JUATIKA
JURNAL AGRONOMI TANAMAN TROPIKA
VOL.
6 NO.
1 January 2024 DOI :https://doi.
org/10.
36378/juatika.
eissn 2656-1727 pissn 2684-785X Hal : 53 Ae 63 Strategy For Controlling Bacterial Leaf Blight (Xanthomonas sp.
) on Eucalyptus Pellita Plants by Administering P.
aeruginosa RE081 Siti Marfungah1*.
Fifi Puspita1.
Budi Tjahjono1,2,Bayo A.
Siregar2, dan Abdul Gafur2 Program pascasarjana Ilmu Pertanian.
Universitas Riau.
Jalan Bina Widya KM 12.
Pekanbaru.
Riau 28293 Indonesia Research and Development PT.
Sinarmas Forestry.
Jalan Raya MinasAePerawang KM 26.
Perawang.
Riau 28772 Indonesia *Email: smarfuah@gmail.
ABSTRACT
The demand for Eucalyptus Pellita seeds as a high-throughput inoculum (HTI) crop commodity in the field is extremely high.
However.
Xanthomonas sp.
, a bacteria that causes bacterial leaf blight, poses a significant challenge in ensuring a steady supply of healthy seedlings in nurseries.
Researchers have explored using the rhizobacteria P.
RE081 as an environmentally friendly biocontrol agent to address this issue.
This study aims to determine the optimal concentration of P.
aeruginosa RE081 that can effectively reduce the incidence and severity of bacterial leaf blight caused by Xanthomonas sp.
The research was conducted using a completely randomized design, with different concentrations of P.
aeruginosa RE081 .
, 106, 107, 108 CFU/mL) being tested.
The findings indicate that the biological agent's concentration significantly impacts plant disease occurrence and severity.
vivo testing revealed that P.
aeruginosa RE081 reduced disease severity by only 2.
13% at a 108 CFU/mL concentration.
Interestingly, concentrations lower than 108 CFU/mL increased disease severity compared to the control group.
Keywords:
Concentration, biological Pseudomonas aeruginosa Copyright A 2024.
The authors.
This is an open access article under the CC BY license .
ttps://creativecommons.
org/licenses/by/4.
Marfungah et.
INTRODUCTION
Eucalyptus Pellita, a prominent plant species, possesses valuable chemical properties that contribute to advancing industrial forest plantations (HTI) in the pulp and paper industry.
(Fatimah et al.
, 2.
(Fatimah et al.
The demand for seeds as a commodity for HTI cultivation is exceedingly high.
However, the presence of Xanthomonas sp.
bacteria, which leads to bacterial leaf blight, poses a significant challenge in ensuring the availability of healthy seedlings in The reduction in leaf crosectional area caused by lesions and photosynthesis, resulting in stunted growth and decreased survival rates of eucalyptus seedlings.
To address this presents a potential solution.
Pseudomonas, commonly found in the rhizosphere, has garnered significant attention as a rhizobacterium with remarkable biological Among mechanisms.
Pseudomonas aeruginosa, in particular, has been extensively researched for its ability to serve as a biological agent.
This species employs multiple strategies to combat plant secondary metabolites like hydrogen cyanide, siderophores, and antibiotics.
Additionally, it induces plant defense responses and competes for nutrients, further enhancing its effectiveness in disease control (Lukkani & Reddy, 2.
Pseudomonas aeruginosa is a Pyocyanin, derivative of the blue-green phenazine compound, is one of these pigments.
Its mechanism of action involves disrupting the metabolism of normal cells, as it is released as an exotoxin compound by P.
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6 No.
aeruginosa, effectively inhibiting bacteria (Moustafa et al.
, 1.
In a previous in vitro study conducted by Marfungah et al.
, it was found that Pseudomonas aeruginosa RE081, isolated from the rhizosphere of Eucalyptus Pellita plants, exhibited Xanthomonas sp.
, resulting in the formation of a highly effective inhibition The research also highlighted the RE081, production of HCN, siderophores, proteases.
IAA, and solvent P.
Similarly.
Sandilya et al.
conducted a study on Pseudomonas aeruginosa strain MAJPIA03, a rhizobacteria derived from the rhizosphere of castor plants in India.
This strain produced NH3.
HCN, siderophores.
ACC deaminase.
IAA, and Gibberellins.
It exhibited the ability to enhance the growth of Ricinus communis plants while inhibiting the growth of five Fusarium oxysporum Ciceri.
Fusarium monoliformes.
Fusarium oxysporum.
Fusarium Rhizoctonia Furthermore.
Ghadamgahi et al.
reported that Pseudomonas aeruginosa strain FG106, isolated from the rhizosphere of tomato plants, demonstrated the ability to inhibit the growth of various pathogens including infestans.
michiganensis subsp.
Michiganensis, and euvesicatoria pv.
Perforance.
Based on the description above, it is necessary to research the right Pseudomonas aeruginosa RE081 bacteria to control bacterial leaf blight (Xanthomonas sp.
through in vivo.
Marfungah et.
Juatika Vol.
6 No.
MATERIAL AND METHODS
Research Time and Place The research was conducted in PT Arara Abadi Research and Development.
Perawang's greenhouse, from August to October Research Implementation The Pseudomonas RE081, sourced from the rhizosphere of the Eucalyptus Pellita plant, isolated by Marfungah Xanthomonas sp.
(Plant pathogen E.
Pellita.
R&D collection of PT.
Arara Abad.
The research was carried out using a completely randomized design by administering P.
aeruginosa RE081 at different concentrations to E.
Pellita plants infected with Xanthomonas sp.
Data analysis was done using variance analysis (ANOVA) and the Duncan Multiple Range Test (DMRT) at the 5% The treatments used as test parameters were differences in the concentration of RE081 rhizobacteria, (No .
oncentration 105 CFU/mL).
P2 .
CFU/mL).
P3 .
CFU/mL), and P4 .
CFU /mL).
The stages of the research carried out are described in the following flow diagram:
Pellita plant Pathogenicity Test P.
RE081 and Xanthomonas.
sp on Pellita plant seeds Application 1 Rizobakteri RE081 Application 2 Rizobakteri RE081 Application 2 Xanthomonas Observation and discussion Application 3 Rizobakteri RE081 Application 3 Rizobakteri RE081 Figure 1: Research Stages Preparing E.
Pellita plant seeds The Eucalyptus Pellita clone EP0361WK seeds were utilized for the in vivo testing of plants.
The plants, which were 28 days old, were randomly Completely Randomized Design.
There were four replications and six treatments in total.
Each replication included four units of plant seeds, while each treatment consisted of 16 units of plant seeds.
total, 96 plant seeds were used for this The experiment occurred in a greenhouse, where the humidity and temperature were maintained at 80-90% and 25-30oC, respectively.
Additionally, 12-hour photosynthesis cycle.
Pathogenicity test of P.
RE081 and Xanthomonas sp.
on E.
Pellita plant seeds Each bacteria was tested for pathogenicity on E.
Pellita plant seeds in a growth chamber .
emperature 27-30oC.
Marfungah et.
humidity 80-90%).
Each 48-hour-old isolate was inoculated on NB medium and incubated on a shaker at 100 rpm for 24 hours.
The bacterial suspension is aged for 24 hours, and the density is adjusted to 108 CFU/mL (OD600= 0.
then sprayed on the surface of the plant leaves until wet (Bophela et al.
, 2.
Plants were observed up to 21 days after bacterial inoculation.
Applying Rhizobacterium Pseudomonas aeruginosa Juatika Vol.
6 No.
according to the specified concentration and sprayed on the leaves until wet.
The treatment was repeated twice before and after pathogen application, with a week's interval between each application.
Applying the Pathogen Xanthomonas Inoculation of the pathogen Xanthomonas sp.
This is done by spraying plant leaves with a suspension of Xanthomonas sp bacteria.
At a density of 108 CFU/mL (A600=0.
until wet.
Inoculation of plants with bacterial isolates of Xanthomonas sp.
was carried out once, 24 hours after the second The pathogen is applied in the afternoon 00 and 18.
00 so that the bacteria are not exposed to too hot After pathogen application, observations were made on days 7, 14.
The concentration of the bacterial solution was determined using a spectrophotometer at a wavelength of 600 nm, with each concentration measured at:
Absorbance .
CFU/mL]= 0.
umber of colonies 3.
1 x 105 CFU/mL) Absorbance .
CFU/mL]= 0.
umber of colonies 4 x 106 CFU/mL) Absorbance .
CFU/mL]= 0.
1 (Number Maintenance of colonies is 3.
25 x 107 CFU/mL) Caring for eucalyptus seedlings in Absorbance .
CFU/mL]= 1 (Number of colonies is 3.
35 x 108 CFU/mL) fertilizing, and pest control.
The fertilizer The roots of 28-day-old eucalyptus used Simplot .
acro plants were soaked in 500 mL of P.
which is given by mixing it aeruginosa RE081 suspension along with into the cocopeat planting medium at the the pot tray according to the treatment for beginning of planting.
Watering is done 30 minutes (Heo et al.
, 2.
Apart from using misting .
utomatic mist waterin.
soaking the roots, the P.
4x a day for 10 minutes per watering.
RE081 suspension is sprayed on the Pest control is carried out by mechanical plant leaves until wet.
Next, once a week, means.
10 mL of the rhizobacteria suspension is poured into the planting medium Observation Variable .
Incidence of Disease Disease incidence is determined by observing external symptoms in plants.
Observations were made on days 7, 14, and 21 after pathogen inoculation.
Disease incidence was calculated using the Abbolt method with the following formula:
yaycE = ycu100% Note.
: Disease incidence rate / Disease incidence.
: Number of symptomatic plants observed.
: Total number of plants observed.
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Disease Severity (I) Observations were made on the upper, middle, and lower leaves .
per plan.
on days 7, 14, and 21 after pathogen inoculation.
Calculations are carried out using the Townsend and Heuberger formula (Santosa and Triyono, 1999 (Rahayu & Nurcahyanti, 2.
as follows:
ya= Oc Note: I : Severity of disease.
: 1st infected plant leaf.
: Score with the 1st transmission category.
: Number of plants observed.
: The highest transmission scale value (Score .
The scoring method is determined based on (Rivera-Zabala et al.
, 2.
Score .
: No symptoms Score .
: Blight area >0-5.
Score .
: Blight area 5.
6-19%
Score .
: Blight area >19Ae 48.
Score .
: Blight area 48.
1 Ae 78.
Score .
: Blight area >78.
RESULT AND DISCUSSION
1 Pathogenicity test of P.
RE081 and Xanthomonas sp.
on E.
Pellita plant seeds Pathogenicity tests on the two bacteria gave different results.
After incubation for 21 days, the rhizobacteria aeruginosa RE081 did not show disease symptoms on E.
Pellita seedlings, while Xanthomonas sp.
showed symptoms of bacterial leaf blight since the 7th day and continued to develop until the 21st day.
This indicates that P.
aeruginosa RE081 is not a pathogenic bacteria, while Xanthomonas is pathogenic on E.
Pellita plants.
2 Test the effect of P.
RE081
biocontrol for Xanthomonas sp.
Pellita seedlings using in Vivo.
aeruginosa RE81 is applied in two areas: the rhizosphere and leaves.
The rhizosphere is the area around the Application to the rhizosphere aims to induce a plant immune response, while in leaves, it is hoped that there will be direct competition with pathogens through antagonism.
The results showed that symptoms of bacterial leaf blight in the form of wet spots parallel to the leaf veins were first observed on the 7th day after pathogen inoculation in all treatments except the control (K.
The analysis of variance showed that administration of P.
RE081
concentrations significantly affected the percentage of bacterial leaf blight The results of the Duncan Multiple Range Test (DMRT) at the 5% level on the percentage of bacterial leaf blight caused by Xanthomonas sp.
be seen in Table 1.
Marfungah et.
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Table 1.
Occurrence of bacterial leaf blight disease by Xanthomonas sp.
Eucalyptus Pellita plant seeds Clone EP0361WK
Treatment
Observation Time
7 HSI (%)
14 HSI (%)
21 HSI (%)
K0 (Without Treatmen.
K1 ( Xanthomonas sp.
P1 .
CFU/mL) P2 .
CFU/mL) P3 .
CFU/mL) P4 .
CFU/mL) Note: Numbers followed by different letters in each column indicate significantly different effects using the DMRT test at the 5% significance level.
Observation results On the 7th day The after pathogen inoculation, the disease incidence incidence in the treatment with the rhizobacteria (K.
administration on day highest concentration of rhizobacteria 14 was the same as the highest (P4=108 CFU/mL) showed the smallest concentration treatment (P.
, namely percentage, namely 50% significantly 94%.
different from all treatments.
Treatments The incidence of bacterial leaf P1.
P2, and P3 were not substantially blight on the 21st day in all treatments different from the treatment without except K0 had reached 100%.
The rhizobacteria (K.
but showed higher incidence of bacterial leaf blight was also numbers .
%) compared to K1 .
%).
observed in the control (K.
at 38%.
The incidence of disease on control Increased incidence of leaf blight plants (K.
was 0%, indicating that of the caused by Xanthomonas sp.
in all 16 plant units, not a single leaf was treatments, including control.
This infected with Xanthomonas sp.
occurred due to disease transmission On observation days 14 to 21 after through contact between leaves or pathogen inoculation, the disease splashing water when watering, an incidence in all treatments increased and environment with high humidity, and the was not significantly different.
The pathogen's virulence.
incidence of disease in treating In the disease severity analysis rhizobacteria with concentrations of P1, results, the percentage also increased P2, and P3 reached 100% at 14 days, from the 7th day to the 21st day after meaning that the pathogen had attacked pathogen inoculation, as shown in Table all plant units.
This incidence was higher 2 below.
(K.
Marfungah et.
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Table 2.
The severity of bacterial leaf blight disease by Xanthomonas sp.
Eucalyptus Pellita plants Clone EP0361WK
Observation Time
Treatment
7 HSI (%)
14 HSI (%)
21 HSI (%)
K0 (Without Treatmen.
K1 ( Xanthomonas sp.
25 bc P1 .
CFU/mL) 80,00 c P2 .
CFU/mL) 00 cd P3 .
CFU/mL) 75 cd P4 .
CFU/mL) Note: Numbers followed by different letters in each column indicate significantly different effects according to the DMRT follow-up test at the 5% level The lowest percentage of bacterial compounds present in pathogens (Sun et leaf blight disease severity at 21 days, , 2.
5%, was observed in the However, treatment with the highest concentration rhizobacterial concentration (P.
of rhizobacteria (P4=108 CFU/mL).
This aeruginosa RE081 reduced disease value is 2.
13% lower than the severity of severity by 2.
It is possible that at the disease in seedlings without this concentration.
aeruginosa RE081 rhizobacteria (K.
, namely 58.
has reached quorum to form compounds The higher severity of disease in that can prevent the formation of the treatment given P.
aeruginosa RE081 virulence factors from Xanthomonas sp.
compared to the control is thought to be so that the severity of plant disease due to increased nutrients, especially P decreases, as reported by Kanugala et and N in the growing media for E.
Pellita al.
Pseudomonas aeruginosa Research conducted in vitro strain CGK-KS-1 produces Chumacin-1 by Marfungah et al.
reported that and Chumacin-2 as inhibitors of quorum the characteristics of P.
sensing signaling for biocontrol of rice RE081 as a biocontrol agent could bacterial blight.
Mechanistic studies produce siderophores.
HCN.
Protease, revealed that both electrolytes inhibited IAA, and solvent P.
This was confirmed the production of quorum sensing by other research reported by Sandilya et .
is-11-methyl-2al.
, who stated that the dodecenoic aci.
, suppressed xanthan Pseudomonas strain gum secretion, and inhibited biofilms MAJPIA03 bacteria is capable of Xanthomonas producing NH3.
HCN, siderophores.
ACC However, in this study, the deaminase.
IAA, and gibberellins.
number of colonizers was still too small.
The increased phosphorus and so the reduction in disease severity was nitrogen resulting from the administration also small.
Another possibility is that the of P.
aeruginosa RE081 in treatments P1, presence of rhizobacteria RE081 in P4 P2, and P3 may negatively affect the can trigger systemic plant resistance spread of the disease.
Nitrogen is the through the mechanism of systemic primary macronutrient needed for plant acquired resistance (SAR) in Eucalyptus Nitrogen can affect the Pellita seedlings.
strength of the plant's physical barrier by This result aligns with the research reducing the thickness of the wax layer results observed visually that in the K1 and lignin content, making it easier for treatment .
dministration pathogens to penetrate.
Nitrogen also Xanthomonas sp.
), the plant leaves were influences the formation of virulent tougher, and the color of the leaves turned yellow, with smaller leaf Marfungah et.
Treatment with P.
RE081 rhizobacteria at all concentrations resulted in plants with greener leaf color, softer and brittle leaf surface structure, and wider leaf diameter.
At a P.
aeruginosa RE081 concentration of Juatika Vol.
6 No.
105,106,107 CFU/mL, around 3-4 pairs of lower leaves experienced loss, while at a concentration of 108 CFU/mL, the old leaves could still be maintained without falling off until the last day of observation (Figure .
Figure 2.
The in-vivo test results on day 21 after inoculation of the pathogen Xanthomonas sp.
The rhizobacteria P.
and severity of bacterial leaf blight on E.
RE081 are able to produce HCN.
HCN is Pellita clone EP0361WK seedlings.
a very toxic volatile compound.
However.
However, it indirectly contributes to Eucalyptus plants are tolerant to HCN increasing disease severity through This is consistent with the increasing rhizosphere nutrition.
pathogenicity test of the rhizobacteria P.
Another possibility is that the aeruginosa RE081 on Eucalyptus Pellita rhizobacteria P.
aeruginosa RE081 plant seeds in the growth chamber.
The cannot colonize the rhizosphere or leaves results of the pathogenicity test showed properly because they are washed away that after RE081 inoculation and incubation for 21 days, the plant seeds environmental conditions, or competition still looked healthy, without any with other microbes.
Thus, these bacteria symptoms of disease or wilting.
Visually, require a carrier medium, adhesive the average growth was better than the material, or even the application of control without rhizobacteria.
Rijavec and technology that has recently been Lapanje .
reported that HCN in the developed, namely microencapsulation, soil does not act as a direct biocontrol so that the rhizobacteria can survive agent but is involved in geochemical unfavorable environments so that they processes in the substrate .
, metal can colonize the rhizosphere and leaves chelatio.
, which indirectly increases the optimally (Fathi et al.
, 2021.
Riseh et al.
availability of nutrients for rhizobacteria and their host plants, such as phosphate.
The ability of rhizobacteria to colonize Thus, in this study.
aeruginosa roots is essential for effective Plant RE081 could not maximally reduce the growth promoting Rhizobacteria (PGPR) incidence and severity of bacterial leaf Heo et al.
reported that blight caused by Xanthomonas sp.
rhizobacteria colonization of Burkholderia According to the author, the presence of contaminants strain AY001 was tested by HCN produced by P.
aeruginosa RE081 quantitative measurements of bacterial does not directly influence the incidence populations in tomato roots infected with Marfungah et.
the pathogen.
The AY001 population increased to 3y107 CFU/g at 14 days post-inoculation, indicating that this bacterium could colonize the tomato root system well.
RT-PCR and real-time qRTPCR tests show that AY001 bacteria can protect tomato plants from pathogen infection by triggering ISR by expressing genes coding for jasmonic acid or Kirtanayasa .
stated that one of the antibacterial activities is influenced by the concentration of the antibacterial extract produced.
The ability environmental factors such as soil type, soil moisture, pH, temperature, and plant age and condition (Reisberg et al.
, 2.
CONCLUSION
The concentration of biological agents influences the incidence and severity of plant diseases.
however, the reduction in disease severity is closely related to the relationship between environment, and the plants themselves.
In vivo test results.
aeruginosa RE081 was able to reduce disease severity by 13% at a concentration of 108 CFU/mL.
At lower concentrations.
RE081 increased bacterial leaf blight
ACKNOWLEDGMENT
A huge appreciation to the Research and Development leadership of PT.
Arara Abadi, thank you for facilitating this research so it can be carried out well.
REFERENCE