Suppression of Bacterial Leaf Blight (Xanthomonas oryzae pv. oryzae) using Local Isolates of Rhizobacteria

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CMUJS Vol.21, No 1 (2017) 2-8

 

Fedencio V. Paslon, Jr.1*, Revelieta B. Alovera2, Carolina D. Amper2, & Myrna G. Ballentes3
1Biological Stress Management Department, R&D, Syngenta Philippines Inc, General Santos City, 9500 Philippines
2Department of Plant Pathology, College of Agriculture, Central Mindanao University, Musuan,
Maramag, Bukidnon, 8710 Philippines
3Department of Entomology, College of Agriculture, Central Mindanao University, Musuan, Maramag,
Bukidnon, 8710 Philippines

 


ABSTRACT

Local isolates of rhizobacteria associated with rice rhizosphere were evaluated against Xanthomonas oryzae pv. oryzae (Xoo) both in vitro and in vivo conditions. Twenty-three rhizobacteria were isolated from the rice-growing provinces of Caraga Region, Philippines, and their antagonistic activity were evaluated against Xoo causing bacterial leaf blight (BLB) on rice using well diffusion assay method. Among the isolates evaluated, three (FVP 08, FVP 09, and FVP 22) were found highly antagonistic against the test pathogen with a clear and strong degree of the zone of inhibition. The three rhizobacterial antagonists were further evaluated for biological control against Xoo under greenhouse condition. The application of these rhizobacterial antagonists on inoculated rice plants showed shorter lesion length, effectively suppressed the bacterial leaf blight and managed to reduce the severity of BLB.

Keywords: Antagonistic effect, Rhizospheric bacteria, Rice

 


INTRODUCTION

 

Rice crop is susceptible to a large num­ber of diseases (Muneer, Rafi, & Akhtar, 2007) and among the most destructive bacterial dis­eases of rice which belongs to the genus Xan­thomonas. It mostly comprises of phytopatho­genic bacteria and is a member of the family Pseudomonaceae. Among the xanthomonads, Xanthomonas oryzae pv. oryzae causes bacte­rial blight of rice and is one of the most impor­tant diseases of rice in most of the rice growing countries (Hopkins, White, Choi, Guo, & Leach, 1992). It is found worldwide and is particularly destructive and epidemic potential in Asia to high-yielding cultivars during the heavy rains of the monsoon season in both temperate and tropical regions especially in Asia (JIRCAS, 2006). Synthetic antibacterial chemicals mostly copper based, although often effective, are a sig­nificant expense for rice farmers. Their use, how­ever, has many drawbacks and serious effects including destruction of beneficial micro-flora, an excessive level of unmonitored chemical residues to the crop, environmental contamina­tion, health hazards, development of resistance of the bacterium to active compounds, and the potential to high phytotoxicity (Janse, 2005). It is, therefore, appropriate that an alternative and compatible measure for bacterial blight disease be introduced and applied.

The promising performances of the local rhizobacteria isolates have a great potential in the future to reduce the damage due to bacterial blight (Velusamy, Immanuel, Gnanamanickam, & Thomashow, 2006). The recent discoveries of Thangavelu and Mustafa (2012) on different isolates of rhizobacteria showed great antagonis­tic potential to bacterial leaf blight disease and have the capability to protect and promote plant growth by colonizing and multiplying in both rhizosphere and plant system.

This study was undertaken to assess the antagonistic capacity of local rhizobacteria applied as a seed treatment, seedling root-dipped, and foliar spray against Xanthomonas oryzae pv. oryzae.

 


METHODOLOGY

 

Isolation and Purification of the Pathogen

Diseased samples of rice at panicle initiation stage showing typical bacterial blight symptoms were collected from Philippine Rice Research Institute-Agusan Experimental Station. Diseased leaf sections (2×7 mm) were surface sterilized with 1% sodium hypochlorite solutions then rinsed thoroughly with sterile distilled water. These sections were transferred into the steril­ized distilled water in test tubes to allow bacterial oozing. A loopful of the suspension was streaked on previously plated peptone sucrose agar (PSA) and was incubated at room condition for 24 to 48 hours. Yellow, mucoid, dome-shaped colo­nies with entire margins that grow on the culture plates were further sub-cultured.

 

Isolation of the Potential Rhizobacterial Antagonists

Figure 1 illustrates the potential rhizobacte­rial antagonists that were sampled from differ­ent rice-growing provinces in Caraga Region, Philippines, namely: Agusan del Norte, Agusan del Sur, Socorro Islands, Surigao del Norte, and Surigao del Sur. About 1 gram of the rice soil rhizosphere was suspended in 10 mL sterile dis­tilled water. The suspension was shaken using a thermo rotator at 150 rpm for 30 minutes and was heated at 80oC water bath for 30 minutes. A serial dilution was prepared by adding 1 mL of suspension to 9 mL sterile distilled water un­til at 106 dilutions were obtained and 0.1 mL of sample was spread on PSA plates and incubated at 30°C for 24 hrs. The growth of colonies was further subcultured and purified using the same medium (Beric et al., 2012).

Figure 1. Map of Caraga Region showing the Rice-growing Provinces where the Antagonist Rhizobacteria were Isolated

 

In vitro Screening of the Potential Rhizobacterial Antagonists

Rhizobacterial isolates were screened against Xoo using the well diffusion assay. Pure cultures of the different isolates were grown in a liquid containing peptone and sucrose for 48 hours. In separate plated medium, Xoo suspensions (ap­prox. 106cfu/mL) were seeded uniformly using pour plate method. Five equidistant wells were made in the previously plated medium seeded with Xoo by using a sterilized cork borer and us­ing a sterile syringe of a 0.1 mL of rhizobacteria (108cfu/mL) was placed on the wells. The as­sayed plates were incubated for 24 to 48 hours and the zones of growth inhibition of Xoo around the rhizobacterial antagonists were measured (Beric et al., 2012).

 

In vivo Screening of the Potential Rhizobacterial Antagonists

            Different isolates of the rhizobacteria that inhibit the growth of Xoo under in vitro test were separately applied to rice seeds of NSIC Rc224. The seeds were soaked in 0.01% polyoxyeth­ylene–sorbitan monolaurate (Tween20, Sigma Aldrich) containing 108cfu/mL of the rhizobac­terial antagonist, and then kept at 28°C for 48 hours. The control seeds (without rhizobacteria) were soaked in sterile water. Subsequently, both control and treated seeds were sown in seedbeds using natural field soil. Seedlings were trans­planted into plastic pots after 21 days. At the time of transplanting, the seedlings were root-dipped into rhizobacterial antagonist suspension containing 108cfu/mL and sterile distilled water for control. Foliar sprays of each rhizobacterial antagonist were applied 28 and 35 days after transplanting (DAT). Inoculation of bacterial leaf blight (BLB) pathogen was done at 40 and 45 DAT using leaf cutting method (Gnanaman­ickam, Velusamy, & Immanuel, 2013). Percent of disease suppression was calculated by mea­suring the mean lesion length of the inoculated control plants minus the mean lesion length of the rhizobacterial antagonist treated plants over the mean lesion length of the inoculated control plants multiply by 100. There were 20 random plants with three replications in these experi­ments. Table 1 shows the disease severity that was evaluated 18 days after inoculation (DAI) up to 56 DAI with seven days interval following the modified rating scale of International Rice Research Institute –Standard Evaluation System for BLB percent infection following the formula:

Where:

n = number of samples showing the scale of 0, 1, 2, 3 and

4, 5 to 9, respectively

N = total number of samples

9 = represents the highest scale

 

Statistical Analysis

Both in vitro and in vivo screening of the po­tential rhizobacterial antagonists were conducted using Complete Randomized Design (CRD) with three replications. Statistical analyses were car­ried out using Stata/IC 12.1. Experimental data were subjected to analysis of variance (ANOVA) in which mean values of the different variables were compared, and statistical interactions were investigated. To determine which means were significantly different (p < 0.01), multiple com­parisons tests Duncan’s new multiple range test (DMRT) were used.

 

Table 1. Modified Disease Severity Rating Scale Used to Assess Symptoms caused by Bacterial Leaf Blight (Xanthomonas oryzae pv. oryzae) in Rice Plants in Greenhouse Test (IRRI- SES 1996)

INDEX PERCENT  LEAF INFECTION
0

1

0%

1-3 %

2 4-6 %
3 7-12 %
4 13-25 %
5 26-50 %
6 51-75 %
7 76-87 %
8 88-94 %
9 95-100 %

 


RESULTS AND DISCUSSION

 

In vitro Screening of the Potential Rhizobacterial Antagonists

Table 2 illustrates that seven out of twenty-three rhizobacterial isolates inhibited the growth of Xoo. However, only three showed a strong de­gree of antagonism exhibiting a wide, pronounce zone of inhibition. The three isolates, namely: FVP 08 (31. 23 mm), FVP 09 (41 mm), and FVP 22 (54.7 mm) continued to inhibit the growth of Xoo at 72 hours incubation as shown in Figure 2. The inhibition of growth could have been due to the metabolites produced by these isolates. Beric and co-workers (2012), found out that large a number of rhizobacteria produced bacteriocin, contained genes involved in the biosynthesis of lipopeptides of the iturin and surfactin classes, carry the sfp gene, responsible for the biosynthe­sis of surfactin with potential for biological con­trol against Xoo.

 

Table 2.  Zone of inhibition (mm) of the rhizobacterial antagonists against Xanthomonas oryzae pv. oryzae at 24, 48 and 72 hours after incubation

RHIZOBACTERIA ISOLATES HOURS AFTER INCUBATION
24 48 72
FVP08 14.43ab 25.27bc 31.23b
FVP09 19.33ab 33.67c 41.00c
FVP11 13.33ab 14.03ab 14.03a
FVP14 11.23a 11.43a 11.43a
FVP15 14.70ab 14.87ab 14.87a
FVP17 11.77a 11.83a 11.83a
FVP22 21.27b 49.10d 54.70d
F-test ** ** **
C.V. 4.63 3.88 2.53

** Duncan’s multiple range test (P < 0.01)

     Figure 2. In vitro test for antagonism of rhizobacterial isolates  (a) FVP 08 and FVP 09 and (b) FVP 22 against Xanthomonas oryzae pv. oryzae at 72 hours after incubation

 

In vivo Screening of the Rhizobacterial Antagonists.

The mean BLB lesion length in the rhizobac­terial antagonists-treated plants shows signifi­cant difference. Table 3 reveals that among the isolates, FVP 22 treated plants showed the short­est mean lesions with only 22.32 cm followed by FVP 09 and FVP 08 treated plants with 27.25 cm and 31.63 cm respectively, compared to the control plants with 46.97 cm. The control plants showed severe BLB disease symptoms with pronounced, long, and spreading blight lesions ex­tending to the near base of the infected leaves, while the plants treated with the rhizobacterial antagonists have relatively shorter lesions. Ta­ble 3 further shows that the three isolates sig­nificantly suppressed BLB lesions with FVP 22 by 52. 48% followed by FVP 09, and FVP 08 with 42.30% and 32.36% respectively. These findings conform with the relative results from Gnanamicknam et al., (2013) on the different rhizobacteria isolated in rice ecosystem that sup pressed the rice BLB by 58.83% and 51.88% un­der glasshouse and field conditions, respectively.

Thus, different species of these rhizobacte­ria are applied to rice plants as a seed treatment before sowing, root dip prior to transplanting and two foliar sprays prior to inoculation can suppress Xoo by up to 59% (Islam, Pamplona, Atkinson, & Azucena, 2013). Moreover, figure 3 illustrates that the rhizobacterial antagonists significantly reduced the percent BLB severity. Table 3 and Figure 4 both show that from 18 to 56 DAI, plants treated with FVP 22 got the lowest BLB severity with 42.59% followed by FVP 09 and FVP 08 with 44.44% and 48.89% re­spectively compared to the control plants with of 60.74% BLB severity. The Antagonistic potential of different rhizobacteria were studied by several workers including Gnanamanickam (2013) and Gangwar (2013). They found that bacterization of rice with these rhizobacteria followed by its foliar sprays caused 40 to 60% reduction in bac­terial leaf blight. The possibilities of Induced Systemic Resistance (ISR) were also considered for the management of the bacterial leaf blight in this study. Kloepper, Ryu, and Zhang (2007) noted in their review on the ISR of some rhizo­spheric bacteria like Bacillus spp. and Serratia spp. that the protection resulting from elicitation has been reported against major plant pathogens. Several specific strains of B.amyloliquifaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B.mycoides, and B. sphaericus.

In most cases, Bacillus spp. that elicits ISR also elicits plant growth promotion. Studies on mechanisms indicate that elicitation is associ­ated with ultrastructural changes in plants during pathogen attack and with cytochemical altera­tions. Investigation into the signal transduction pathways of elicited plants suggests that Bacil­lus spp. activate some of the same pathways as Pseudomonas spp. and some additional path­ways (Choudhary, Prakash, & Johri, 2007).

 

Table 3. Effects of rhizobacterial antagonists on the lesion length, disease suppression and severity on inoculated rice plants at 56 days after inoculation

TREATMENTS Lesion Length (cm) Percent Disease Suppression Percent Disease Severity
FVP08 31.63c 32.36a 48.89a
FVP09 27.25b 42.30b 44.44a
FVP22 22.32a 52.48c 42.59a
CONTROL 46.97d 60.74b
F-test ** ** **
CV 0.38 0.99 0.82

** Duncan’s multiple range test (P < 0.01)

Figure 3. Suppression of bacterial leaf blight (Xanthomonas oryzae pv. oryzae as affected by the different rhizobacterial antagonists at 56 days after inoculation

Figure 4. Percent bacterial leaf blight severity as affected by different rhizobacterial antagonists at 18 to 56 days after inoculation

 


CONCLUSIONS

It can, therefore, be concluded that the three, antagonist rhizobacterial isolates effectively in­hibited the growth of the bacterial leaf blight pathogen (X. oryzae pv. oryzae) and suppressed bacterial leaf blight severity when applied as seed soaking, root dipping prior to transplanting, and foliar sprays at 40 and 45 days after trans­planting.

 


RECOMMENDATIONS

The in vitro and in vivo screening of the rhi­zobacterial isolates showed great success in in­hibiting and suppressing the bacterial leaf blight pathogen. Thus, the success in these two sets of conditions suggests further evaluation and tests. The morphological and biochemical tests should be undertaken to confirm the identities of the three local rhizobacterial antagonists. Further studies on the isolation and characterization of the possible antibiotic volatiles/secondary me­tabolites produced by these antagonist rhizobac­terial isolates capable of inhibiting the growth of the bacterial leaf blight pathogen may be con­ducted. Further tests and investigation on the possible growth-promoting hormones produced by the antagonist rhizobacterial isolates associ­ated with rice may be done.

 


ACKNOWLEDGEMENT

This work is part of the Master’s Thesis of the corresponding author. We are thankful to the Philippine Rice Research Institute – Agusan Experimental Station for allowing us to conduct and use their research facilities.

 


REFERENCES

Beric T., Koji M., Stankovi S., Topisirovi L., Degrassi G., Myers M., Venturi V., Fira D. (2012). Antimicrobial Activity of Bacillus sp. Natural Isolates and Their Potential Use in the Biocontrol of Phytopathogenic Bacteria. Food                  Technology and Biotechnology. 50 (1):25-26.

Choudhary, D. K., Prakash, A., & Johri, B. N. (2007). Induced systemic resistance (ISR) in plants: mechanism of action. Indian Journal of Microbiology, 47, 289.

Gangwar G.P. (2013). Efficacy of different Isolates of Fluorescent Pseudomonads against Bacterial Leaf Blight of Rice. African Journal of Agricultural Research. 8(37):4588-4589.

Gnanamanickam S.S., Velusamy P., Immanuel, J. E. (2013). Rhizosphere Bacteria for Biocontrol of Bacterial Blight and Growth Promotion of Rice. Rice Science, 20(5):356−362.

Hopkins C.M., White F.F., Choi S., Guo H.A., Leach J.E. (1992). Identification of a Family of Avirulence Genes from Xanthomonas oryzae pv. oryzae. Molecular Plant-Microbe Interactions. 5(6):451-495.

International Rice Research Institute. (1996). Standard Evaluation System for Rice. Manila, Philippines. 4th Edition, pp. 11 and 35.

Islam Z., Pamplona R.A., Atkinson D., Azucena E.J. (2013). Control of Rice Diseases. International Rice Research Institute. Los Banos, Laguna. pp 9-11. Retrieved October 2013.

Janse J. D. (2005). Phytobacteriology Principles and Practice. Department of Bacteriology Plant Protection Service, Wageningen, the Netherlands. p. 189.

Japan International Research Centre for Agricultural Sciences (JIRCAS). (2006).  Xanthomonas oryzae pv. oryzae generic database.

Kloepper, J. W., Ryu, C.M., & Zhang, S. (2007). The nature and application of biocontrol microbes: Bacillus spp. induced systemic resistance and promotion of plant growth by Bacillus spp. The American Phytopatho­logical Society, 94(11), 1259.

Muneer, N., Rafi, A., Akhtar M.A. (2007). Isolation and Characterization of Xanthomonas oryzae pv. oryzae isolates from North West Frontier Province (NWFP) Pakistan. Sarhad J. Agric. 23(3):743.

Thangavelu R., Mustaffa M.M. (2012). Current Advances in the Fusarium Wilt Disease Management in Banana with Emphasis on Biological Control. National Research Centre for Banana, Trichirapalli, India. Retrieved: December 26, 2006.

Velusamy P., Immanuel J.E., Gnanamanickam S.S., Thomashow L. (2006). Biological Control of Rice Bacterial Blight by Plant-associated Bacteria Producing 2,4diacetylphloroglucinol. Canadian Journal of Microbiology 52(1):56.

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Central Mindanao University Journal of Science

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