculated by F. graminearum Treatments with different letters are significantly different according to the protected least significant difference (LSD) test at P =0.05 using the models procedure in SAS. Healthy control: The spikes were treated with sterilized water only; Infected control: The spikes were sprayed with pathogen F. graminearum ; Chemical control: The spikes were previously sprayed with F. graminearum and treated with carbendazim a day later Treatment Mean percentage of disease (%) Mean percentage of disease suppression (%) Pre Pre ZJY-1 16.01 58.60 A Post Post ZJY-1 17.50 54.72 B Pre Pre ZJY-116 18.46 52.23 B post post ZJY-116 21.20 45.15 C Infected control 38.65 0.00 D Chemical control 18.00 53.40 B Healthy control 0.00 / DISCUSSION 1 A constitutively expressing gfp plasmid was constructed to monitor the colonization of two BCAs on barley spikes. The constructed plasmid was successfully transformed into strains B. brevi s ZJY-1 and B. subtilis ZJY-116. GFP fluorescence from bacterial transformants was easily observed under UV radiation by fluorescence microscopy. Although the pRP22-GFP replicon was not very stably maintained in the two BCAs, and only 55.3% of strain ZJY-1G cells and 47.6% of strain ZJY-116G cells were C m resistant and showed GFP fluorescence under laboratory condition, significant stability of the plasmid was achieved in the field. The abundance of GFP-tagged bacteria colonizing the spikes after 37 d of inoculation confirmed this. The reason for this may be that the nutrition in medium is more abundant than that in spikes for strains to grow, so the generation time in the medium is shorter than that in spikes in the field and the pRP22-GFP plasmid can be maintained a long time in the BCAs. Tracking the bacterial strains introduced on the barley spikes revealed that the two strains showed good survival on both infected and healthy spikes in the field. When the biocontrol agents were inoculated during mid-anthesis, these bacterial strains still maintained a numerical level (×10 5 CFU/grain of barley) that could be detected until the spikes matured. Although the cells applied on the spikes were initially vegetative, Bennett et al.( 2003 2003 )’s study showed that introduced organism might survive as spores in the environment afterwards. Presumably, the strains’ extensive adaptability to the environment is due to their ability of producing heat- and desiccation-resistant spores. Results from the field tests also suggested that on Fusarium infected spikes, the pathogen may compete with BCAs for micro-niches and nutrition. To obtain a higher population of the biocontrol bacteria on barley spikes, B. subtilis , and B. brevi s should be sprayed on the barley spikes before Fusarium infection. Fig. 2 2 and Fig. 3 3 showed that the number of the two bacterial strains can be dramatically affected by rain events. Each period of rain caused a decrease in the population of the introduced strains, although the cell number recovered several days later. This study on the distribution of GFP-tagged strains on barley spikes showed that the inoculated strains were mostly distributed on the husks, least on the kernel, even though the number of total indigenous microorganisms was highest on the kernels and lowest on the awns, probably because the dominant indigenous bacteria’s occupation of space and assimilation of nutrition inhibit the biocontrol agents from colonizing the kernel. A better biocontrol effect was obtained when barley spikes were sprayed with biocontrol agents before F. graminearum inoculation. Our results indicated that these biocontrol strains might compete for space and resources with the fugal pathogen, which inhibit fungal establishment on barley spikes. Both biocontrol agents strongly inhibited colonial growth of F. graminearum and produced clear inhibition zones in vitro. In the field, both strains induced significant disease suppression. One feature involved in suppressing disease might be that the biocontrol agents released secondary metabolites that inhibited the growth of F. graminearum . Although production of secondary metabolites is most often associated with fungal suppression by fluorescent Pseudomonas (Handelsman and Stabb, 1996 1996 ; Mavrodi et al., 2000 2000 ; Thomashow and Weller, 1988 1988 ), the reports on the sporulating gram-positive bacteria producing antifungal compounds are not rare (Yu et al., 2002 2002 ; Cho et al., 2003 2003 ; Kim and Chung, 2004 2004 ). B. brevis is now well established as a biocontrol agent in Europe showing efficacy against Botrytis and powdery mildew disease (Edwards and Seddon, 1992 1992 ). By comparing the activity of B. brevis Nagano against Botrytis cinerea with that of pure gramicidin S and the antibiotic-negative mutant B. brevis E-1, Edwards and Seddon ( 2001 2001 ) showed that the mode of antagonism exhibited was antibiosis due to the presence of gramicidin S. There are some other antibiotics (for example tyrocidins and gramicidin D) reported be produced by B. brevis (Saito et al., 1970 1970 ), so further studies are required to identify the metabolites produced by the two bacteria strains and their effects on biocontrol. Under natural conditions, climatic conditions fluctuate naturally, Studies are also needed to explore how these factors influence the survival and the biocontrol effects of these two bacterial strains. References MAV Araujo , LC Mendonca-Hagler , AN Hagle . Survival of genetically modified Pseudomonas fluorescences introduced into subtropical soil microcosms . FEMS Microbiol Ecol , 1994 . 13 205 - 216 . . Survival of genetically modified Pseudomonas fluorescences introduced into subtropical soil microcosms AJ Bennett , C Leifert , JM Whipps . Survival of the biological agents Coniothyrium minitans and Bacillus subtilis MBI600 introduced into pasteurized, sterilised and non-sterile soils . Soil Biol Biochem , 2003 . 35 1565 - 1573 . . Survival of the biological agents Coniothyrium minitans and Bacillus subtilis MBI600 introduced into pasteurized, sterilised and non-sterile soils J Boelens , MV Woestyne , W Verstraele . Ecological importance of motility for the plant growth-promoting rhizopseudomonas strain ANP15 . Soil Biol Biochem , 1994 . 26 269 - 277 . . Ecological importance of motility for the plant growth-promoting rhizopseudomonas strain ANP15 F Capettini , DC Rasmusson , R Dill-Macky , E Schiefelbein , A Elakkad . Inheritance of resistance to fusarium head blight in four populations of barley . Crop Sci , 2003 . 43 1960 - 1966 . . Inheritance of resistance to fusarium head blight in four populations of barley S Chang , SN Cohen . High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA . Molec Gen Genet , 1979 . 168 111 - 115 . . High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA WL Chao , EB Nelson , GE Harman , HC Hoch . Colonization of the rhizosphere by biological control agents applied to seeds . Phytopathology , 1986 . 76 60 - 65 . . Colonization of the rhizosphere by biological control agents applied to seeds SJ Cho , SK Lee , BJ Cha , YH Kim , KS Shin . Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03 . FEMS Microbiol Lett , 2003 . 223 47 - 51 . . Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03 E Douka , A Christogianni , AI Koukkou , AS Afendra , C Drainas . Use of green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata . FEMS Microbiol Lett , 2001 . 201 221 - 227 . . Use of green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata SG Edwards , B Seddon . Bacillus brevis as a Biocontrol Agent against Botrytis cinerea on Protected Chinese Cabbage . Recent Advances in Botrytis Research , 1992 . : The Netherlands Pudoc Scientific Publications , 267 - 271 . . Bacillus brevis as a Biocontrol Agent against Botrytis cinerea on Protected Chinese Cabbage SG Edwards , B Seddon . Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro . J Appl Microbiol , 2001 . 91 652 - 659 . . Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro AB Elizabeth , HJ Emmert . Biocontrol of plant disease: A (Gram-)positive perspective . FEMS Microbiol Lett , 1999 . 171 1 - 9 . . Biocontrol of plant disease: A (Gram-)positive perspective J Handelsman , E Stabb . Biocontrol of soilborne plant pathogen . Plant Cell , 1996 . 8 1855 - 1869 . . Biocontrol of soilborne plant pathogen JR Ingalls . Influence of deoxynivalenol on feed consumption by dairy cows . Anim Feed Sci , 1996 . 60 297 - 300 . . Influence of deoxynivalenol on feed consumption by dairy cows P Kim , KC Chung . Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908 . FEMS Microbiol Lett , 2004 . 234 177 - 183 . . Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908 Z Klement , K Rudolph , DC Sands . Methods in Phytobacteriology , 1990 . : Budapest Academiai Kiado , . Methods in Phytobacteriology L Kragelund , B Christoffersen , O Nybroe , FJ de Bruijn . Isolation of lux report gene fusions in Pseudomonas fluorescences DF57 inducible by nitrogen or phosphorus starvation . FEMS Microbiol Ecol , 1995 . 17 95 - 106 . . Isolation of lux report gene fusions in Pseudomonas fluorescences DF57 inducible by nitrogen or phosphorus starvation PJ Lewis , AL Marston . GFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilis . Gene , 1999 . 227 101 - 109 . . GFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilis W Mao , JA Lewis , PK Hebbar , RD Lumsden . Seed treatment with a fungal or a bacterial antagonist for reducing corn damping-off caused by species of Pythium and Fusarium . Plant Dis , 1997 . 81 450 - 454 . . Seed treatment with a fungal or a bacterial antagonist for reducing corn damping-off caused by species of Pythium and Fusarium OV Mavrodi , BBM Gardener , DV Mavrodi , RF Bonsall , DM Weller , LS Thomashow . Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing Fluorescent pseudomonas spp. . Phytopathology , 2000 . 91 35 - 43 . . Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing Fluorescent pseudomonas spp. JH Miller . Determination of Viable Cell Count: Bacterial Growth Curves . Experiments in Molecular Genetics , 1972 . 2nd Ed : New York, USA Cold Spring Harbour Laboratory , 31 - 36 . . Determination of Viable Cell Count: Bacterial Growth Curves CS Nautiyal , JK Johri , HB Singh . Survival of the rhizosphere-competent biocontrol strains Pseudomonas fluorescence s NBR12650 in the soil and phytosphere . Can Microbiol , 2002 . 48 ( 7 ): 588 - 601 . . Survival of the rhizosphere-competent biocontrol strains Pseudomonas fluorescences NBR12650 in the soil and phytosphere M O’Callaghan , EM Gerard , VW Johnson . Effect of soil moisture and temperature on survival of microbial control agents . New Zeal Plant Protect , 2001 . 54 128 - 135 . . Effect of soil moisture and temperature on survival of microbial control agents J Olsson , T Bőrjesson , T Lundstedt , J Schnürer . Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose . Int J Food Microbiol , 2002 . 72 203 - 214 . . Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose Y Onji , Y Aoki , N Tani , K Umebayashi , Y Kitada , Y Dohi . Direct analysis of several Fusarium mycotoxins in cereals by capillary gas chromatography-mass spectrometry . J Chromatogr A , 1998 . 815 59 - 65 . . Direct analysis of several Fusarium mycotoxins in cereals by capillary gas chromatography-mass spectrometry CM Placinta , JPF D’Mello , AMC Macdonald . A view of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins . Anim Feed Sci Tech , 1999 . 78 21 - 37 . . A view of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins HJO Ramos , LDB Roncato-Maccari , EM Souza , JRL Soares-Ramos , M Hungria , FO Pedrosa . Monitoring Azospirillum -wheat interaction using the gfp and gusA genes constitutively expressed from a new broad-host range vector . J Biotechnology , 2002 . 97 243 - 252 . . Monitoring Azospirillum-wheat interaction using the gfp and gusA genes constitutively expressed from a new broad-host range vector Y Saito , S Otani , S Otani . Biosynthesis of gramicidin S . Adv Enzymol , 1970 . 33 337 - 380 . . Biosynthesis of gramicidin S KP Scott , DK Mercer , LA Glover , HJ Flint . The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems . FEMS Microbiol Ecol , 1998 . 26 219 - 230 . . The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems P Somasegaran , HJ Hoben . Handbook for Rhizobia: Method in Legume-Rhizobium Technology , 1994 . : New York, USA Springer Publications , . Handbook for Rhizobia: Method in Legume-Rhizobium Technology LS Thomashow , DM Weller . Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. . J Bacteriology , 1988 . 170 3499 - 3508 . . Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. CA Urrea , RD Horsley , BJ Steffenson , PB Schwarz . Heritability of fusarium head blight resistance and deoxynivalenol accumulation from barley accession CIho 4196 . Crop Sci , 2002 . 42 1404 - 1408 . . Heritability of fusarium head blight resistance and deoxynivalenol accumulation from barley accession CIho 4196 KJ Wilson . Molecular technology for the study of rhizobial ecology in the field . Soil Biol Biochem , 1995 . 27 501 - 514 . . Molecular technology for the study of rhizobial ecology in the field CW Xi , M Lambrecht , J Vanderleyden , J Michiels . Bi-functional gfp -and gusA -containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies . J Microbiol Meth , 1999 . 35 85 - 92 . . Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies GY Yu , JB Sinclair , GL Hartman , BL Bertagnolli . Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctoria solani . Soil Biol Biochem , 2002 . 34 955 - 963 . . Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctoria solani We thank professor Zhi-Yi Chen for providing us the plasmid pRP22, the Weather Bureau of Hangzhou City for providing the climate data and the Environment Bureau of Hangzhou City for permission the field trial with genetically manipulated organisms. We thank Diane Keen for reviewing the manuscript and offering valuable comments. 10.1631/jzus.2005.B0770 Project supported by the National Natural Science Foundation of China (No. 30230250) and Science and Technology Committee of Zhejiang Province (No. 2003C22029), China 2005-03-10 2005-04-30 2005-07-29 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 6B