Nguyen Thi Thu Nga, Department of Plant Protection, College of Agriculture and Applied Biology, Can Tho University, Can Tho City, Vietnam. E-mail: email@example.com
Aims: To identify rhizobacteria from the Mekong Delta of Vietnam, which can systemically protect watermelon against Didymella bryoniae and elucidate the mechanisms involved in the protection conferred by isolate Pseudomonas aeruginosa 231-1.
Methods and Results: Bacteria were isolated from watermelon roots and their antagonistic ability tested in vitro. Of 190 strains, 68 were able to inhibit D. bryoniae by production of antibiotics. Four strains were able to reduce foliar infection by D. bryoniae when applied to watermelon seeds before sowing. Strain Ps. aeruginosa 231-1 was chosen for investigations of the mechanisms involved in protection and ability to control disease under field conditions. In the field, the bacterium was able to significantly reduce disease in two consecutive seasons and increase yield. Furthermore, it colonized watermelon plants endophytically, with higher numbers in plants infected by D. bryoniae than in noninoculated plants. To elucidate the mechanisms involved in protection, the infection biology of the pathogen was studied in bacterially treated and control plants. Pseudomonas aeruginosa 231-1 treatment inhibited pathogen penetration and this was associated with hydrogen peroxide accumulation, increased peroxidase activity and occurrence of new peroxidase isoforms, thus indicating that resistance was induced.
Conclusions: The endophytic bacterium Ps. aeruginosa 231-1 can control D. bryoniae in watermelon by antibiosis and induced resistance under greenhouse and field conditions.
Significance and Impact of the Study: These findings suggest that rhizobacteria from native soils in Vietnam can be used to control gummy stem blight of watermelon through various mechanisms including induction of resistance.
Gummy stem blight, caused by Didymella bryoniae (Auersw.) Rehm., is a very destructive disease on watermelon [Citrullus lanatus (Thunb.) Matssumura and Nakai] and other species in Cucurbitaceae (Sherf and MacNab 1986). Under favourable conditions such as high humidity and a warm climate, the disease can cause serious losses in the field as well as after harvest (Sherf and MacNab 1986). No highly resistant cultivars are available (Gusmini et al. 2005), so the main disease control method is application of fungicides (Keinath 2001; Trung et al. 2005). However, resistance to fungicides in the pathogen has been observed (Keinath and Zitter 1998). To reduce the usage of pesticides, biological control is an environmentally friendly method with a large potential to control diseases (Vallad and Goodman 2004). Plant growth–promoting rhizobacteria (PGPR), which are a group of bacteria living freely in the rhizosphere, are promising organisms for control of phytopathogens (Siddiqui 2006). Besides promoting plant growth by fixing nitrogen, solubilizing phosphorus and producing phytohormones etc. (Tien et al. 1979; Pandey and Palni 1998; Thakuria et al. 2004), PGPR can directly suppress phytopathogens by producing substances or antibiotics toxic to the pathogens or by producing enzymes lysing the fungal cell walls such as chitinases and β-1,3-glucanases (Siddiqui 2006). Furthermore, they may induce resistance in the host against the pathogens (Liu et al. 1995a,b; Siddiqui 2006) and indeed, there have been several studies on induced systemic resistance by PGPR (e.g. Van Peer et al. 1991; Liu et al. 1995a,b; Van Loon 2007).
The aim of the present study was to explore the prospects for controlling gummy stem blight by seed treatment combined with soil drenching of PGPR isolated from the rhizosphere of watermelon under controlled and field conditions. Furthermore, we investigated the mechanisms of disease protection by the most efficient bacterial strain in order to clarify if the observed disease reductions involved induced resistance.
Materials and methods
Isolation and characterization of rhizobacteria
Rhizobacteria were isolated from roots of watermelon from 33 fields from four provinces in the Mekong Delta of Vietnam (i.e. Can Tho, Vinh Long, Tien Giang and Tra Vinh). Well-growing, nearly 1-month-old plants were chosen for isolation of bacteria. The plants were dug out and their root systems rinsed in tap water followed by washing with sterilized distilled water twice. Subsequently, the roots were cut into 1-cm pieces and transferred into sterilized tubes containing 10 ml distilled water. The tubes were shaken for 10 min at room temperature to release bacteria from the roots, and the bacterial suspension was spread on Luria Bertani (LB) medium (Shurtleff and Averre 1997). Series of bacterial dilutions from 101 to 103 were spread on each plate for obtaining separate bacterial colonies. The plates were incubated for 24–48 h at room temperature and from single and well-separated colonies, bacteria were transferred to tubes containing LB medium. A total of 190 bacterial strains were isolated.
Gram status of each bacterial isolate was tested by the method of Shurtleff and Averre (1997) by applying 3% KOH. For the Gram-negative bacteria, it was tested whether they belonged to the genus Pseudomonas, i.e. if they grew on the selective Gould S1 medium (Gould et al. 1985). Furthermore, the bacterial cultures were tested for fluorescence under UV light (λ = 366 nm).
For the Gram-positive bacteria, it was tested whether they belonged to the genus Bacillus using the method of Walker et al. (1998) except that the bacterial suspensions were incubated for 30 min instead of 10 min at 80°C. Each bacterial strain was cultured in a test tube containing sterile liquid LB medium with continuous shaking at 200 rev min−1 for 24 h at 28°C. Subsequently, the tubes were incubated at 80°C for 30 min, and the bacterial suspension was spread on LB plates. The plates were incubated for 24–48 h at 25°C, and if the bacteria formed colonies, they were considered to belong to the genus Bacillus.
Dual culture experiments of Didymella bryoniae and rhizobacteria
In vitro studies of the antagonistic ability of 190 strains of rhizobacteria against D. bryoniae were carried out by inoculating potato dextrose agar (Difco) plates in the middle with a 5-mm disk from a 7-day-old culture of isolate I16 of D. bryoniae (isolated from an infected watermelon leaf collected in the Can Tho province, Vietnam). One day later, the rhizobacteria were inoculated in the margin of each plate. An inoculation loop, which was dipped in a bacterial culture, was subsequently pressed lightly against the agar plate (where the pathogen was grown for 24 h) to deposit bacteria. After that, the bacteria were allowed to grow and inhibition zones observed. Two bacterial strains were cultured in each plate, and each strain was cultured at two points on opposite sides of D. bryoniae, serving as two replicates. The plates were incubated at 28°C for 4–5 days, and the diameter of the inhibition zone in D. bryoniae mycelial growth was measured for each bacterium.
Preparation of inoculum of rhizobacteria
All bacterial strains were cultured in LB broth for 24 h at 28°C with continuous shaking (300 rev min−1). Subsequently, bacterial cultures were centrifuged twice for removing growth medium and metabolites therein (4000 rev min−1 for 10 min at 25°C). The pellet of bacterial cells was re-suspended in 0·9% NaCl. The concentration of bacterial cells was determined by measuring the absorbance spectrophotometrically at λ = 600 nm. Based on a standard curve, expressing the correlation between optical density (OD600 nm) and the number of bacterial cells, the actual bacterial concentration was calculated.
Bacterial treatment and plant growth conditions
Two cultivars of watermelon were used in the experiments: the moderately resistant cv. PI189225 from USDA, ARS (United States Department of Agriculture, Agricultural Research Service), National Genetic Resources Program (originally collected from Zaire) and the susceptible cv. 232-0125/B from India. Seeds of watermelon were soaked in bacterial suspensions (108 CFU ml−1) for 45 min with continuous shaking at 75 rev min−1 at room temperature. Seeds soaked in distilled water served as controls. Subsequently, seeds were sown in pots (10 × 10 cm) containing the soil mix Pindstrup Substrate (Pindstrup Mosebrug A/S, Pindstrup, Denmark). The pots were transferred to a growth chamber with alternating cycles of 12 h light and 12 h darkness. Light was supplied by fluorescent tubes (Osram L 36W/11-860 Luminux plus Eco Daylight, 200 μE m−2 s−1; Osram GmbH, Augsburg, Germany). Temperature and relative humidity (RH) were approx. 25°C/50–60% RH and 20°C/80–90% in light and darkness, respectively. Seven days after sowing (DAS), the soil was furthermore drenched with 5 ml of bacterial suspension (5 × 108 CFU ml−1) for each seedling by pipetting. Controls were drenched with water.
Preparation of inoculum of Didymella bryoniae, inoculation and assessment of disease
Inoculum of D. bryoniae, isolate I16, was cultured on sterilized potato cubes in 100-ml conical flasks, placed in a growth chamber with cycles of 12 h light (28°C) and 12 h darkness (25°C) for 5–7 days. Spores were harvested by lightly scraping the surface of the potato cubes with a spatula and washing them with sterilized distilled water. The spore suspension was filtered through four layers of cheese-cloth, and the concentration of inoculum was determined using a haemocytometer and adjusted to 106 spores ml−1 for the moderately resistant cv. PI 189225 and 105 spores ml−1 for the susceptible cv. 232-0125/B.
When plants had the first true leaf fully developed (approx. 15–16 day-old seedlings), they were inoculated with D. bryoniae by atomizing the spore suspension onto the leaves until run-off. After inoculation, the plants were sealed in plastic bags with 100% humidity for 48 h in darkness. After 48 h, the bags were opened and the plants were placed under the normal light regime again. Disease was assessed on inoculated leaves by recording the percentage of leaf area covered with symptoms 4 days after inoculation (dai) for cv. 232-0125/B and 7 dai for cv. PI 189225.
Screening of rhizobacteria for ability to protect watermelon against foliar infection by Didymella bryoniae
A first screening of the potential disease reducing ability of bacteria (compared to treatment with water) included 33 of the 190 rhizobacterial strains chosen based on their ability to produce inhibition zones in dual culture experiments. Screening took place under greenhouse conditions using the susceptible cv. 232-0125/B and bacteria were applied by seed soaking (108 CFU ml−1). A second screening was conducted to confirm the effect of the most promising rhizobacterial strains selected from the first screening. The number of plants for each treatment in each screening was at least five. A third test included only Pseudomonas aeruginosa strain 231-1, which was selected as the most effective strain in the previous screenings. This screening was conducted using the moderately resistant cv. PI 189225 and the susceptible cv. 232-0125/B. Bacteria were applied by both seed soaking and soil drenching (108 CFU ml−1). For both cultivars, at least 16 plants were used for each treatment.
The Ps. aeruginosa 231-1 strain was selected for further studies as it gave significant disease reductions in more than five experiments.
Species identification of Pseudomonas strain 231-1 using 16S rDNA sequencing
fD1 (forward primer: 5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (reverse primer: 5′- ACGGCTACCTTGTTACGACTT-3′) (Weisburg et al. 1991) were used in a PCR amplification assay. One microlitre of bacterial culture (a 25-fold dilution of a single picked colony) together with 1·5 μl of each of the primers was run in a standard PCR reaction with an annealing temperature of 55°C for 30 cycles with 1 min at each temperature step. After amplification, the PCR product was checked by gel electrophoresis and purified using the Qiagen PCR purification kit (Qiagen, Hilden, Germany). The purified products were sequenced in both directions with the fD1 and rP2 primers using the services at Eurofins MWG Operon (Ebersberg, Germany). The obtained sequences were assembled and submitted to the Blast programme available at http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Experiments were conducted in two consecutive crops in 2008, i.e. the spring–summer (April–July) and the autumn–winter (September–November) crops at the same site for both crops in a field plot located at the experimental area of Can Tho University, Can Tho, Vietnam. The weather in the spring–summer crop is dry and hot in the beginning season, with rain starting from the middle of the season. In the autumn–winter season, rain appears throughout the season.
The experiments were designed as a completely randomized block experiment with four treatments and three replications of each treatment (plots 3·5 × 3·0 m, each with ten plants). The treatments were: (i) Seed soaking and soil drenching with bacterial suspensions (108 CFU ml−1, starting at 2 days after transplanting and then at 7 days intervals until harvest); (ii) Foliar spraying with bacterial suspensions (108 CFU ml−1) starting at 2 days after transplanting and then at 7 days intervals until harvest; (iii) Seed soaking, soil drenching and foliar spraying with bacterial suspensions (108 CFU ml−1) at same starting time and at same intervals as before; (iv) Controls (untreated seeds). Disease arose through inoculum naturally present in the field. All practical operations in the experiments such as application of fertilizers, control of insects, cutting etc. were similar between all treatments. Plants were sown in a tray (1st April for the first crop and 1st September for the second crop) containing a mixture of rice husk ash and soil. At 5–7 DAS, seedlings were transplanted into the field in rows at a distance of 45 cm in the row and 250 cm between rows. The soil was covered with mulch. Pruning took place when the plants had four true leaves, by leaving the main shoot in order to induce two subvines. Subsequent pruning was conducted regularly until near harvest time. Plants were fertilized before sowing with lime and manure compost (50 and 30 kg per 1000 m2, respectively). During plant growth, 110 kg NPK (16–16–8), 6 kg urea and 10 kg KNO3 were applied per 1000 m2. The NPK fertilizer was split in three applications at the ratio (30 : 40 : 30%) before sowing, 30 and 50 DAS, respectively. Urea was sprayed to the plants at 26 and 33 DAS, and KNO3 was applied to the soil at 58 and 65 DAS. Pollination was conducted during the main flowering stage at 30–45 DAS. Each plant was allowed to raise only one fruit. Assessment of disease was carried out once a week by recording the number of infected leaves and the total number of leaves. In the first season, scoring took place at 28, 35, 42, 49 and 56 DAS and in the second season at 19, 26, 33, 40, 47, 54 and 61 DAS. Yield of watermelons (tonnes ha−1) was recorded at harvesting time (72 DAS) in the second growth season.
Transformation of Pseudomonas aeruginosa 231-1 with gfp
A gfp delivery plasmid (pBK-miniTn7-gfp2) carrying the gfp gene and the gentamycin resistance gene (GmR) as well as a helper plasmid (pUX-BF13) were obtained from Dr Ole Nybroe, Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen. The construction of these plasmids was described by Koch et al. (2001). The plasmids were introduced into Ps. aeruginosa 231-1 by electroporation as described by Choi et al. (2006). Insertion of the gfp gene in the strain was checked by PCR using specific primers GFPF (5′-ATGGTGAGCAAGGGCGAGG-3′) and GFPR (5′-TTCTGCTGGTAGTGGTCGGC-3′) amplifying approx. a 600-bp fragment (Sarrocco et al. 2006). PCR amplification was carried out as described for amplification of the 16S rDNA mentioned earlier.
Determination of the colonization pattern of Pseudomonas aeruginosa 231-1
For all the following experiments, bacteria were applied to the plants by both seed soaking and soil drenching as before (108 CFU ml−1). Experiments with two replications were performed, each replication consisting of five plants. In each replication, the five plants were pooled and the bacterial population size was determined.
A culture plate method was used to determine whether Ps. aeruginosa 231-1 colonized endophytically in the upper parts of watermelon plants. Gfp-transformed Ps. aeruginosa 231-1 was applied to cv. 232-0125/B by seed soaking and subsequently by soil drenching to 7-day-old seedlings. When the first leaves were fully expanded (15 DAS), they were either inoculated with D. bryoniae or left untreated. The population of Ps. aeruginosa 231-1 was determined in hypocotyls at 1, 3, 5, 9, 10 and 11 days after soil drenching and in the first true leaves at 9, 10 and 11 days after soil drenching (9, 10 and 11 days corresponding to 1, 2 and 3 dai, respectively). Hypocotyls and leaves were harvested 1, 2 and 3 dai in order to determine if Ps. aeruginosa 231-1 was present. Plants treated with bacteria, but not inoculated with the pathogen, were used as controls. At each time point, five plants were used for each of the two treatments. The method described by Wulff et al. (2003) was followed for determination of the population size of bacteria inside the plant tissue. Hypocotyl and leaf tissue were weighed, surface sterilized by rinsing in 1% NaOCl for 1 min, washed 3–4 times with sterilized distilled water and ground using a sterile mortar and pestle. Subsequently, 0·9% sterile NaCl was added to the paste (1 ml g−1 tissue), followed by filtering through four layers of sterilized cheese-cloth for removing the plant tissues. One hundred microlitre of the solution was spread on LB medium containing gentamycin (25 μg ml−1) for specifically determining the presence of Ps. aeruginosa 231-1. The number of bacteria per ml was subsequently calculated. To test if surface disinfection was completely effective, a sterility control was performed. Thus, 100 μl from the last wash was transferred to LB plates and incubated at 28°C for 48 h. If contamination was detected, the sample was discarded.
Infection biology and accumulation of H2O2 at penetration sites in leaves
Infection biology of D. bryoniae and accumulation of H2O2 was studied in the first true leaves of cv. 232-0125/B pretreated with Ps. aeruginosa 231-1 (seed treatment and soil drenching with 108 CFU ml−1) and control plants treated with water. All plants were inoculated with D. bryoniae. The accumulation of H2O2 was studied by staining with 3,3′-diaminobenzidine (DAB; Sigma) (Thordal-Christensen et al. 1997). Inoculated leaves were harvested at 12, 24 and 36 hai (hours after inoculation), and the petioles were immediately immersed in the DAB solution for 8 h. The leaves were cleared at 20, 32 and 44 hai using a mixture of absolute ethanol : glacial acetic acid (3 : 1 v/v) as described by Shetty et al. (2003). For easy identification of the fungal structures, leaves were stained with 0·1% Evans Blue in lactoglycerol. For each treatment and time point, four leaves were examined. At 20 and 32 hai, infection biology of D. bryoniae and accumulation of H2O2 was studied for 50 randomly germinated spores on each leaf (200 germinated spores per time point in total). For each germinated spore, it was recorded whether the spore formed appressoria, whether penetration took place from here and whether H2O2 accumulated beneath the appressoria. At 44 hai, 50 randomly selected appressoria were examined on each leaf (200 appressoria per treatment per time point) as it was not possible to distinguish from where the individual superficial hyphae originated any more. For each appressorium, it was recorded whether successful penetration took place, whether appressoria with successful penetration yielded hyphae which colonized the intercellular spaces, whether H2O2 accumulated beneath the appressoria and whether H2O2 accumulated around hyphae originating from the appressoria with successful penetration.
Peroxidase activity and native-PAGE for detection of peroxidase isozymes
Total peroxidase activity was determined in plants treated with Ps. aeruginosa 231-1 and control plants treated with water, either with or without inoculation with D. bryoniae (noninoculated plants were only studied for cv. 232-0125/B). Experiments were performed with both cvs. PI189225 and 232-0125/B and each experiment had two replications, each consisting of five to six plants. Within each replication, the plants were pooled and examined for peroxidase. Leaves were harvested at 0, 12, 24, 36, 48, 72 and 96 hai. In cv. PI189225, additional samples were harvested at 6 and 9 hai, as there was an early, high activity of peroxidase in cv. PI 189225 compared to cv. 232-0125/B (Nga 2007). Leaves were immediately frozen in liquid nitrogen and stored at −80°C. Leaf samples were ground in liquid nitrogen and proteins were extracted in 0·1 mol l−1 potassium phosphate buffer (pH 7·0). Protein concentration in the extract was determined using the method of Bradford (1976) with bovine serum albumin (Sigma) as standard.
Peroxidase activity was determined as described by Kapchina-Toteva and Yakimova (1997). The reaction mixture contained 0·25% (v/v) guaiacol (Sigma) and 10 mmol l−1 H2O2 (Sigma) in 0·1 mol l−1 potassium phosphate buffer (pH 7·0). Protein extract (5 μl) of each sample were added into wells of a microtitre plate and the reaction started by adding 255 μl of the reaction mixture. The linear increase in absorbance at 470 nm resulting from the formation of the coloured product tetraguaiacol was monitored in an ELISA reader (Spectra MAX 190; SoftMax Pro; Molecular Devices, Sunnyvale, CA) for 3 min. Peroxidase activity was calculated in μmol guaiacol dehydrogenated product (GDHP) mg−1 protein per second using the molar extinction coefficient of GDHP (ε = 26·6 (mmol l−1)−1 cm−1).
Peroxidase isozymes were determined by native-PAGE according to the method of Schrauwen (1966) following electrophoresis of 40 μg protein in a 10% polyacrylamide gel. Basic gels were used for detection and separation of acidic isoforms and acidic gels for separation of basic isoforms. To detect the different isoforms of peroxidase, the gels were stained after electrophoresis in a solution containing 10 mmol l−1 H2O2 and 0·2% (w/v) benzidine (Sigma) dissolved in 0·1% (v/v) glacial acetic acid for approx. 20 min under continuous shaking until all the isoforms appeared. The reaction was stopped by adding distilled water and the gel was washed several times for elimination of excess stain. After running the gels and studying the isozyme patterns, the same gels were stained with Coomassie Brilliant Blue to check for equal loading of protein.
Data from studies of infection biology and H2O2 accumulation as well as disease assessments under field conditions represent discrete variables since it was recorded whether or not a certain event took place (e.g. whether a germinated spore formed an appressorium and whether an appressorium caused penetration). These data were assumed to follow a binomial distribution and therefore analysed by logistic regression (corrected for overdispersion when present) (Collett 1991). For comparison of discrete variables (percentages), odds ratios (OR) (Collett 1991) were calculated using the control treatment as reference (OR = 1·00). For example, OR for per cent appressoria with successful penetration and causing further colonization at 44 hai is 0·39 (Table 4). This means that odds [P/(1 − P), in which P is the probability of an appressorium with successful penetration causing further colonization] in plants treated with Ps. aeruginosa 231-1 is approx. two and a half time less than odds for control plants treated with water.
Table 4. Comparison of the population size of Pseudomonas aeruginosa 231-1 in hypocotyls and the first true leaves of cv. 232-0125/B with and without inoculation with Didymella bryoniae*
Days after soil drenching
Days after inoculation
*Plants were treated with bacteria by seed soaking and soil drenching with a bacterial suspension to 7-day-old seedlings. When the first true leaves were fully expanded (15 day-old seedlings), these were inoculated with D. bryoniae. The hypocotyls and the first true leaves were harvested for determination of the population size of Ps. aeruginosa 231-1.
†The number in brackets indicates days after soil drenching with bacteria.
‡The bacterial population size is presented as log 10 CFU g−1 tissue.
¶P-value for comparison of the two treatments for each time point.
Without inoculation with pathogen
Inoculation with pathogen
First true leaf
Without inoculation with pathogen
Inoculation with pathogen
Data on percentages of leaf infection under controlled conditions and enzyme activities and yield under field conditions represent continuous variables and were analysed by analysis of variance assuming a normal distribution. Variances were stabilized by appropriate transformations when necessary. Means were separated by least significant difference (LSD)-values. All data were analysed by PC-SAS (release 8.2; SAS Institute, Cary, NC, USA).
All experiments were performed at least twice with similar results, and representative results from individual experiments are presented.
Isolation of rhizobacteria and in vitro screening for inhibition of Didymella bryoniae
A total of 190 strains of rhizobacteria were isolated from roots of watermelon from 33 fields in four provinces in the Mekong Delta. Of these, nine strains belonged to the genus Bacillus (4·7%), and 52 to the genus Pseudomonas (27·4%) of which 32 were found to be fluorescent (16·8%). The remaining strains were not classified to genus level (data not shown).
Screening of the antagonistic ability of the 190 strains of rhizobacteria in dual culture experiments indicated that 68 strains (35·7%) were able to inhibit the growth of D. bryoniae mycelium with inhibition zones ranging from 0·5 to 14 mm on agar plates (data not shown). A total of 17 of the 32 fluorescent Pseudomonas strains (53·1%) and two of nine Bacillus strains (22·2%) possessed antagonistic ability against D. bryoniae.
Screening of rhizobacterial strains for ability to protect watermelon against Didymella bryoniae
A total of 33 strains of rhizobacteria were chosen for further experiments, i.e. 19 fluorescent Pseudomonas strains, 3 nonfluorescent Pseudomonas strains and 5 strains of Bacillus. The remaining six were not determined to genus level. Of these, 11 strains were able to inhibit growth of D. bryoniae in dual culture experiments (data not shown). Of the 33 strains tested in the first screening, eight strains showed a significant protection of watermelon leaves from disease, the percentages protection ranging from 30 to 70% (data not shown). This group included one fluorescent strain of Pseudomonas, three nonfluorescent strains, one strain of Bacillus sp. and three unknown strains.
When the eight most promising rhizobacteria found in the first series of tests were retested, only four strains, i.e. three strains of fluorescent Pseudomonas (71, 231-1 and 232-1) and Bacillus 8, were able to protect watermelon from disease. Protection ranged between 44 and 62%, with Ps. aeruginosa 231-1 giving the highest protection (data not shown). Therefore, this strain was chosen for further studies.
The third test included only Ps. aeruginosa 231-1. Seed soaking combined with soil drenching with a bacterial suspension protected both the moderately resistant cv. PI 189225 and the susceptible cv. 232-0125/B from foliar infection caused by D. bryoniae (Table 1, Fig. 1). There was a significant reduction of leaf infection by 69·7% and 49·2% in cv. PI 189225 and cv. 232-0125/B, respectively (Table 1).
Table 1. Ability of strain Pseudomonas aeruginosa 231-1 to protect watermelon leaves from gummy stem blight in cvs. PI 189225 and 232-0125/B*
Percent infected leaf area†
*Bacteria were applied by seed soaking and soil drenching with a bacterial suspension to 7-day-old seedlings.
†Disease was assessed at 7 and 4 days after inoculation of cv. PI 189225 and cv. 232-0125/B, respectively.
Cv. PI 189225
Subsequently, more than five experiments have confirmed that application of Ps. aeruginosa 231-1 to watermelon by seed treatment and soil drenching was able to reduce infection of D. bryoniae (data not shown).
Ability of Pseudomonas aeruginosa 231-1 to control gummy stem blight in the field
In the spring-summer season (Table 2), no treatment significantly reduced disease at the first assessment (28 DAS). From the second assessment (35 DAS), the triple treatment with bacteria (seed soaking, soil drenching and foliar spraying) significantly reduced disease compared to the control. From the third assessment time, (42 DAS) all three bacterial treatments significantly reduced disease. In the autumn–winter season (Table 3), the double (seed soaking and soil drenching) and triple bacterial treatment (seed soaking, soil drenching and foliar spraying) gave significant protection against disease already at the first assessment (19 DAS), whereas the single treatment (foliar spraying) was only able to significantly reduce disease compared to the control from 47 DAS. Yield of watermelons in the autumn–winter crop was significantly increased by all treatments with bacteria, especially the triple treatment (Table 3).
Table 2. Percent watermelon leaves infected by Didymella bryoniae in the spring–summer crop (April–July, 2008)
Days after sowing
†PI, percentage of infection.
‡OR, odd ratios. ORs for comparison of the plants treated with Pseudomonas aeruginosa 231-1 and controls without bacterial treatment. In the analysis, the control was used as a reference, OR = 1·00. The number of asterisks indicates the degree of significance. NS, nonsignificant difference, ***significant at P ≤ 0·001, **significant at P ≤ 0·01, *significant at P ≤ 0·05.
Seed soaking + soil drenching with Pa 231-1
Foliar spraying with Pa 231-1
Seed soaking + soil drenching + foliar spraying with Pa 231-1
Control without bacteria
Table 3. Per cent watermelon leaves infected by Didymella bryoniae in the autumn–winter crop (September–November, 2008)
Days after sowing
Yield (tonnes ha−1)§
†PI, percentage of infection.
‡OR, odd ratios. OR for comparison of the plants treated with Pseudomonas aeruginosa 231-1 and controls without bacterial treatment. In the analysis, the control was used as a reference, OR = 1·00. The number of asterisks indicates the degree of significance. NS, nonsignificant difference, ***significant at P ≤ 0·001, **significant at P ≤ 0·01.
§Continuous variable, analysed by Analysis of Variance. P = 0·0002, LSD95 = 3·0.
Seed soaking + soil drenching with Pa 231-1
Foliar spraying with Pa 231-1
Seed soaking + soil drenching + foliar spraying with Pa 231-1
Control without bacteria
Colonization pattern of Pseudomonas aeruginosa 231-1 in watermelon
Pseudomonas aeruginosa 231-1 transformed with gfp were detected in the hypocotyls of cv. 232-0125/B plants not inoculated with D. bryoniae, at day 1 after drenching the soil with a bacterial suspension (Table 4). At 1 dai, the density was log10 2·3 and the population fluctuated between log10 1·7 and log10 3·2 during the course of the experiment (until 11 days after drenching). In the first true leaves, no bacteria could be recovered from noninoculated plants. When plants were inoculated with D. bryoniae on the first true leaf of 15-day-old seedlings (8 days after drenching the soil with bacteria), the population of Ps. aeruginosa 231-1 in the hypocotyls and first true leaves were determined at 1, 2 and 3 dai (equivalent to 9, 10 and 11 days after soil drenching). In hypocotyls, the density of bacteria increased from log10 1·4 at 1 dai to log10 4·9 at 3 dai and in the first true leaves, it increased from log10 1·2 to log10 6·4 (Table 4). All differences between inoculated and control plants were significant.
Infection biology and accumulation of H2O2
Didymella bryoniae conidia germinated with one to three germ tubes. Most often, a single appressorium formed terminally on a germ tube. Penetration took place from the appressoria and most often, this occurred directly into the lumen of an epidermal cell, and penetration also took place through stomata. After successful penetration, the invading hypha developed into thick intra- and intercellular hyphae which colonized the leaf.
Infection biology of D. bryoniae and H2O2 accumulation was compared in cv. 232-0125/B either pretreated with Ps. aeruginosa 231-1 or water (control) (Table 5). The percentage of spores forming appressoria could only be determined at 20 hai because at the later time points, it was not possible to determine from which spore the appressoria were produced. At 20 hai, there was no significant difference in appressorial formation between the two pretreatments. Fungal penetration occurred already at 20 hai in plants treated with water, but only at 32 hai in plants treated with Ps. aeruginosa 231-1. At 20 hai, the penetration frequency was significantly higher in the controls, than after the bacterial pretreatment, whereas no significant differences were seen at 32 and 44 hai. Fungal colonization after penetration occurred to a significantly lower extent at 32 hai in the controls than after bacterial pretreatment, whereas at 44 hai, the reverse situation occurred. The result at 32 h reflects that there was only one appressorium with successful penetration and tissue colonization in the bacteria-treated plants, but none in the controls, so even though the difference is significant, it is based on a very low number of interaction sites.
Table 5. Infection biology of Didymella bryoniae and accumulation of hydrogen peroxide (H2O2) at the penetration sites in the first leaves of watermelon cv. 232-0125/B treated with Pseudomonas aeruginosa 231-1 (B) or water (C)
†OR, odds ratio. ORs for comparison of the plants treated with Ps. aeruginosa 231-1 (B) and water (C). In the analysis, (C) is used as a reference, OR = 1·00. The number of asterisks indicates the degree of significance. NS, nonsignificant difference, ***significant at P ≤ 0·001, **significant at P ≤ 0·01, *significant at P ≤ 0·05.
Infection biology (percentage of)
Spores forming appressoria
Appressoria causing penetration
Appressoria with successful penetration and tissue colonization
Accumulation of H2O2 (percentage of)
Appressoria with accumulation of H2O2
Appressoria with failed penetration and H2O2 accumulation
Appressoria with successful penetration and H2O2 accumulation
Appressoria with successful penetration, tissue colonization and H2O2 accumulation
Where penetration attempts by D. bryoniae took place, H2O2 accumulated beneath appressoria as a response to infection (Table 5, Fig. 2a,d). The percentage of appressoria that accumulated H2O2 after treatment with Ps. aeruginosa 231-1 was rather high, but only significantly higher than in the controls at 44 hai (Table 5). On the other hand, the percentage of appressoria with failed penetrations and H2O2 accumulation was only significantly higher in the bacteria-treated than in the control plants at 20 hai. Furthermore, it was found that at 32 hai, all appressoria with successful penetration also had accumulation of H2O2 after both pretreatments. However, at 44 hai, there was a significantly higher percentage of appressoria with successful penetration and accumulation of H2O2 after bacterial pretreatment than in the control plants (Table 5, Fig. 2b,e). H2O2 accumulation in relation to tissue colonization was only seen at 44 hai. Again, a higher percentage of appressoria giving successful penetration and tissue colonization with H2O2 accumulation occurred after bacterial pretreatment than in the control plants (Table 5, Fig. 2c,f).
Peroxidase activity and isoforms
Total peroxidase activity (Fig. 3) and isozyme analysis (Figs 4 and 5) were studied in both cv. PI189225 and cv. 232-0125/B.
In cv. PI189225 (Fig. 3a), peroxidase activity was rather stable until 48 hai, but from 48 hai, it rapidly increased and attained significantly higher levels at 96 hai in the control plants than in plants treated with bacteria. Looking closer at the period 0–48 hai (Fig. 3b) revealed that at 9 hai, peroxidase activity was significantly higher in plants treated with bacteria than in the controls, whereas there was no significant difference at the other time points until 48 hai. Peroxidase activity in cv. 232-0125/B inoculated with D. bryoniae started to increase dramatically from 48 hai both in plants pretreated with bacteria and water (Fig. 3c). At 72 hai, there was a significantly higher peroxidase activity in the controls treated with water compared to the plants treated with bacteria, whereas this was not seen at 96 hai. Looking closer at the period 0–48 hai (Fig. 3d) revealed that at 36–48 hai, there was a significantly higher peroxidase activity following pretreatment with bacteria than with water. Peroxidase activity was not significantly different between the four treatment combinations (bacterial pretreatment + no pathogen inoculation, bacterial pretreatment + pathogen inoculation, control pretreatment + no pathogen inoculation, control pretreatment + pathogen inoculation) until 48 hai (data not shown). After this time, the two noninoculated samples retained the same level of peroxidase activity (data not shown), whereas activity for the inoculated samples increased.
Basic native-PAGE acryl amide gel electrophoresis for detecting acidic peroxidase isoforms in cv. PI189225 revealed ten isoforms (Fig. 4). At the early stages of infection (0–36 hai), only five isoforms (POX1, POX7, POX8, POX9 and POX10) were detected. Four new isoforms started to appear from 48 to 72 hai (POX3, POX4, POX5 and POX6). Some acidic isoforms (POX1, POX8, POX9 and POX10) had the same intensity in plants pretreated with water or Ps. aeruginosa 231-1. On the other hand, POX7 (45 kDa) from plants pretreated with bacteria showed higher band intensity at 0–36 hai compared to the controls. Furthermore, a new band POX4 (Fig. 4b, lane 3) appeared only at 9 hai at a very light intensity in plants pretreated with bacteria but not in the control plants. Basic isoforms of peroxidase were detected in an acidic gel. Four basic isoforms were detected in plants treated with water as well as in plants treated with Ps. aeruginosa 231-1 and appeared with the same intensity (data not shown).
In cv. 232-0125/B, only acidic isoforms of peroxidase were studied (Fig. 5). Six isoforms were detected after the two pretreatments. Four isoforms, i.e. POX2, POX3, POX5 and POX6 had the same band intensity after the two pretreatments. POX4 (45 kDa) accumulated from 24 hai and to higher intensity in plants pretreated with Ps. aeruginosa 231-1 than in the controls.
A total of 190 bacterial strains were initially isolated from the roots of watermelon and they mainly belonged to the genera Pseudomonas and Bacillus. Based on in vitro tests as well as tests in planta, one strain, Ps. aeruginosa 231-1, was found to be able to consistently reduce foliar infection by D. bryoniae under controlled conditions by up to 70% in two watermelon cultivars following seed treatment and soil drenching, and this strain was therefore studied in more detail. Furthermore, this bacterial strain was also able to control the disease in the field and significantly increase the harvested yield.
To our knowledge, this is the first report on protection of watermelon using indigenous bacteria isolated from the rhizosphere in Vietnam. Interestingly, application of rhizobacteria to seeds/soil could reduce a foliar disease. This has been reported in some cases before, e.g. in cucumber (Wei et al. 1991; Liu et al. 1995a,b) and tomato (Silva et al. 2004).
Pseudomonas spp. have been reported to inhibit pathogens directly by production of antibiotics, enzymes lysing fungal cell walls, siderophores and hydrogen cyanide (Défago and Keel 1995; Bano and Musarrat 2003; Siddiqui 2006) and indirectly by inducing systemic resistance against diseases (Liu et al. 1995a,b; De Meyer et al. 1999; Van Loon 2007). Bacillus spp. have also been reported to produce antibiotic substances and lytic enzymes, which could directly inhibit pathogens (Raaijmakers et al. 2002; Siddiqui 2006) or induce resistance against foliar diseases (Silva et al. 2004). In the present study, the protection exerted by the four selected bacteria likely involves antibiosis as all strains tested gave inhibition zones in dual culture tests. Potentially, such antibiotic substances could systemically be translocated to the upper plant parts where they could inhibit the pathogen. Alternatively, the rhizobacteria could grow endophytically to the site where the pathogen attacks and inhibit it directly or induce resistance. In order to investigate the possibility of endophytic growth, a gfp-transformant of Ps. aeruginosa 231-1 was produced for determination of the bacterial colonization pattern. A gfp-transformed strain of the rhizobacterium is a very useful tool for studies of the colonization by culture plating and for in planta studies by confocal laser scanning microscopy (Normander et al. 1999; Hallmann et al. 2001; Poonguzhali et al. 2008). By using a culture plate method, bacteria were detected inside the hypocotyls after application by seed treatment and soil drenching. This indicates that Ps. aeruginosa 231-1 is able to live endophytically. Interestingly, the bacterial density increased dramatically in hypocotyls and first true leaves only in plants inoculated with the pathogen, whereas no bacteria were detected in the first true leaves in plants where D. bryoniae was absent. These results indicate that Ps. aeruginosa 231-1 is able to live and proliferate in the plant and rapidly increase in population size, thus being able to directly affect the pathogen. The recovery of rhizobacteria from aerial plant parts after application to roots has previously been reported, e.g. in broccoli (Lamb et al. 1996) and cabbage (Wulff et al. 2003), whereas in other cases, it has not been examined. Nevertheless, it is important to determine this when evaluating whether the protection is local or systemic. The rapid increase of the bacterial population in the hypocotyls and first true leaves in plants after infection by the pathogen but not in noninfected plants found in this study is interesting. It could be hypothesized that in the plants not infected by D. bryoniae, Ps. aeruginosa 231-1 is also present in the first true leaf but at such a low population level that it cannot be detected by culture plating. When the pathogen infects the first true leaf, the tissues are degraded and the components liberated from here could be beneficial for bacterial proliferation. Alternatively, D. bryoniae could produce metabolites that serve to attract the bacteria, and subsequently they will proliferate, as observed for the biological control agent Pseudomonas fluorescens WCS365 in the interaction between tomato and Fusarium oxysporum f.sp. radicis-lycopersici (De Weert et al. 2004).
Studies of the colonization pattern of bacteria are important for determining and understanding the mechanisms involved in disease protection. If the bacteria are localized at the same place as the pathogen, protection is local and not systemic. Antibiosis may be involved if the bacterium produces metabolites toxic to the pathogen (Vidhyasekaran et al. 1997). Alternatively, the protection exerted by rhizobacteria may involve induced plant resistance (Liu et al. 1995a,b) or both mechanisms may be involved (Ramamoorthy et al. 2002; Someya et al. 2002). Rhizobacteria have often been found to protect the plant by activating defence responses in the host (Chen et al. 2000; Ongena et al. 2000; Nandakumar et al. 2001; Sari et al. 2007) and this possibility was examined for Ps. aeruginosa 231-1. Studies of the infection biology of D. bryoniae in cv. 232-0125/B showed that there was a significant reduction in fungal penetration at 20 hai and tissue colonization at 44 hai after bacterial pretreatment compared to the controls, resulting in less disease of the first true leaves at 4 dai. Penetration was accompanied by more frequent and prominent accumulation of H2O2 at the fungal penetration sites and during early colonization after bacterial pretreatment than in the controls. Furthermore, a higher percentage of appressoria failing to cause penetration had H2O2 accumulation after bacterial treatments than in the controls. This pattern of accumulation could suggest that H2O2 plays a role in inhibition of D. bryoniae penetration and restrict the initial stages of the infection. H2O2 has many roles in plant resistance against pathogens (reviewed by Shetty et al. 2008). Accumulation of H2O2 was also found to inhibit the penetration and initial growth of D. bryoniae in watermelon (Nga 2007), and the present results therefore suggest that an early accumulation of H2O2 is one of the mechanisms associated with bacterially induced protection of watermelon by Ps. aeruginosa 231-1 against D. bryoniae. However, data also indicate that even though arrest of pathogen ingress correlated with H2O2 accumulation, this response was only able to inhibit the pathogen during the very early stages of the interaction (20 hai). At later stages of the interaction, the response was too weak or overwhelmed by the pathogen which very aggressively colonized the host. Nevertheless, even at these stages, H2O2 accumulation was seen more frequently at interaction sites in plants treated with bacteria than in control plants. Usually, H2O2 is considered to be important in inhibiting growth and development of biotrophic pathogens, whereas necrotrophic pathogens have often been reported to benefit from H2O2 accumulation and even sometimes stimulate H2O2 production (reviewed by Shetty et al. 2008). However, the general nature of this conclusion has been questioned (Shetty et al. 2008). Didymella bryoniae is considered a facultative necrotrophic pathogen (Svedelius 1990), but often such pathogens have a short biotrophic phase where they, in fact, may be inhibited by H2O2 as found by Kumar et al. (2001) for Bipolaris sorokiniana infecting barley. This corresponds to the situation observed for D.bryoniae (Nga 2007). In addition, even though D. bryoniae can penetrate directly through intact tissue surfaces, it has also been observed that it can grow intercellularly without causing tissue damage in flower infections (De Neergaard 1989). Collectively, these observations suggest that D. bryoniae has a biotrophic element in its life style.
Fluorescent pseudomonads have previously been reported to induce resistance in many plants by activating the accumulation of, e.g. phytoalexins, PR-proteins and different defence-related enzymes including phenylalanine ammonia lyase and polyphenol oxidase (De Meyer et al. 1999; Ongena et al. 2000; , Nandakumar et al. 2001; Ramamoorthy et al. 2002). In the present study, we showed that seed treatment and soil drenching with Ps. aeruginosa 231-1 could induce early peroxidase activity and alter the peroxidase isozyme profile during the early stages of infection by D. bryoniae. Peroxidases have been reported to participate in many defence responses such as modification of plant cell walls and the hypersensitive reaction (Brisson et al. 1994; Thordal-Christensen et al. 1997; Quiroga et al. 2000), but the specific role of peroxidase in the watermelon-D. bryoniae interaction is currently not known and further studies are needed to elucidate this issue. In both watermelon cultivars, total peroxidase activity started to increase and there was a larger accumulation of a 45-kDa acidic isoform (POX7 in cv. PI189225, identical to POX4 in cv. 232-0125/B) during the early stages of infection after bacterial pretreatment than in the control plants. The early increase of peroxidase activity correlated with a larger accumulation of H2O2 in plants treated with Ps. aeruginosa 231-1. Similarly, an early increase of peroxidase activity and accumulation of an acidic 45-kDa peroxidase isoform was also found to be involved in defence as well as in induced resistance by sodium tetraborate in watermelon against D. bryoniae (Nga 2007). Likewise, increased peroxidase activity was found to be involved in induced resistance by Ps. fluorescens in tomato against damping-off caused by Pythium aphanidematum (Ramamoorthy et al. 2002) and in rice against R. solani (Nandakumar et al. 2001).
The large increase of total peroxidase activity during the late stages of infection in the control plants, correlating with increased fungal colonization, was seen in both cultivars. A similar pattern of accumulation was observed in the watermelon-D. bryoniae interaction, where a higher peroxidase activity was seen in a susceptible than in a moderately resistant cultivar at the late stage of infection and in induced resistant watermelons pretreated with Na2B4O7 against D. bryoniae (Nga 2007) as well as in induced resistance in cucumber plants pretreated with K2HPO4 against Colletotrichum lagenarium (Irving and Kuć 1990). The large increase of peroxidase activity in control plants in the present study could be a very late defence response which is not effective in stopping the pathogen. There are two aspects of this point. First of all, different isoforms of POX accumulate during the different stages of the interaction. Some of these are probably involved in defence (typically the early occurring isoforms, c.f. Figs 4 and 5). Other isoforms accumulate as a response to stress occurring during the late stages of the interaction, where the tissue collapses. Therefore, peroxidase may well be involved in defence during the early stages of the interaction, whereas there is no correlation during the late stages.
The late accumulation of POX occurred to a similar level in both cultivars and for both pretreatments at 96 hai and this accumulation reflects a stress response, e.g. accumulation of ascorbate peroxidase to remove H2O2 at this stage of infection (c.f. Arias et al. 2005; Nga 2007). At this time, the pathogen has colonized and destroyed the tissue in both bacterially treated and control plants. Our results indicate that protection against the pathogen occurs early in the interaction. However, the pathogen is so aggressive that it eventually will overcome any defence under controlled conditions and this has taken place at 96 hai.
Collectively, our results show that rhizobacteria have a potential in biological control of gummy stem blight in watermelon as seed treatment combined with soil drenching with a suspension of Ps. aeruginosa 231-1 could protect watermelon from foliar infection caused by D. bryoniae. The disease protection appears to involve both antibiosis and induced resistance as defence responses were activated. In the field, all three application methods of Ps. aeruginosa 231-1 were efficient in reducing disease and increase yield. The reason for the big differences between the two seasons is that the spring–summer experiment was performed in the beginning of the rainy season (where humidity is not high) and the autumn–winter experiment was performed in the rainy season (where the humidity is very high). Thus, in the autumn–winter season, it rains almost every day and this leaves the moisture level very high, which is beneficial for the pathogen. Even though Ps. aeruginosa 231-1 did not have a spectacular effect on symptom expression in the spring–summer season because of the lower moisture level, the effect which was observed here is, in fact, very important. Thus, watermelons are often grown in the same field for several consecutive seasons and if it is possible to reduce the amount of fungal inoculum in the autumn–winter season, it will be an advantage for the following crops where the increased moisture levels will cause more severe disease problems. Pseudomonas aeruginosa 231-1 can grow endophytically and this is a very useful characteristic under field conditions because such bacteria can escape the adverse physical and chemical conditions in the soil. The endophytic behaviour might also help explain the efficiency of all the application methods. However, it could also be envisaged that inoculum surviving from the spring–summer crop could have been involved the suppression of D. bryoniae inoculum in the subsequent crop, thus helping in explaining the very efficient protection against disease observed in the autumn–winter crop.
Further studies should include testing the effect of the bacterium against other relevant pathogens of watermelon such as F. oxysporum f.sp. niveum, Phytophthora capsici, C. lagenarium and Pseudoperonospora cubensis. In addition, it is important to investigate whether the bacterium might have any adverse effects on humans, animals or the environment before it potentially could be released for wider use to control diseases in Vietnam. This is especially important as some strains of Ps. aeruginosa are known to be able to cause disease in humans (Lyczak et al. 2002).
This research was supported by the ‘Enhancement of Research Capacity’ (ENRECA) grant: ‘Systemic Acquired Resistance – an eco-friendly strategy for managing diseases in rice and pearl millet’ financed by DANIDA. We thank Ole Nybroe for providing plasmids pBK-miniTn7-gfp2 and pUX-PF13; Lisa Biørnlund, Susanne Iversen and Karin Olesen for their help in the transformation work; Julie Torp and Helle Thers for their help in isolating the rhizobacteria and Susanne Kromann Jensen for her help in the experiments to determine the colonization pattern of Ps. aeruginosa 231-1. Finally, we thank the International Foundation for Science (Sweden) for financial support for the field experiments.