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Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1
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Biological Control 45 (2008) 288–296 www.elsevier.com/locate/ybcon
Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1 Guang-Hai Ji a,*, Lan-Fang Wei b, Yue-Qiu He a, Ya-Peng Wu a, Xue-Hui Bai a a
Key Laboratory of Agricultural Biodiversity for Plant Disease Management under the Ministry of Education, Yunnan Agricultural University, Kunming 650201, PR China b Faculty of Resource and Environment, Yunnan Agricultural University, Kunming 650201, PR China Received 17 November 2006; accepted 6 January 2008 Available online 16 January 2008
Abstract Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (108 CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Xanthomonas oryzae pv. oryzae; Rice bacterial blight; Lysobacter antibioticus; Biocontrol
1. Introduction Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae [(Ishiyama) Swings] (Xoo) is one of the most devastating diseases of rice. The bacterial pathogen infects the host-plant at the maximum tillering stage, resulting in 20–40% reduction in yields. The disease was first observed by the Japanese farmers in 1884 (Tagami and Mizukami, 1962). Since then, it has been reported in Asia, Northern Australia, Africa, and the United States (Mew, 1987; Ou, 1985). In China, BB has become endemic occurring mainly in southeast coastal plain and Yangtze River delta. Since *
Corresponding author. Fax: +86 871 5228024. E-mail address: [email protected] (G.-H. Ji).
1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.01.004
2001, annual epidemics of this disease have become common in the Yunnan Province of China (Ji et al., 2003). Various disease management practices, such as chemical control, host-plant resistance, modification of cropping systems, and biological control have been employed to reduce damage caused by BB. Chemical control and hostplant resistance, two of the most common management practices, have their limitations. Chemical pesticides harm the environment, and host-plant resistance, which is based on a single gene, may not be durable in the field leading to frequent resistance breakdowns. It is imperative to develop environmental-friendly and sustainable control strategies. Biological control is an ecology-conscious, cost-effective, and sustainable alternative method in BB management. This approach can also be integrated with other manage-
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ment practices to afford greater levels of protection and sustain rice yields. Antagonistic bacteria are considered as ideal biological control agents with obvious advantages. They are easy to handle, grow rapidly, and colonize the rhizosphere aggressively (Weller, 1988). Certain strains of Bacillus spp. and Pseudomonas spp., have been used as biocontrol agents to suppress rice BB (Vasudevan et al., 2002). A novel plant growth-promoting strain of Delftia tsuruhatensis HR4 has been shown to be a promising biocontrol agent against BB (Han et al., 2005). Lysobacter sp. (Christensen and Cook), a Gram-negative bacterium with gliding motility has long been studied for its ability to suppress plant pathogens or otherwise promote plant growth (Christensen and Cook, 1978). L. enzymogens strain C3 suppressed a number of plant diseases incited by fungal pathogens, including Bipolaris sorokintana, Fusarium graminearum, and Rhizoctonia solani, in field experiments (Giesler and Yuen, 1998; Zhang and Yuen, 1999; Yuen and Zhang, 2001; Jochum et al., 2006). This bacteria controls fungal pathogens by various mechanisms, such as production of chitinases and b-1,3-glucanases (Zhang and Yuen, 2000b; Zhang et al., 2001; Palumbo et al., 2003), antibiotics (Zhang and Yuen, 2000a; Islam et al., 2005) or by induction of systemic resistance (Kilic-Ekici and Yuen, 2004). The antifungal activity of another strain of L. enzymogens, 3.1 T8, against Pythium aphanidermatum was attributed to a polyketide synthase (Folman et al., 2003, 2004). In this study, we investigated Lysobacter antibioticus strain 13-1, which occurs in the plant rhizosphere in China, as a potential biocontrol agent against rice BB. The objectives of this study were (i) to evaluate the efficacy of L. antibioticus strain 13-1 against BB under greenhouse and field condition, and (ii) to determine the main factors that affect the efficiency of L. antibioticus strain 13-1 in reducing the severity of rice BB. 2. Materials and methods 2.1. Isolation of bacteria Samples were collected from rhizospheres of various crops including rice, maize, potato, millet, and wheat at different locations in Yunnan Province. The rhizosphere samples were serially diluted (from 106 to 108) and plated on nutrient agar (NA) (Fang, 1998). After incubation at 28 °C for 2 days, bacterial colonies were isolated and tentatively identified. The candidate Lysobacter spp. were stored in 30% glycerol at 30 °C. 2.2. In vitro antagonistic activity assay Lysobacter antibioticus strain 13-1 was inoculated in nutrient-poor R2A medium and incubated at 25 °C for 5 days with constant shaking at 180 rpm. The resultant yield of ca. 109 CFU/mL was used for study of antagonistic activity against several plant pathogenic fungi and bacteria listed in Table 1.
289
Antibacterial activity was determined by agar diffusion technique. Suspensions (10 mL) of plant pathogenic bacteria (around 109 CFU/mL) were mixed with NA (100 mL) prior to pouring into plates. After solidification, 2 lL suspensions of L. antibioticus strain 13-1 was plated on the agar surface and incubated at 27 ± 1 °C for 48 h. Antibacterial activity was measured as the diameter of inhibition zone around the colony of strain 13-1 (Monterio et al., 2005). The inhibition of mycelial growth of a range of the pathogenic fungi by strain 13-1 was measured in vitro (Table 1). Three 2 lL drops of strain 13-1 suspensions were placed equidistant at the margins of potato dextrose agar (PDA) (BioLab) plates and incubated at 27 ± 1 °C for 24 h. A 5mm diameter block of phytopathogenic fungi from fresh PDA culture was inoculated in the center of the PDA plate and incubated at 27 ± 1 °C. After one week, zones of inhibition of mycelial growth around colonies of strain 13-1 were scored (Folman et al., 2003). 2.3. Greenhouse experiments Strain 13-1 was inoculated in R2A medium and incubated at 25 °C for 5 days with constant shaking at 180 rpm yielding ca. 109 CFU/mL. The BB pathogen, Xoo strain 53 was cultured on NA for 3 days at 28 °C. The bacterial cells were collected from plates and suspended in distilled water, and cell density was determined using a spectrophotometer. A BB susceptible rice cultivar Huangkenuo was used as the host-plant. It was planted in 15 cm 30 cm pots, four plants in each pot, and each treatment was replicated three times. Strain 13-1 whole broth culture (WBC) was used as the standard treatment, and the distilled water as the control. At the maximum tillering stage, Plants were sprayed with either WBC or water using laboratory atomizer. On the 3rd day post inoculation, the treated pots were inoculated with BB pathogen by spraying leaves with cell suspensions (around 109 CFU/mL). Treated pots were kept at constant humidity moisture and temperature for 2 days and then returned to the greenhouse bench. The pots were placed in a randomized complete block design and the experiment was repeated three times. The suppression effect of WBC and various components of strain 13-1 culture on rice BB were also studied. The WBC was separated into cellular and supernatant fractions by centrifuging suspensions at 12,000 rpm for 5 min. The supernatant was further filtered through 0.22 lm filter, and the filtrate was designated as cell-free culture fluid (CFC). The cells were washed three times with distilled water, and re-suspended in sterile distilled water, designated as cells re-suspended in water (CRW). WBC was also inactivated by heating at 70 °C for 30 min, and designated as heated whole culture (HWC). Furthermore, disease reduction of WBC, CCF, and CRW was also evaluated against strain Y14, a non-sensitive strain of BB pathogen
290
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Table 1 Antagonistic activity of strain 13-1 against various plant pathogenic bacteria and fungi tested in plate assays Strain code
Diameter of inhibition zone (mm)1
Host-plants
14–25
Rice
Xathomonas oryzae pv. oryzicola Xanthomonas axonopodis pv. dieffenbachiae Xanthomonas campestris pv. campestris
53,X13,Y4,A10,Q6-3,9,X6,Y6,57,Q15-1,Q15-2,A1, 63,Y7,Y9,QH3,79,Y8,X5,3-1,39,JL18,X9,Q7-2,, A3,Q8-5,26,30,GD38,Yuan2,Q8-3,5 Y14, X7, A2, A6, 13, 51 Tb23 CX10 T5 My9 ECC2 Wm2 Rs68 XCD-S 8004
0.0 15.6 ± 0.2 11.5 ± 0.2 11.4 ± 0.1 5.0 ± 0.2 7.0 ± 0.5 4.6 ± 0.3 7.4 ± 0.7 20.7 ± 1.3 9.7 ± 0.9
Rice Tobacco Tobacco Oriential tobacco Konnyaku Cabbage Zantedeschia Rice Anthurium Cabbage
Fungi Pyricularia grisea (Cooke) Sacc. Pythium sp. Fusarium orysporum Schlf. sp. Vanillae (Tucker) Gordon F. solani (Mart.) Appel & Wollenw. emend Snyd. & Hans Exserohilum turcicum (pass.) Leonard & Suggs Bipolaris maydis (Nishik & Miyabe) shoem Phytophthora nicotianae Alternaria alternate (Fries) Keissler
SQ1 F1 LH5 DB XB 97007, 97008 CX
4.3 ± 0.2 6.5 ± 0.8 7.5 ± 0.5 6.7 ± 0.4 15.7 ± 0.6 12.0 ± 0.3 28.7 ± 0.2 11.5 ± 0.7
Rice Panax notoginseng Vanilla Aloe barbadensis Maize Maize Tobacco Tobacco
Pathogen
Bacteria Xanthomonas oryzae pv. oryzae
Xanthomonas oryzae pv. oryzae Ralstonia solanacearum P. syringae pv. tabaci P. syringae pv. tabaci Pectobacterium carotovorum spp. carotovorum
1
Note: The inhibition zone is given in mm (± standard deviation).
to antibiotic inhibition by strain 13-1. In the second set of experiments, biocontrol efficacy of WBC on Xoo was evaluated with serial dilutions, ranging from 1/5 to 1/1000. The original WBC and distilled water were the controls. A third set of experiments was designed to examine the effects of timing of application of strain 13-1 on biocontrol efficacy against the BB pathogen. WBC was applied once to rice leaves at maximum tillering stage 1, 3, or 7 days prior to inoculation with Xoo strain 53. Efficacy was compared with a water control applied one day prior to inoculation of pathogen. Each of these three experiments was repeated two or three times, and were arranged in a completely randomized design (CRD). Five replicates were used per treatment, each replicate containing four rice plants. The fourth experiment was performed in the greenhouse for evaluation of the biocontrol efficacies of WBC of strain 13-1 on 18 different rice cultivars listed in Table 4. Rice cultivars were evaluated for their resistance to Xoo strain 53. In the field, plants were transplanted into experimental plots, and managed using standard cultural practices. At the maximum tillering stage, 10–15 leaves of each cultivar were clip-inoculated with a cell suspension of strain 53 (108 CFU/mL) and lesion length and whole leaf lengths of the 10 inoculated leaves were measured 14 days after inoculation following the method of Kauffman et al. (1973). Data sets of measured lesion lengths from 10 observations were used for analysis. Plants were characterized as resistant or susceptible based on the average lesion length/ leaf lengths (LL) with LL 6 0.25 cm indicating resistant
(R); 0.25–0.50 cm indicating moderately susceptible (MS), and LL P 0.5 cm indicating susceptible (S) cultivars (Fang, 1998). The rice cultivars were grown in a greenhouse as previously described for cultivar Huangkenuo. Huangkenuo was used as the ‘‘susceptible control” to confirm that conditions were favorable for rice BB development. All cultivars were submitted to the treatment with WBC and the water control. The assays were performed in a randomized complete block design with three replicates per treatment. Nine rice plants were used in each treatment plot. Each cultivar was tested twice. 2.4. Field experiments Field trials were conducted in two consecutive consecutive years, 2005 and 2006, at Honghe Agricultural Research Station, Mengzi County in Yunnan Province. Rice cultivar Huangkenuo was used. There were three applied treatments: (1) 5-day-old broth culture of strain 13-1 filtered through cheese cloth, and diluted 100 times with water; (2) 1% Zhongshengmycin; and (3) water check. Each treatment was applied to four plots (4 m 5 m), arranged in randomized complete block design. A single application of each treatment was made at the maximum tillering stage of rice plants. Plots were sprayed with the WBC of strain 13-1 (108 CFU/mL) at a rate of 265 L/ha commonly used for bactericide evaluation against BB. Suspensions (108 CFU/mL) of Xoo strain 53 were sprayed onto rice plants 3 days after application of treatments.
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A field experiment with identical treatments was also repeated in Yuanmou County, Yunnan Province in 2006. Rice cultivar Hexi No.41 was used. There were six replicate plots per treatment arranged in a randomized complete block design. All other procedures were same as those in trials conducted in Mengzi County in 2005 and 2006, except that pathogen was sprayed 1 day and 3 days after application of treatment at the booting stage. 2.5. Scoring of BB disease and evaluation of disease control by strain 13-1 Development of BB lesions was scored 14 days or 21 days after pathogen inoculation. The diseased lesion area and the whole leaf area were measured. And scoring was done based on the average area of diseased lesion/the area of whole leaves (L/W). A disease scale of 0–9 was used, with 0 indicating no infection of leaves; 1, L/W < 0.2; 3, L/W = 0.25; 5, L/W = 0.5; 7, L/W = 0.75; and 9 indicating complete death of leaves. A disease index (DI) was calculated using the formula, DI = 100 sum of individual scores/total leaves observed maximum score. The extent of disease reduction attributed to each treatment was calculated using the following formula: disease suppression efficiency = ([disease index of control disease index of treatment group]/disease index of control) 100%. 2.6. Colonization of strain 13-1 on plant tissue In order to monitor variations in the population of strain 13-1, a Rifampicin-resistant strain of 13-1 (Rifr) was used in the field experiments. Four leaves or stems from each replicate plot were collected at 16 h and intervals of 1, 3, 5, 7, 9, 11, 13, 15, 17, 21 days after treatment. The plant parts were homogenized with a tissue homogenizer in 10 mL sterile phosphate buffer. The extract was used for dilution plating. Colonies of strain 13-1 were counted on R2A agar plates amended with rifampicin and cycloheximide at 100 mg/L. Population levels of strain 13-1 were expressed as: log10 CFU/g fresh weight of leaves and stems. 2.7. Data analysis Analysis of variance (ANOVA) for disease index and suppression efficacy was performed for all data using general linear model procedures of the Statistical Analysis System (SAS institute, version 6, Cary, NC, USA). Mean comparisons were conducted using a least significant difference (Fisher’s LSD) test (P = 0.05 or P = 0.01). Standard error and LSD results were recorded. 3. Results 3.1. Antagonistic activity
16S rDNA sequence (data not shown), and was designated as strain 13-1 of L. antibioticus. Strain 13-1 showed significant antagonistic activity against numerous different plant pathogens, including bacterial pathogens Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas axonopodis, and Xanthomonas campestris, and fungal pathogens Alternaria alternata, Phytophthora nicotiana, Exherohilum turcicum, and Bipolaris maydis (Table 1). Moreover, strain 13-1 inhibited activity of 32 out of 38 isolates of the rice BB pathogen, Xoo. Strain 13-1 was the most highly antagonistic isolate of the 1000 bacterial isolates screened (data not shown). 3.2. Control of rice BB by strain 13-1 under greenhouse conditions 3.2.1. Disease suppression efficacy of different culture components of strain 13-1 All of the culture components of strain 13-1 tested had an adverse effect on development of BB disease in rice (Table 2). The WBC provided the highest disease control efficacy, 69.7%, followed by CCF and CRF, between which there was no significant difference. Heating the whole broth culture (HWC) significantly reduced its inhibitory effect against BB pathogen, but it still provided substantial reduction in development of BB disease relative to the control. Efficacy of WBC and its components differed for two pathogen strains with varying sensitivity to antibiotic inhibition by strain 13-1. When the treatment was used against the antibiotic-sensitive strain 53, all components of strain 13-1, WBC, CRW, CCF, and HWC, significantly reduced rice BB incidence and disease severity compared to the water control, with suppression efficiencies of 48–58% (Table 3). Against non-sensitive strain Y14, all culture treatments still reduced incidence and severity of BB, but to a lesser degree, with suppression efficiencies of 17–32%. 3.2.2. Effect of timing of application of strain 13-1 cultures Applications of 13-1 WBC 1, 3, or 7 days prior to pathogen inoculation, all reduced disease incidence and index of rice BB, with suppression efficiencies of 49.9%, 57.6%, and 31.7%, respectively, relative to the control (Table 4). The Table 2 Effects of culture of Lysobacter antibiotus strain 13-1 and its components in controlling rice bacterial blight in greenhouse experiments Treatment
Disease index (%)
Water Whole 13-1 broth culture (WBC) Cells re-suspended in water (CRW) Cell-free culture fluid (CCF) Heated whole culture (HWC)
51.4 ± 2.9 15.6 ± 1.3 18.5 ± 1.1 19.8 ± 1.5 25.9 ± 0.1
1
A bacterial strain was identified as L. antibioticus based on its physiological and biochemical characteristics, and
291
Suppression efficacy (%) a1 d cd c b
— 69.7 ± 2.6 64.1 ± 2.0 61.6 ± 2.9 49.6 ± 0.1
a b b c
Means within columns followed by the same letter within a column are not significantly different (P = 0.05) by Fisher’s Least Significant Difference (LSD) test.
292
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Table 3 Evaluation of the effects of L. antibioticus strain 13-1 and different culture components for suppression of sensitive strain 53 and non-sensitive strain Y14 of the bacterial blight pathogen Treatment strain 13-1
1
WBC CRW CCF Water 1 2
Diameter of inhibition of BB strain (mm)
Disease incidence (%)
Disease index (%)
Suppression efficacy (%)
53
Y14
53
Y14
53
Y14
53
Y14
0 0 0 0
50 ± 2.5 c 55.7 ± 1.5 b 57.1 ± 1.8 b 89.4 ± 2.3 a
74.3 ± 5.0 62.6 ± 7.3 72.3 ± 5.3 89.4 ± 1.5
14.6 ± 0.9 b 16 ± 1.8 b 18.2 ± 1.9 b 35.2 ± 2.9 a
25.6 ± 5.1 23.8 ± 3.7 29.1 ± 7.3 35.2 ± 5.0
58.5 ± 2.6 a 54.5 ± 5.1 a 48.3 ± 5.5 a —
27.3 ± 3.5 a 32.4 ± 7.1 a 17.3 ± 2.2 b —
2.5 ± 0.01 b 2.5 ± 0.02 b 2.6 ± 0.01 a 0
2
b b b a
c d b a
WBC, whole 13-1 broth culture; CRW, 13-1 cells re-suspended in water; CCF, 13-1 cell-free culture fluid. Means within columns followed by the same letter within a column are not significantly different (P = 0.05).
optimum time of application for strain 13-1 was 3 days prior to pathogen inoculation. Disease index (100%)
70.0
3.2.3. Suppression of rice BB by varying concentrations of 13-1 whole broth culture Dilutions of the whole broth bacterial culture had only a marginal effect on biocontrol efficacy in greenhouse experiments. All serial dilutions, ranging from 5-fold up to 1000fold significantly reduced the BB disease index compared to the control, and disease suppression was comparable to that of the undiluted WBC (Fig. 1). However, DI did increase somewhat from 12% for WBC to 20% for the 5-fold to 100-fold dilutions, and 30% for the 1000-fold dilution treatment. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments (Fig. 1).
60.0 50.0 40.0 30.0 20.0 10.0 0.0 1
2
3
4
5
6
7
Dilution factor
Fig. 1. Effects of dilutions of broth cultures of L. antibioticus strain 13-1 on the disease index of rice bacterial blight in a greenhouse experiment (1, undiluted; 2, 1/5; 3, 1/10; 4, 1/50; 5, 1/100; 6, 1/1000; 7, control).
disease control was 37.8%. These results suggest that biocontrol efficacy of strain 13-1 was somewhat affected by the genetic variability of different rice cultivars, but efficacy was generally not related to the susceptibility of the cultivar to Xoo. Overall, Suppression efficacy of BB by 13-1 was similar on both the susceptible and moderately susceptible cultivars.
3.2.4. Influence of rice cultivar on biocontrol efficacy Biocontrol efficacy of strain 13-1 varied significantly among different rice cultivars, with disease suppression ranging from 0.6% to 62.8% across the 18 cultivars (Table 5). Of the 18 rice cultivars, five cultivars were classified as moderately susceptible to the Xoo strain 53, and all the others were considered susceptible. The greatest efficacy was achieved with susceptible cultivars II-You725, Huangkkenuo, and IRBB5, and moderately susceptible Yunhui290 (47–63% BB suppression). However, strain 13-1 was not effective in reducing disease on six rice cultivars (<10% BB suppression), including susceptible cultivars Hongyou No.3, X-21, IRBB1, and IRBB3, and moderately susceptible Hongyou No. 6 and Diantun 502 (Table 5). Average BB suppression across the 12 cultivars showing effective
3.3. Field experiments In Mengzhi County field trials, strain 13-1 reduced rice BB disease index by 78.3% and 59.1% in 2005 and 2006, respectively, relative to the water control (Table 6). This was better than the control provided by Zhongshengmycin, with suppression efficacies of 69.4% and 47.7% in 2005 and 2006, respectively. In Yuanmou County in 2006, after two inoculations with the BB pathogen, the untreated plants
Table 4 Suppression of rice bacterial blight in the greenhouse by culture of Lysobacter antibiotus 13-1 applied at different times relative to pathogen inoculation Treatment Distilled water 13-1 WBC 13-1 WBC 13-1 WBC 1 2
Time of application1 1 1 3 7
% Infected leaves 63.0 ± 9.0 42.9 ± 5.3 36.3 ± 2.5 47.4 ± 0.9
2
a bc c b
Disease index
Disease suppression efficacy (%)
38.5 ± 8.0 20.8 ± 2.9 16.3 ± 1.7 26.3 ± 1.3
— 45.9 ± 3.1 b 57.6 ± 1.9 a 31.7 ± 2.0 c
a b c b
Days before pathogen inoculation. Means within columns followed by the same letter are not significantly different (P = 0.05) according to Fisher’s LSD test.
G.-H. Ji et al. / Biological Control 45 (2008) 288–296
293
Table 5 Evaluation of Lysobacter antibiotus strain 13-1 in biological suppression of rice bacterial blight on different rice cultivars in greenhouse trials Cultivar
BB susceptible level
Disease incidence (%)
Disease index (%)
BB suppression (%)
Strain 53
Treatment
Control
Treatment
Control
IR-24 IR64 Huangkenuo Zhinuo Yunhui290 Zhong413 Zhong419 II You725 Fenjin Xiaohua Nuo IRBB5 Hongyou No.3 Hongyou No.5 Hongyou No.6 Diantun 502 X-21 IRBB1 IRBB3
S S S S MS MS MS S S MS S S S MS MS S S S
32.9 ± 4.1 23.7 ± 3.5 43.5 ± 2.8 63.2 ± 3.9 41.0 ± 2.9 55.3 ± 5.3 52.6 ± 9.9 57.1 ± 7.2 83.1 ± 1.4 55.4 ± 3.2 41.8 ± 2.1 72.4 ± 3.1 53.4 ± 4.6 68.9 ± 4.8 26.34 ± 5.8 68.3 ± 2.0 33.3 ± 2.0 47.2 ± 4.8
53.5 ± 2.8 43.2 ± 4.9 58.9 ± 6.3 69.8 ± 8.1 53.6 ± 5.8 62.1 ± 2.2 52.9 ± 3.4 55.3 ± 4.6 80.3 ± 9.1 60.2 ± 7.7 58.1 ± 6.3 68.8 ± 5.4 63.6 ± 7.6 72.7 ± 5.8 32.1 ± 6.8 69.9 ± 7.8 27.7 ± 2.1 40.8 ± 5.1
15.6 ± 3.7**1 13.2 ± 2.0** 15.3 ± 3.2** 27.1 ± 5.5** 14.7 ± 1.8** 14.6 ± 2.0** 11.5 ± 2.0** 14.5 ± 2.2** 24.3 ± 3.2** 16.5 ± 3.8** 14.5 ± 2.0** 26.5 ± 3.3 ns 28.8 ± 2.1** 28.2 ± 2.0 ns 17.5 ± 3.3* 28.8 ± 2.0 ns 10.8 ± 2.4 ns 17.8 ± 2.0 ns
27.6 ± 1.1 19.8 ± 3.2 38.8 ± 1.2 32.2 ± 2.2 26.2 ± 1.2 21.5 ± 1.4 15.9 ± 2.0 39.0 ± 2.5 42.1 ± 4.1 23.0 ± 2.4 27.4 ± 3.3 28.0 ± 1.1 32.5 ± 4.4 30.1 ± 2.4 19.1 ± 1.2 29.0 ± 3.0 11.9 ± 1.4 18.6 ± 2.5
43.5 ± 5.2 33.3 ± 3.8 60.6 ± 4.1 15.8 ± 5.2 48.9 ± 1.0 32.1 ± 7.3 27.7 ± 4.0 62.8 ± 2.5 42.2 ± 4.9 28.2 ± 1.1 47.1 ± 3.1 5.4 ± 0.1 11.4 ± 0.4 6.3 ± 0.3 8.5 ± 0.2 0.6 ± 0.1 9.2 ± 1.1 4.3 ± 1.4
Note: Experiments were performed in a screen house at the agricultural research station, YAU. Kunming, Yunnan Province, China. BB, bacterial blight. Reduction in disease index: **, significant at the 1% level between treatment and the control; *, significant at the 5% level between treatment and the control; ns, not significant. 1 Each value is a mean of 30 observations.
showed severe BB disease symptoms with long and spreading lesions, while plants treated with 13-1 strain were relatively healthy with small lesions (Table 6). Strain 13-1 and 1% Zhongshengmycin both reduced disease incidence and disease index compared to the control in these field experiments, but strain 13-1 was more effective than Zhongshengmycin, with a suppression efficacy of 73.5% vs. 34.9% for Zhonshengmycin (see Table 7). Strain 13-1 was able to colonize not only on rice leaf, but also on rice stems three weeks after application. The survival level of strain 13-1 from treated rice leaves and stems, however, declined gradually after treatment (Fig. 2). 4. Discussion Some strains of Lysobacter spp., such as 3.1T8, SB-K88, and C3, had been reported to be effective in controlling both soilborne and foliar diseases (Folman et al., 2004; Islam et al., 2005; Jochum et al., 2006). In the present work, antagonistic effects of L. antibioticus strain 13-1 against rice
BB were studied under laboratory, greenhouse, and field conditions. And then, disease suppression efficacy of different individual culture components, different concentrations, techniques and timing of application, as well as the influence of rice cultivar on biocontrol efficacy, were also extensively evaluated. The results suggested the potential of L. antibioticus strain 13-1 to be developed as a promising commercial biological control agent in the future. The whole bacterial broth culture of L. antibioticus strain 13-1 was the most effective form for control of rice BB. However, the heated bacterial broth culture was almost as effective as the living 13-1 cells, suggesting that the primary antagonistic component was heat-stable. In further analysis of the biocontrol factors of strain 13-1 potentially responsible for the inhibitory role against BB pathogen, culture supernatant of strain 13-1 was extracted with excess ethyl acetate and concentrated in vacuum. Thin-layer chromatography with silica gel plate was used to further extract the main active compounds from all fractions towards Xoo. In Agar (NA) well diffusion assays, the
Table 6 Efficacy of broth culture of L. antibioticus 13-1 for control of rice bacterial blight in 2005 and 2006 field on ‘‘Huangkenuo” rice cultivars at Mengzhi county of Yunnan province Treatment
L. antibioticus 13-1 1% Zhongshengmycin Water 1
2005
2006
Incidence (%)
Disease index (%)
Biocontrol efficacy (%)
Disease incidence (%)
Disease index (%)
Suppression efficacy (%)
28.0 ± 2.8 c1 40.6 ± 3.7 b 76.1 ± 9.2 a
7.6 ± 1.1 c 10.7 ± 2.1 b 34.7 ± 8.7 a
78.3 ± 2.3 a 69.4 ± 3.1 b —
34.0 ± 5.2 b 55.7 ± 6.8 a 63.0 ± 8.0 a
15.8 ± 4.3 c 22.1 ± 2.2 b 38.6 ± 5.22 a
59.1 ± 5.1 a 42.7 ± 3.4 b
Means followed by the same letter within a column are not significantly different (P = 0.05) according to Fisher’s LSD test.
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Table 7 Evaluation of strain 13-1 in the biological control of BB in rice cultivar II You 725 at Yuanmu County of Yunnan Province (2006) Treatment
Incidence (%) 1
Lysobacter antibiotus 13-1 1% Zhongshengmycin Water-treated control
Biocontrol efficacy (%)
22 ± 6.3 c 54 ± 5.5 b 83 ± 7.8 a
73.5 ± 8.1 a 34.9 ± 3.6 b —
Means followed by the same letter within a column are not significantly different (P = 0.05) according to Fisher’s LSD test.
6 5
2006 2005 2006 2005
4 3 2
leaf leaf stem stem
21d
18d
15d
9d
11d
7d
5d
3d
2d
0
1d
1 16h
13-1 cell density (Log CFU/g)
1
24.2 ± 3.7 c 46.6 ± 7.5 b 62.2 ± 10.1 a
Index (%)
Incubation time
Fig. 2. Population levels of L. antibioticus strain 13-1 detected on rice leaves and stems in 2005 and 2006 field experiments on rice cultivar ‘‘Huangkenuo”.
five fractions inhibited the growth of rice BB pathogen, whereas in control plates containing acetone, there was no inhibition (unpublished data). Interestingly, in greenhouse trials, when strain 13-1 was used in conjunction with BB pathogen strain Y14, which was insensitive to strain 131 in the previous inhibitory zone tests, disease suppression decreased greatly compared to results with a more sensitive pathogen isolate. This further supports the role of antibiotics in the suppression of BB by strain 13-1. Although the mechanism of action of strain 13-1 has not been definitively determined, the results suggest the active compound responsible for the inhibitory role against the BB pathogen mainly comes from the culture supernatant of strain 13-1, is heat-stable, and can provide protection against BB when applied in highly diluted form or up to one week prior to pathogen inoculation. In greenhouse and field assays, the amount of strain 13-1 inoculum presented on rice leaves differed greatly between 2005 and 2006, as indicated by the recovery of this strain from rice leaves (log 5.5 CFU/g in 2005 and log 4 CFU/g in 2006). It is questionable whether the densities of the biocontrol strain were sufficient to be effective in 2006. For many biocontrol bacterial species, it has been shown that a certain threshold population size must be reached before pathogen populations can be suppressed (Dunny and Winans, 1988). Pseudomonas fluorescens was only effective at densities of log 5–6 CFU/g root (Raaijmakers et al., 1999). Moreover, concentrations of antimicrobial compounds produced by the antagonistic bacteria in situ may be too low at the site of pathogen infection, and antimicrobial activity of the inhibitory compounds was not sufficient to inhibit pathogen populations (Folman et al., 2004; Kim et al., 2001). Moreover, populations of most biocontrol
agents introduced into the environment decline with time, which affects the synthesis of inhibitory compounds (Acea et al., 1988). In our study, population level of strain 13-1 on treated rice leaves gradually declined with time after treatment (Fig. 2). Therefore, population size, inhibitory compounds produced, and their activity in situ on rice leaves may have contributed to the difference in biocontrol efficacy of strain 13-1 in 2005 and 2006. Application methods of the bacterial biocontrol agents and inoculation methods of Xoo may also affect biocontrol against rice BB. In our experiment, strain 13-1 and the pathogen Xoo strain 53 were applied by foliar spray, and provided significant control of BB under field conditions, with mean reduction in disease severity of 70%. This reduction was greater than that provided by other bacterial biocontrol agents, such as P. fluorescens, P. putida, and Bacillus spp. (producing maximum reductions of 64%, 57%, and 60%, respectively) in previous published reports (Sivamani et al., 1987; Gnanamanickam et al., 1999; Johri et al., 2003; Vasudevan et al., 2002; Velusamy et al., 2006). These reports also demonstrated that application of the individual biocontrol agents by seed-soaking, root-dipping, or foliar spraying was not as effective in reducing BB severity compared with application of all three methods together. Our studies revealed that foliar spraying of strain 13-1 on rice once or twice followed by inoculation with Xoo strain 53 resulted in greater biocontrol efficacy against BB. So, the application method of strain 13-1 for suppressing BB in our research was apparently more efficient and practical than that in some earlier works. However, in our research, when the pathogen Xoo strain 53 was inoculated by the leaf clipping method, there was no obvious reduction in lesion length compared with that in untreated control plants (data not shown), which is not in agreement with other previous reports (Velusamy et al., 2006). Therefore, future studies should establish a greater understanding of the effects of different application methods of strain 13-1 on its biocontrol efficacy, and optimize its application methods and timing for practical utilization in the field. Deployment of resistant cultivars is, generally, the most economical strategy to control BB, but has only been partially successful due to the development of race variation in the pathogen that overcomes resistance (Prashant et al., 2006). In our studies, rice cultivars with differing genetic backgrounds and levels of resistance were evaluated for their effects on biocontrol efficacy. The disease resistance levels of the 18 rice cultivars tested were found to be mod-
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erately susceptible or susceptible to strain 53. However, among these, IRBB5, Hongyou Nos. 3, 5, and 6, Diantun 502, and Yunhui 290 are known to be resistant to other specific races of Xoo (Qian et al., 2003). In our tests, strain 13-1 resulted in comparable suppression of disease on most cultivars, regardless of their susceptibility to strain 53. However, since strain 13-1 was not effective on some cultivars, and these included cultivars with known resistance to other races, such as, Hongyou Nos. 3 and 6, and Diantun 502, there may be some relationship between disease susceptibility and biocontrol efficacy with some resistance factors. A similar phenomenon was found by Jochum et al. (2006), when L. enzymogenes C3 applied to different wheat cultivars in the greenhouse was effective in reducing the severity of Fusarium head blight in six wheat cultivars, but was not effective in two other cultivars; Biocontrol effects were also not directly related to reported levels of resistance/susceptibility to FHB. Strain 13-1 was highly effective on resistant cultivars Yunhui290 and IRBB5, so any relationship between biocontrol efficacy and cultivar resistance is not clear at this point and requires further investigation. Leaf surface structure, such as hydathodes and the other mesophyll tissue of different rice cultivars, might have affected colonization of strain 13-1 on the leaf surface and its biocontrol efficacy on different cultivars, Recently, Islam et al. (2005) reported that characteristic colonization of Lysobacter sp. strain SB-K88 on both rhizoplane and phylloplane of sugar beet were different to that of other plants such as spinach and tomato. For example, SB-K88 densely colonized both the root and cotyledon surface and a semitransparent bacterial film was formed on the root surface of sugar beet, but not on the root surface of spinach and the other plant species (Islam et al., 2005). Good biocontrol agents must either be effective in reducing the initial inoculum of pathogen population, or be active and persistent in suppressing disease development. In our study, strain 13-1 effectively protected rice leaves against BB during the initial stages of BB epidemics whether they occurred naturally or by inoculating with a highly virulent form of Xoo pathogen (strain 53). Further research on 13-1 as a potential biocontrol agent to BB is needed. One suggestion is to integrate the use of different biological control agents with distinct modes of action. Strain 13-1 may be a good candidate for use in a mixture of antagonistic microorganisms. Another suggestion is to formulate commercial biocontrol agents with compounds that would enhance growth or activity of the biocontrol agents when applied to plant surfaces. A third suggestion is to integrate biological controls with chemical bactericides, improve spray application technologies or develop new delivery technologies specific for microorganisms. In addition, future studies should evaluate the integration of two major approaches by combining host resistance and biocontrol for management of BB, and understanding their mechanisms for BB management.
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Acknowledgments We thank Dr. Baoyu Tian for his help and advice in preparing the manuscript. This research work was funded by projects from the Natural Science Foundations of China and Yunnan (No. 30760139 and 2003C006Z), Ministry of Agriculture (No. nyhyzx07-056), and 863 High-tech. Program (No. 2002AA245041) of China. References Acea, M.J., Moore, C.R., Alexander, M., 1988. Survival and growth of bacteria introduced into soil. Soil Biology and Biochemistry 20, 209– 515. Christensen, P., Cook, D., 1978. Lysobacter, a new genus of non-fruiting, gliding bacteria with a high base ratio [soil and water organisms]. International Journal of Systematic Bacteriology 28, 367–393. Dunny, G.M., Winans, S.C. (Eds.), 1988. Cell–cells signaling in bacteria. American Society for Microbiology, p. 367. Fang, Z.D., 1998. Methods of Plant Pathology. China Agriculture Press, Beijing. Folman, L.B., Postma, J., van Veen, J.A., 2003. Characterization of Lysobacter enzymogenes (Christensen and Cook, 1978) strain 3.1 T8, a powerful antagonist of fungal disease of cucumber. Microbiology Research 158, 107–115. Folman, L.B., De Klein, M.J.E.M., Postma, J., van Veen, J.A., 2004. Production of antifungal compounds by Lysobacter enzymogenes isolate 3.1 T8 under different conditions in relation to its efficacy as a biocontrol agent of Pythium aphanidermatum in cucumber. Biological Control 31, 145–154. Giesler, L.J., Yuen, G.Y., 1998. Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection 17, 509–513. Gnanamanickam, S.S., Brindha, P.V., Narayanan, N.N., 1999. An overview of bacterial blight disease of rice and strategies for its management. Current Science 77, 1435–1443. Han, J., Sun, L., Dong, X., 2005. Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens. Systematic Applied Microbiology 28, 66–76. Islam, M.T., Hashidoko, Y., Deora, A., Ito, T., Tahara, S., 2005. Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne peronosporomycetes. Applied and Environmental Microbiology 71, 3786–3796. Ji, G., Zhang, S., Qian, J., 2003. Preliminary analysis on races of Xanthomonas oryzae pv. oryzae in Yunnan Province. Plant Protection 29, 19–21 (in Chinese). Jochum, C.C., Osborne, L.E., Yuen, G.Y., 2006. Fusarium head blight biological control with Lysobacter enzymogenes strain C3. Biological Control 39, 336–344. Johri, G.H., Sharma, A., Virdi, J.S., 2003. Rhizobacterial diversity in India and its influence on soil and plant health. Advanced Biochemical Engineering Biotechnology 84, 49–89. Kauffman, H.E., Reddy, A.P.K., Hsieh, S.P.Y., Merca, S.D., 1973. An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Disease Report 57, 537–541. Kilic-Ekici, O., Yuen, G.Y., 2004. Comparison of strains of Lysobacter enzymogenes and PGPR for induction of resistance against Bipolaris sorokiniana in tall fescue. Biological Control 30, 446–455. Kim, S.B., Shin, B.S., Choi, S.K., Kim, C.K., Park, S.H., 2001. Involvement of acetyl phosphate in the in vivo activation of response regulator ComA in Bacillus subtilis. FEMS Microbiology Letters 195, 179–183. Mew, T.W., 1987. Current status and future prospects of research on bacterial blight of rice. Annual Review of Phytopathology 25, 359–382.
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Biological Control 45 (2008) 288–296 www.elsevier.com/locate/ybcon
Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1 Guang-Hai Ji a,*, Lan-Fang Wei b, Yue-Qiu He a, Ya-Peng Wu a, Xue-Hui Bai a a
Key Laboratory of Agricultural Biodiversity for Plant Disease Management under the Ministry of Education, Yunnan Agricultural University, Kunming 650201, PR China b Faculty of Resource and Environment, Yunnan Agricultural University, Kunming 650201, PR China Received 17 November 2006; accepted 6 January 2008 Available online 16 January 2008
Abstract Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (108 CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Xanthomonas oryzae pv. oryzae; Rice bacterial blight; Lysobacter antibioticus; Biocontrol
1. Introduction Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae [(Ishiyama) Swings] (Xoo) is one of the most devastating diseases of rice. The bacterial pathogen infects the host-plant at the maximum tillering stage, resulting in 20–40% reduction in yields. The disease was first observed by the Japanese farmers in 1884 (Tagami and Mizukami, 1962). Since then, it has been reported in Asia, Northern Australia, Africa, and the United States (Mew, 1987; Ou, 1985). In China, BB has become endemic occurring mainly in southeast coastal plain and Yangtze River delta. Since *
Corresponding author. Fax: +86 871 5228024. E-mail address: [email protected] (G.-H. Ji).
1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.01.004
2001, annual epidemics of this disease have become common in the Yunnan Province of China (Ji et al., 2003). Various disease management practices, such as chemical control, host-plant resistance, modification of cropping systems, and biological control have been employed to reduce damage caused by BB. Chemical control and hostplant resistance, two of the most common management practices, have their limitations. Chemical pesticides harm the environment, and host-plant resistance, which is based on a single gene, may not be durable in the field leading to frequent resistance breakdowns. It is imperative to develop environmental-friendly and sustainable control strategies. Biological control is an ecology-conscious, cost-effective, and sustainable alternative method in BB management. This approach can also be integrated with other manage-
G.-H. Ji et al. / Biological Control 45 (2008) 288–296
ment practices to afford greater levels of protection and sustain rice yields. Antagonistic bacteria are considered as ideal biological control agents with obvious advantages. They are easy to handle, grow rapidly, and colonize the rhizosphere aggressively (Weller, 1988). Certain strains of Bacillus spp. and Pseudomonas spp., have been used as biocontrol agents to suppress rice BB (Vasudevan et al., 2002). A novel plant growth-promoting strain of Delftia tsuruhatensis HR4 has been shown to be a promising biocontrol agent against BB (Han et al., 2005). Lysobacter sp. (Christensen and Cook), a Gram-negative bacterium with gliding motility has long been studied for its ability to suppress plant pathogens or otherwise promote plant growth (Christensen and Cook, 1978). L. enzymogens strain C3 suppressed a number of plant diseases incited by fungal pathogens, including Bipolaris sorokintana, Fusarium graminearum, and Rhizoctonia solani, in field experiments (Giesler and Yuen, 1998; Zhang and Yuen, 1999; Yuen and Zhang, 2001; Jochum et al., 2006). This bacteria controls fungal pathogens by various mechanisms, such as production of chitinases and b-1,3-glucanases (Zhang and Yuen, 2000b; Zhang et al., 2001; Palumbo et al., 2003), antibiotics (Zhang and Yuen, 2000a; Islam et al., 2005) or by induction of systemic resistance (Kilic-Ekici and Yuen, 2004). The antifungal activity of another strain of L. enzymogens, 3.1 T8, against Pythium aphanidermatum was attributed to a polyketide synthase (Folman et al., 2003, 2004). In this study, we investigated Lysobacter antibioticus strain 13-1, which occurs in the plant rhizosphere in China, as a potential biocontrol agent against rice BB. The objectives of this study were (i) to evaluate the efficacy of L. antibioticus strain 13-1 against BB under greenhouse and field condition, and (ii) to determine the main factors that affect the efficiency of L. antibioticus strain 13-1 in reducing the severity of rice BB. 2. Materials and methods 2.1. Isolation of bacteria Samples were collected from rhizospheres of various crops including rice, maize, potato, millet, and wheat at different locations in Yunnan Province. The rhizosphere samples were serially diluted (from 106 to 108) and plated on nutrient agar (NA) (Fang, 1998). After incubation at 28 °C for 2 days, bacterial colonies were isolated and tentatively identified. The candidate Lysobacter spp. were stored in 30% glycerol at 30 °C. 2.2. In vitro antagonistic activity assay Lysobacter antibioticus strain 13-1 was inoculated in nutrient-poor R2A medium and incubated at 25 °C for 5 days with constant shaking at 180 rpm. The resultant yield of ca. 109 CFU/mL was used for study of antagonistic activity against several plant pathogenic fungi and bacteria listed in Table 1.
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Antibacterial activity was determined by agar diffusion technique. Suspensions (10 mL) of plant pathogenic bacteria (around 109 CFU/mL) were mixed with NA (100 mL) prior to pouring into plates. After solidification, 2 lL suspensions of L. antibioticus strain 13-1 was plated on the agar surface and incubated at 27 ± 1 °C for 48 h. Antibacterial activity was measured as the diameter of inhibition zone around the colony of strain 13-1 (Monterio et al., 2005). The inhibition of mycelial growth of a range of the pathogenic fungi by strain 13-1 was measured in vitro (Table 1). Three 2 lL drops of strain 13-1 suspensions were placed equidistant at the margins of potato dextrose agar (PDA) (BioLab) plates and incubated at 27 ± 1 °C for 24 h. A 5mm diameter block of phytopathogenic fungi from fresh PDA culture was inoculated in the center of the PDA plate and incubated at 27 ± 1 °C. After one week, zones of inhibition of mycelial growth around colonies of strain 13-1 were scored (Folman et al., 2003). 2.3. Greenhouse experiments Strain 13-1 was inoculated in R2A medium and incubated at 25 °C for 5 days with constant shaking at 180 rpm yielding ca. 109 CFU/mL. The BB pathogen, Xoo strain 53 was cultured on NA for 3 days at 28 °C. The bacterial cells were collected from plates and suspended in distilled water, and cell density was determined using a spectrophotometer. A BB susceptible rice cultivar Huangkenuo was used as the host-plant. It was planted in 15 cm 30 cm pots, four plants in each pot, and each treatment was replicated three times. Strain 13-1 whole broth culture (WBC) was used as the standard treatment, and the distilled water as the control. At the maximum tillering stage, Plants were sprayed with either WBC or water using laboratory atomizer. On the 3rd day post inoculation, the treated pots were inoculated with BB pathogen by spraying leaves with cell suspensions (around 109 CFU/mL). Treated pots were kept at constant humidity moisture and temperature for 2 days and then returned to the greenhouse bench. The pots were placed in a randomized complete block design and the experiment was repeated three times. The suppression effect of WBC and various components of strain 13-1 culture on rice BB were also studied. The WBC was separated into cellular and supernatant fractions by centrifuging suspensions at 12,000 rpm for 5 min. The supernatant was further filtered through 0.22 lm filter, and the filtrate was designated as cell-free culture fluid (CFC). The cells were washed three times with distilled water, and re-suspended in sterile distilled water, designated as cells re-suspended in water (CRW). WBC was also inactivated by heating at 70 °C for 30 min, and designated as heated whole culture (HWC). Furthermore, disease reduction of WBC, CCF, and CRW was also evaluated against strain Y14, a non-sensitive strain of BB pathogen
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Table 1 Antagonistic activity of strain 13-1 against various plant pathogenic bacteria and fungi tested in plate assays Strain code
Diameter of inhibition zone (mm)1
Host-plants
14–25
Rice
Xathomonas oryzae pv. oryzicola Xanthomonas axonopodis pv. dieffenbachiae Xanthomonas campestris pv. campestris
53,X13,Y4,A10,Q6-3,9,X6,Y6,57,Q15-1,Q15-2,A1, 63,Y7,Y9,QH3,79,Y8,X5,3-1,39,JL18,X9,Q7-2,, A3,Q8-5,26,30,GD38,Yuan2,Q8-3,5 Y14, X7, A2, A6, 13, 51 Tb23 CX10 T5 My9 ECC2 Wm2 Rs68 XCD-S 8004
0.0 15.6 ± 0.2 11.5 ± 0.2 11.4 ± 0.1 5.0 ± 0.2 7.0 ± 0.5 4.6 ± 0.3 7.4 ± 0.7 20.7 ± 1.3 9.7 ± 0.9
Rice Tobacco Tobacco Oriential tobacco Konnyaku Cabbage Zantedeschia Rice Anthurium Cabbage
Fungi Pyricularia grisea (Cooke) Sacc. Pythium sp. Fusarium orysporum Schlf. sp. Vanillae (Tucker) Gordon F. solani (Mart.) Appel & Wollenw. emend Snyd. & Hans Exserohilum turcicum (pass.) Leonard & Suggs Bipolaris maydis (Nishik & Miyabe) shoem Phytophthora nicotianae Alternaria alternate (Fries) Keissler
SQ1 F1 LH5 DB XB 97007, 97008 CX
4.3 ± 0.2 6.5 ± 0.8 7.5 ± 0.5 6.7 ± 0.4 15.7 ± 0.6 12.0 ± 0.3 28.7 ± 0.2 11.5 ± 0.7
Rice Panax notoginseng Vanilla Aloe barbadensis Maize Maize Tobacco Tobacco
Pathogen
Bacteria Xanthomonas oryzae pv. oryzae
Xanthomonas oryzae pv. oryzae Ralstonia solanacearum P. syringae pv. tabaci P. syringae pv. tabaci Pectobacterium carotovorum spp. carotovorum
1
Note: The inhibition zone is given in mm (± standard deviation).
to antibiotic inhibition by strain 13-1. In the second set of experiments, biocontrol efficacy of WBC on Xoo was evaluated with serial dilutions, ranging from 1/5 to 1/1000. The original WBC and distilled water were the controls. A third set of experiments was designed to examine the effects of timing of application of strain 13-1 on biocontrol efficacy against the BB pathogen. WBC was applied once to rice leaves at maximum tillering stage 1, 3, or 7 days prior to inoculation with Xoo strain 53. Efficacy was compared with a water control applied one day prior to inoculation of pathogen. Each of these three experiments was repeated two or three times, and were arranged in a completely randomized design (CRD). Five replicates were used per treatment, each replicate containing four rice plants. The fourth experiment was performed in the greenhouse for evaluation of the biocontrol efficacies of WBC of strain 13-1 on 18 different rice cultivars listed in Table 4. Rice cultivars were evaluated for their resistance to Xoo strain 53. In the field, plants were transplanted into experimental plots, and managed using standard cultural practices. At the maximum tillering stage, 10–15 leaves of each cultivar were clip-inoculated with a cell suspension of strain 53 (108 CFU/mL) and lesion length and whole leaf lengths of the 10 inoculated leaves were measured 14 days after inoculation following the method of Kauffman et al. (1973). Data sets of measured lesion lengths from 10 observations were used for analysis. Plants were characterized as resistant or susceptible based on the average lesion length/ leaf lengths (LL) with LL 6 0.25 cm indicating resistant
(R); 0.25–0.50 cm indicating moderately susceptible (MS), and LL P 0.5 cm indicating susceptible (S) cultivars (Fang, 1998). The rice cultivars were grown in a greenhouse as previously described for cultivar Huangkenuo. Huangkenuo was used as the ‘‘susceptible control” to confirm that conditions were favorable for rice BB development. All cultivars were submitted to the treatment with WBC and the water control. The assays were performed in a randomized complete block design with three replicates per treatment. Nine rice plants were used in each treatment plot. Each cultivar was tested twice. 2.4. Field experiments Field trials were conducted in two consecutive consecutive years, 2005 and 2006, at Honghe Agricultural Research Station, Mengzi County in Yunnan Province. Rice cultivar Huangkenuo was used. There were three applied treatments: (1) 5-day-old broth culture of strain 13-1 filtered through cheese cloth, and diluted 100 times with water; (2) 1% Zhongshengmycin; and (3) water check. Each treatment was applied to four plots (4 m 5 m), arranged in randomized complete block design. A single application of each treatment was made at the maximum tillering stage of rice plants. Plots were sprayed with the WBC of strain 13-1 (108 CFU/mL) at a rate of 265 L/ha commonly used for bactericide evaluation against BB. Suspensions (108 CFU/mL) of Xoo strain 53 were sprayed onto rice plants 3 days after application of treatments.
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A field experiment with identical treatments was also repeated in Yuanmou County, Yunnan Province in 2006. Rice cultivar Hexi No.41 was used. There were six replicate plots per treatment arranged in a randomized complete block design. All other procedures were same as those in trials conducted in Mengzi County in 2005 and 2006, except that pathogen was sprayed 1 day and 3 days after application of treatment at the booting stage. 2.5. Scoring of BB disease and evaluation of disease control by strain 13-1 Development of BB lesions was scored 14 days or 21 days after pathogen inoculation. The diseased lesion area and the whole leaf area were measured. And scoring was done based on the average area of diseased lesion/the area of whole leaves (L/W). A disease scale of 0–9 was used, with 0 indicating no infection of leaves; 1, L/W < 0.2; 3, L/W = 0.25; 5, L/W = 0.5; 7, L/W = 0.75; and 9 indicating complete death of leaves. A disease index (DI) was calculated using the formula, DI = 100 sum of individual scores/total leaves observed maximum score. The extent of disease reduction attributed to each treatment was calculated using the following formula: disease suppression efficiency = ([disease index of control disease index of treatment group]/disease index of control) 100%. 2.6. Colonization of strain 13-1 on plant tissue In order to monitor variations in the population of strain 13-1, a Rifampicin-resistant strain of 13-1 (Rifr) was used in the field experiments. Four leaves or stems from each replicate plot were collected at 16 h and intervals of 1, 3, 5, 7, 9, 11, 13, 15, 17, 21 days after treatment. The plant parts were homogenized with a tissue homogenizer in 10 mL sterile phosphate buffer. The extract was used for dilution plating. Colonies of strain 13-1 were counted on R2A agar plates amended with rifampicin and cycloheximide at 100 mg/L. Population levels of strain 13-1 were expressed as: log10 CFU/g fresh weight of leaves and stems. 2.7. Data analysis Analysis of variance (ANOVA) for disease index and suppression efficacy was performed for all data using general linear model procedures of the Statistical Analysis System (SAS institute, version 6, Cary, NC, USA). Mean comparisons were conducted using a least significant difference (Fisher’s LSD) test (P = 0.05 or P = 0.01). Standard error and LSD results were recorded. 3. Results 3.1. Antagonistic activity
16S rDNA sequence (data not shown), and was designated as strain 13-1 of L. antibioticus. Strain 13-1 showed significant antagonistic activity against numerous different plant pathogens, including bacterial pathogens Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas axonopodis, and Xanthomonas campestris, and fungal pathogens Alternaria alternata, Phytophthora nicotiana, Exherohilum turcicum, and Bipolaris maydis (Table 1). Moreover, strain 13-1 inhibited activity of 32 out of 38 isolates of the rice BB pathogen, Xoo. Strain 13-1 was the most highly antagonistic isolate of the 1000 bacterial isolates screened (data not shown). 3.2. Control of rice BB by strain 13-1 under greenhouse conditions 3.2.1. Disease suppression efficacy of different culture components of strain 13-1 All of the culture components of strain 13-1 tested had an adverse effect on development of BB disease in rice (Table 2). The WBC provided the highest disease control efficacy, 69.7%, followed by CCF and CRF, between which there was no significant difference. Heating the whole broth culture (HWC) significantly reduced its inhibitory effect against BB pathogen, but it still provided substantial reduction in development of BB disease relative to the control. Efficacy of WBC and its components differed for two pathogen strains with varying sensitivity to antibiotic inhibition by strain 13-1. When the treatment was used against the antibiotic-sensitive strain 53, all components of strain 13-1, WBC, CRW, CCF, and HWC, significantly reduced rice BB incidence and disease severity compared to the water control, with suppression efficiencies of 48–58% (Table 3). Against non-sensitive strain Y14, all culture treatments still reduced incidence and severity of BB, but to a lesser degree, with suppression efficiencies of 17–32%. 3.2.2. Effect of timing of application of strain 13-1 cultures Applications of 13-1 WBC 1, 3, or 7 days prior to pathogen inoculation, all reduced disease incidence and index of rice BB, with suppression efficiencies of 49.9%, 57.6%, and 31.7%, respectively, relative to the control (Table 4). The Table 2 Effects of culture of Lysobacter antibiotus strain 13-1 and its components in controlling rice bacterial blight in greenhouse experiments Treatment
Disease index (%)
Water Whole 13-1 broth culture (WBC) Cells re-suspended in water (CRW) Cell-free culture fluid (CCF) Heated whole culture (HWC)
51.4 ± 2.9 15.6 ± 1.3 18.5 ± 1.1 19.8 ± 1.5 25.9 ± 0.1
1
A bacterial strain was identified as L. antibioticus based on its physiological and biochemical characteristics, and
291
Suppression efficacy (%) a1 d cd c b
— 69.7 ± 2.6 64.1 ± 2.0 61.6 ± 2.9 49.6 ± 0.1
a b b c
Means within columns followed by the same letter within a column are not significantly different (P = 0.05) by Fisher’s Least Significant Difference (LSD) test.
292
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Table 3 Evaluation of the effects of L. antibioticus strain 13-1 and different culture components for suppression of sensitive strain 53 and non-sensitive strain Y14 of the bacterial blight pathogen Treatment strain 13-1
1
WBC CRW CCF Water 1 2
Diameter of inhibition of BB strain (mm)
Disease incidence (%)
Disease index (%)
Suppression efficacy (%)
53
Y14
53
Y14
53
Y14
53
Y14
0 0 0 0
50 ± 2.5 c 55.7 ± 1.5 b 57.1 ± 1.8 b 89.4 ± 2.3 a
74.3 ± 5.0 62.6 ± 7.3 72.3 ± 5.3 89.4 ± 1.5
14.6 ± 0.9 b 16 ± 1.8 b 18.2 ± 1.9 b 35.2 ± 2.9 a
25.6 ± 5.1 23.8 ± 3.7 29.1 ± 7.3 35.2 ± 5.0
58.5 ± 2.6 a 54.5 ± 5.1 a 48.3 ± 5.5 a —
27.3 ± 3.5 a 32.4 ± 7.1 a 17.3 ± 2.2 b —
2.5 ± 0.01 b 2.5 ± 0.02 b 2.6 ± 0.01 a 0
2
b b b a
c d b a
WBC, whole 13-1 broth culture; CRW, 13-1 cells re-suspended in water; CCF, 13-1 cell-free culture fluid. Means within columns followed by the same letter within a column are not significantly different (P = 0.05).
optimum time of application for strain 13-1 was 3 days prior to pathogen inoculation. Disease index (100%)
70.0
3.2.3. Suppression of rice BB by varying concentrations of 13-1 whole broth culture Dilutions of the whole broth bacterial culture had only a marginal effect on biocontrol efficacy in greenhouse experiments. All serial dilutions, ranging from 5-fold up to 1000fold significantly reduced the BB disease index compared to the control, and disease suppression was comparable to that of the undiluted WBC (Fig. 1). However, DI did increase somewhat from 12% for WBC to 20% for the 5-fold to 100-fold dilutions, and 30% for the 1000-fold dilution treatment. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments (Fig. 1).
60.0 50.0 40.0 30.0 20.0 10.0 0.0 1
2
3
4
5
6
7
Dilution factor
Fig. 1. Effects of dilutions of broth cultures of L. antibioticus strain 13-1 on the disease index of rice bacterial blight in a greenhouse experiment (1, undiluted; 2, 1/5; 3, 1/10; 4, 1/50; 5, 1/100; 6, 1/1000; 7, control).
disease control was 37.8%. These results suggest that biocontrol efficacy of strain 13-1 was somewhat affected by the genetic variability of different rice cultivars, but efficacy was generally not related to the susceptibility of the cultivar to Xoo. Overall, Suppression efficacy of BB by 13-1 was similar on both the susceptible and moderately susceptible cultivars.
3.2.4. Influence of rice cultivar on biocontrol efficacy Biocontrol efficacy of strain 13-1 varied significantly among different rice cultivars, with disease suppression ranging from 0.6% to 62.8% across the 18 cultivars (Table 5). Of the 18 rice cultivars, five cultivars were classified as moderately susceptible to the Xoo strain 53, and all the others were considered susceptible. The greatest efficacy was achieved with susceptible cultivars II-You725, Huangkkenuo, and IRBB5, and moderately susceptible Yunhui290 (47–63% BB suppression). However, strain 13-1 was not effective in reducing disease on six rice cultivars (<10% BB suppression), including susceptible cultivars Hongyou No.3, X-21, IRBB1, and IRBB3, and moderately susceptible Hongyou No. 6 and Diantun 502 (Table 5). Average BB suppression across the 12 cultivars showing effective
3.3. Field experiments In Mengzhi County field trials, strain 13-1 reduced rice BB disease index by 78.3% and 59.1% in 2005 and 2006, respectively, relative to the water control (Table 6). This was better than the control provided by Zhongshengmycin, with suppression efficacies of 69.4% and 47.7% in 2005 and 2006, respectively. In Yuanmou County in 2006, after two inoculations with the BB pathogen, the untreated plants
Table 4 Suppression of rice bacterial blight in the greenhouse by culture of Lysobacter antibiotus 13-1 applied at different times relative to pathogen inoculation Treatment Distilled water 13-1 WBC 13-1 WBC 13-1 WBC 1 2
Time of application1 1 1 3 7
% Infected leaves 63.0 ± 9.0 42.9 ± 5.3 36.3 ± 2.5 47.4 ± 0.9
2
a bc c b
Disease index
Disease suppression efficacy (%)
38.5 ± 8.0 20.8 ± 2.9 16.3 ± 1.7 26.3 ± 1.3
— 45.9 ± 3.1 b 57.6 ± 1.9 a 31.7 ± 2.0 c
a b c b
Days before pathogen inoculation. Means within columns followed by the same letter are not significantly different (P = 0.05) according to Fisher’s LSD test.
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293
Table 5 Evaluation of Lysobacter antibiotus strain 13-1 in biological suppression of rice bacterial blight on different rice cultivars in greenhouse trials Cultivar
BB susceptible level
Disease incidence (%)
Disease index (%)
BB suppression (%)
Strain 53
Treatment
Control
Treatment
Control
IR-24 IR64 Huangkenuo Zhinuo Yunhui290 Zhong413 Zhong419 II You725 Fenjin Xiaohua Nuo IRBB5 Hongyou No.3 Hongyou No.5 Hongyou No.6 Diantun 502 X-21 IRBB1 IRBB3
S S S S MS MS MS S S MS S S S MS MS S S S
32.9 ± 4.1 23.7 ± 3.5 43.5 ± 2.8 63.2 ± 3.9 41.0 ± 2.9 55.3 ± 5.3 52.6 ± 9.9 57.1 ± 7.2 83.1 ± 1.4 55.4 ± 3.2 41.8 ± 2.1 72.4 ± 3.1 53.4 ± 4.6 68.9 ± 4.8 26.34 ± 5.8 68.3 ± 2.0 33.3 ± 2.0 47.2 ± 4.8
53.5 ± 2.8 43.2 ± 4.9 58.9 ± 6.3 69.8 ± 8.1 53.6 ± 5.8 62.1 ± 2.2 52.9 ± 3.4 55.3 ± 4.6 80.3 ± 9.1 60.2 ± 7.7 58.1 ± 6.3 68.8 ± 5.4 63.6 ± 7.6 72.7 ± 5.8 32.1 ± 6.8 69.9 ± 7.8 27.7 ± 2.1 40.8 ± 5.1
15.6 ± 3.7**1 13.2 ± 2.0** 15.3 ± 3.2** 27.1 ± 5.5** 14.7 ± 1.8** 14.6 ± 2.0** 11.5 ± 2.0** 14.5 ± 2.2** 24.3 ± 3.2** 16.5 ± 3.8** 14.5 ± 2.0** 26.5 ± 3.3 ns 28.8 ± 2.1** 28.2 ± 2.0 ns 17.5 ± 3.3* 28.8 ± 2.0 ns 10.8 ± 2.4 ns 17.8 ± 2.0 ns
27.6 ± 1.1 19.8 ± 3.2 38.8 ± 1.2 32.2 ± 2.2 26.2 ± 1.2 21.5 ± 1.4 15.9 ± 2.0 39.0 ± 2.5 42.1 ± 4.1 23.0 ± 2.4 27.4 ± 3.3 28.0 ± 1.1 32.5 ± 4.4 30.1 ± 2.4 19.1 ± 1.2 29.0 ± 3.0 11.9 ± 1.4 18.6 ± 2.5
43.5 ± 5.2 33.3 ± 3.8 60.6 ± 4.1 15.8 ± 5.2 48.9 ± 1.0 32.1 ± 7.3 27.7 ± 4.0 62.8 ± 2.5 42.2 ± 4.9 28.2 ± 1.1 47.1 ± 3.1 5.4 ± 0.1 11.4 ± 0.4 6.3 ± 0.3 8.5 ± 0.2 0.6 ± 0.1 9.2 ± 1.1 4.3 ± 1.4
Note: Experiments were performed in a screen house at the agricultural research station, YAU. Kunming, Yunnan Province, China. BB, bacterial blight. Reduction in disease index: **, significant at the 1% level between treatment and the control; *, significant at the 5% level between treatment and the control; ns, not significant. 1 Each value is a mean of 30 observations.
showed severe BB disease symptoms with long and spreading lesions, while plants treated with 13-1 strain were relatively healthy with small lesions (Table 6). Strain 13-1 and 1% Zhongshengmycin both reduced disease incidence and disease index compared to the control in these field experiments, but strain 13-1 was more effective than Zhongshengmycin, with a suppression efficacy of 73.5% vs. 34.9% for Zhonshengmycin (see Table 7). Strain 13-1 was able to colonize not only on rice leaf, but also on rice stems three weeks after application. The survival level of strain 13-1 from treated rice leaves and stems, however, declined gradually after treatment (Fig. 2). 4. Discussion Some strains of Lysobacter spp., such as 3.1T8, SB-K88, and C3, had been reported to be effective in controlling both soilborne and foliar diseases (Folman et al., 2004; Islam et al., 2005; Jochum et al., 2006). In the present work, antagonistic effects of L. antibioticus strain 13-1 against rice
BB were studied under laboratory, greenhouse, and field conditions. And then, disease suppression efficacy of different individual culture components, different concentrations, techniques and timing of application, as well as the influence of rice cultivar on biocontrol efficacy, were also extensively evaluated. The results suggested the potential of L. antibioticus strain 13-1 to be developed as a promising commercial biological control agent in the future. The whole bacterial broth culture of L. antibioticus strain 13-1 was the most effective form for control of rice BB. However, the heated bacterial broth culture was almost as effective as the living 13-1 cells, suggesting that the primary antagonistic component was heat-stable. In further analysis of the biocontrol factors of strain 13-1 potentially responsible for the inhibitory role against BB pathogen, culture supernatant of strain 13-1 was extracted with excess ethyl acetate and concentrated in vacuum. Thin-layer chromatography with silica gel plate was used to further extract the main active compounds from all fractions towards Xoo. In Agar (NA) well diffusion assays, the
Table 6 Efficacy of broth culture of L. antibioticus 13-1 for control of rice bacterial blight in 2005 and 2006 field on ‘‘Huangkenuo” rice cultivars at Mengzhi county of Yunnan province Treatment
L. antibioticus 13-1 1% Zhongshengmycin Water 1
2005
2006
Incidence (%)
Disease index (%)
Biocontrol efficacy (%)
Disease incidence (%)
Disease index (%)
Suppression efficacy (%)
28.0 ± 2.8 c1 40.6 ± 3.7 b 76.1 ± 9.2 a
7.6 ± 1.1 c 10.7 ± 2.1 b 34.7 ± 8.7 a
78.3 ± 2.3 a 69.4 ± 3.1 b —
34.0 ± 5.2 b 55.7 ± 6.8 a 63.0 ± 8.0 a
15.8 ± 4.3 c 22.1 ± 2.2 b 38.6 ± 5.22 a
59.1 ± 5.1 a 42.7 ± 3.4 b
Means followed by the same letter within a column are not significantly different (P = 0.05) according to Fisher’s LSD test.
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Table 7 Evaluation of strain 13-1 in the biological control of BB in rice cultivar II You 725 at Yuanmu County of Yunnan Province (2006) Treatment
Incidence (%) 1
Lysobacter antibiotus 13-1 1% Zhongshengmycin Water-treated control
Biocontrol efficacy (%)
22 ± 6.3 c 54 ± 5.5 b 83 ± 7.8 a
73.5 ± 8.1 a 34.9 ± 3.6 b —
Means followed by the same letter within a column are not significantly different (P = 0.05) according to Fisher’s LSD test.
6 5
2006 2005 2006 2005
4 3 2
leaf leaf stem stem
21d
18d
15d
9d
11d
7d
5d
3d
2d
0
1d
1 16h
13-1 cell density (Log CFU/g)
1
24.2 ± 3.7 c 46.6 ± 7.5 b 62.2 ± 10.1 a
Index (%)
Incubation time
Fig. 2. Population levels of L. antibioticus strain 13-1 detected on rice leaves and stems in 2005 and 2006 field experiments on rice cultivar ‘‘Huangkenuo”.
five fractions inhibited the growth of rice BB pathogen, whereas in control plates containing acetone, there was no inhibition (unpublished data). Interestingly, in greenhouse trials, when strain 13-1 was used in conjunction with BB pathogen strain Y14, which was insensitive to strain 131 in the previous inhibitory zone tests, disease suppression decreased greatly compared to results with a more sensitive pathogen isolate. This further supports the role of antibiotics in the suppression of BB by strain 13-1. Although the mechanism of action of strain 13-1 has not been definitively determined, the results suggest the active compound responsible for the inhibitory role against the BB pathogen mainly comes from the culture supernatant of strain 13-1, is heat-stable, and can provide protection against BB when applied in highly diluted form or up to one week prior to pathogen inoculation. In greenhouse and field assays, the amount of strain 13-1 inoculum presented on rice leaves differed greatly between 2005 and 2006, as indicated by the recovery of this strain from rice leaves (log 5.5 CFU/g in 2005 and log 4 CFU/g in 2006). It is questionable whether the densities of the biocontrol strain were sufficient to be effective in 2006. For many biocontrol bacterial species, it has been shown that a certain threshold population size must be reached before pathogen populations can be suppressed (Dunny and Winans, 1988). Pseudomonas fluorescens was only effective at densities of log 5–6 CFU/g root (Raaijmakers et al., 1999). Moreover, concentrations of antimicrobial compounds produced by the antagonistic bacteria in situ may be too low at the site of pathogen infection, and antimicrobial activity of the inhibitory compounds was not sufficient to inhibit pathogen populations (Folman et al., 2004; Kim et al., 2001). Moreover, populations of most biocontrol
agents introduced into the environment decline with time, which affects the synthesis of inhibitory compounds (Acea et al., 1988). In our study, population level of strain 13-1 on treated rice leaves gradually declined with time after treatment (Fig. 2). Therefore, population size, inhibitory compounds produced, and their activity in situ on rice leaves may have contributed to the difference in biocontrol efficacy of strain 13-1 in 2005 and 2006. Application methods of the bacterial biocontrol agents and inoculation methods of Xoo may also affect biocontrol against rice BB. In our experiment, strain 13-1 and the pathogen Xoo strain 53 were applied by foliar spray, and provided significant control of BB under field conditions, with mean reduction in disease severity of 70%. This reduction was greater than that provided by other bacterial biocontrol agents, such as P. fluorescens, P. putida, and Bacillus spp. (producing maximum reductions of 64%, 57%, and 60%, respectively) in previous published reports (Sivamani et al., 1987; Gnanamanickam et al., 1999; Johri et al., 2003; Vasudevan et al., 2002; Velusamy et al., 2006). These reports also demonstrated that application of the individual biocontrol agents by seed-soaking, root-dipping, or foliar spraying was not as effective in reducing BB severity compared with application of all three methods together. Our studies revealed that foliar spraying of strain 13-1 on rice once or twice followed by inoculation with Xoo strain 53 resulted in greater biocontrol efficacy against BB. So, the application method of strain 13-1 for suppressing BB in our research was apparently more efficient and practical than that in some earlier works. However, in our research, when the pathogen Xoo strain 53 was inoculated by the leaf clipping method, there was no obvious reduction in lesion length compared with that in untreated control plants (data not shown), which is not in agreement with other previous reports (Velusamy et al., 2006). Therefore, future studies should establish a greater understanding of the effects of different application methods of strain 13-1 on its biocontrol efficacy, and optimize its application methods and timing for practical utilization in the field. Deployment of resistant cultivars is, generally, the most economical strategy to control BB, but has only been partially successful due to the development of race variation in the pathogen that overcomes resistance (Prashant et al., 2006). In our studies, rice cultivars with differing genetic backgrounds and levels of resistance were evaluated for their effects on biocontrol efficacy. The disease resistance levels of the 18 rice cultivars tested were found to be mod-
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erately susceptible or susceptible to strain 53. However, among these, IRBB5, Hongyou Nos. 3, 5, and 6, Diantun 502, and Yunhui 290 are known to be resistant to other specific races of Xoo (Qian et al., 2003). In our tests, strain 13-1 resulted in comparable suppression of disease on most cultivars, regardless of their susceptibility to strain 53. However, since strain 13-1 was not effective on some cultivars, and these included cultivars with known resistance to other races, such as, Hongyou Nos. 3 and 6, and Diantun 502, there may be some relationship between disease susceptibility and biocontrol efficacy with some resistance factors. A similar phenomenon was found by Jochum et al. (2006), when L. enzymogenes C3 applied to different wheat cultivars in the greenhouse was effective in reducing the severity of Fusarium head blight in six wheat cultivars, but was not effective in two other cultivars; Biocontrol effects were also not directly related to reported levels of resistance/susceptibility to FHB. Strain 13-1 was highly effective on resistant cultivars Yunhui290 and IRBB5, so any relationship between biocontrol efficacy and cultivar resistance is not clear at this point and requires further investigation. Leaf surface structure, such as hydathodes and the other mesophyll tissue of different rice cultivars, might have affected colonization of strain 13-1 on the leaf surface and its biocontrol efficacy on different cultivars, Recently, Islam et al. (2005) reported that characteristic colonization of Lysobacter sp. strain SB-K88 on both rhizoplane and phylloplane of sugar beet were different to that of other plants such as spinach and tomato. For example, SB-K88 densely colonized both the root and cotyledon surface and a semitransparent bacterial film was formed on the root surface of sugar beet, but not on the root surface of spinach and the other plant species (Islam et al., 2005). Good biocontrol agents must either be effective in reducing the initial inoculum of pathogen population, or be active and persistent in suppressing disease development. In our study, strain 13-1 effectively protected rice leaves against BB during the initial stages of BB epidemics whether they occurred naturally or by inoculating with a highly virulent form of Xoo pathogen (strain 53). Further research on 13-1 as a potential biocontrol agent to BB is needed. One suggestion is to integrate the use of different biological control agents with distinct modes of action. Strain 13-1 may be a good candidate for use in a mixture of antagonistic microorganisms. Another suggestion is to formulate commercial biocontrol agents with compounds that would enhance growth or activity of the biocontrol agents when applied to plant surfaces. A third suggestion is to integrate biological controls with chemical bactericides, improve spray application technologies or develop new delivery technologies specific for microorganisms. In addition, future studies should evaluate the integration of two major approaches by combining host resistance and biocontrol for management of BB, and understanding their mechanisms for BB management.
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