Characteristics of Soil Transmission of Ralstonia syzygii subsp. syzygii, the Cause of Sumatra Disease of Clove in Indonesia

MATERIALS AND METHODS

Study area: Observation of symptoms of Sumatra disease was carried out in the clove plantation of PT. Cengkeh, Kendal, Central Java, Indonesia. Molecular detection and transmission experiment of R. syzygii subsp. syzygii was conducted at the Laboratory of Plant Pathology and Greenhouse, Department of Plant Protection, Faculty of Agriculture, University of Gadjah Mada. The research was carried out from July, 2022 to June, 2023.

Observation and sampling of plants showing symptoms of Sumatra disease in the field: Observations were made on two-year-old seedling plants planted in areas infected with Sumatra disease (replanting). Sampling included three plants with size of 50-80 cm height showing symptoms of Sumatra disease, rhizosphere soil in the infected area and remaining infected plant roots. Detection of R. syzygii subsp. syzygii in the sample was carried out using the Polymerase Chain Reaction (PCR) technique⁹.

Hypersensitivity and pathogenicity test of Ralstonia syzygii subsp. syzygii: Hypersensitivity and pathogenicity tests were conducted on isolates suspected to be R. syzygii subsp. syzygii. A hypersensitivity test was carried out on 3 month-old tobacco plants using the method referred to Sharma¹⁰. The observation of hypersensitive reactions was carried out until there were necrotic symptoms on the injected tobacco leaves using the isolates obtained. Pathogenicity assay was carried out on one-year old Zanzibar clove seedlings. As much as 10-20 μL of bacterial suspension with a density of 1×10⁸ CFU mL^–1 was injected into the part of the plant stem close to the shoot that had been perforated using a microtip¹¹. The inoculated plants were incubated in a suitable environment for 2 months and the symptoms and development of Sumatra disease were observed.

Molecular detection of Ralstonia syzygii subsp. syzygii : Extraction of isolates suspected as R. syzygii subsp. syzygii was carried out using the CTAB method (Cetyltrimethylammonium bromide). Confirmation of isolates of R. syzygii subsp. syzygii applied a specific primer UGMRss-F/UGMRss-R (5'-GCTCACCATCGCCAAGGACAGCG-3'/5'TTCGATCGAAC GCCTGGTTGAGC-3') with a total reaction volume of 10 μL consisting of 2 μL DNA template, 1 μL reverse primer, 1 μL forward primer, 1 μL Nuclease Free Water and 5 μL 2x MyTaq Red Mix (Bioline) on a PCR machine (Bio-Rad T100, Germany). The cycle used consisted of pre-denaturation at 96°C for 5 min, followed by denaturation at 94°C for 15 sec, annealing at 59°C for 30 sec, extension at 72°C for 30 sec and equipped with a final extension at 72°C for 10 min, the cycle was repeated 35 times. A total of 4 μL of PCR product was visualized using electrophoresis (Bio-Rad DNA Electrophoresis Cell) at 60 volts for 50 min on 1% agarose gel stained with ethidium bromide. Gel visualization was carried out under UV transilluminator⁹.

Sumatra disease transmission experiment on clove seedlings: The experiment was carried out using a completely randomized design (CRD) with 5 treatments and 3 replications, each consisting of 3 plant units. The treatments used consisted of P1 = Control, P2 = Shoot titration, P3 = Root immersion, P4 = Root watering and P5 = Root wounding. The clove seedlings used were one-year-old Zanzibar variety with uniform height (±50 cm). The R. syzygii subsp. syzygii was cultured on casein peptone glucose (CPG) broth medium and the density was measured using a spectrophotometer (Spectronic GENESYS 10 UV-Vis) at OD₆₀₀ nm until the value OD = 0.1 (1×10⁸ CFU mL^–1) was obtained.

The shoot titration inoculation method was carried out by punching holes in the internode of the clove plant (d = 1 mm) using a sterile needle and then injecting 20 μL of suspension of R. syzygii subsp. syzygii. The suspension was allowed to settle and enter slowly into the exposed vascular tissue with the help of microtips. Clove seedlings were covered using a clear plastic for 24 hrs. The inoculation method by root immersion was carried out by soaking the clove plant seedlings (including the roots and planting medium in polybags) in a suspension of R. syzygii subsp. syzygii for 2 hrs. The root watering method was carried out by watering ±500 mL suspension of R. syzygii subsp. syzygii on the media and clove plant roots until the media conditions turned into field capacity or saturated. The root wounding method was carried out by wounding the clove plant roots using a sterile scalpel and then a suspension of R. syzygii subsp. syzygii was poured until the media became saturated.

Observation of incubation period, disease severity and Area Under the Disease Progress Curve (AUDPC) of Sumatra disease
Incubation period: The incubation period indicates the length of time it takes for the pathogen to cause symptoms in plants. The incubation period of Sumatra disease in each treatment was observed every day. The initial emergence of Sumatra disease symptoms was a benchmark in determining the incubation period of R. syzygii subsp. syzygii in each treatment.

Disease severity: Disease severity of Sumatra disease was observed every 7 days since the emergence of the first symptom in one of the treatments. The results of the disease severity assessment were calculated using the following formula¹²:

The scoring method was based on the condition of the clove canopy which referred to the assessment by Widodo et al.¹³ with slight modification (Table 1).

Area Under the Disease Progress Curve (AUDPC) of Sumatra disease: Disease severity progression was calculated using the AUDPC based formula by Cooke¹⁴ with the following formula:

Confirmation of plant disease symptoms using the PCR method: The DNA extraction of R. syzygii subsp. syzygii was carried out on the roots, stems and leaves of clove seedlings with symptoms of Sumatra disease using the ZymoBIOMICS^TM DNA Miniprep KIT based on an existing protocol. Overall, the amplification and visualization program used was the same as the DNA amplification and visualization method of R. syzygii subsp. syzygii used in this study.

Statistical analysis: Data obtained from observations were analyzed using Analysis of Variance (ANOVA) with a significance level of 5%. If there was a significant difference between the treatments, a further test would be carried out using the Duncan’s Multiple Range Test (DMRT) with a significance level of 5% with the help of the IBM SPSS Statistics software version 20.

RESULTS

Symptoms of Sumatra disease in the field: Observations in the field showed that there was still root debris from plants that had previously been cut down due to infection with Sumatra disease. Two-year-old plants planted at a distance of 1-2 m from diseased plant sites showed symptoms of Sumatra disease. The incubation period required for symptoms of Sumatra disease to emerge in replanting seedlings varied between 5-7 months after transplanting.

Symptoms of Sumatra disease began with the drying of the branches and leaves located at the very bottom of the plant canopy. Symptoms then developed vertically towards the branches and leaves located at the top. One month after the emergence of the initial symptoms, the entire plant experienced changes in the color of the leaves and stems to brown and dried and the shoots of the plants became scorched (Fig. 1a). After a month, some of the dried leaves were shed, while others remained on the stems and branches of the plants (Fig. 1b).

Detection of R. syzygii subsp. syzygii using the PCR technique on rhizosphere soil and clove roots aged ±40 years which had been logged for two years showed results in the form of the emergence of a 378 bp DNA band (Fig. 2).

Fig. 1(a-b):

Clove seedlings showed initial symptoms of (a) Sumatra disease at 5 months after transplantation and (b) Clove seedlings were died at 6 months after transplantation

Fig. 2:

Visualization of soil and root DNA amplicons using the specific primers UGMRss-F/UGMRss-R PCR products were analyzed using 1% agarose gel electrophoresis in 1x TBE buffer. (M: 100 bp DNA Marker), Lane 1: Positive control using Ralstonia syzygii subsp. syzygii isolate, Lane 2: Soil sample and Lane 3: Root sample

Fig. 3:

Hypersensitive reaction of Ralstonia syzygii subsp. syzygii on tobacco leaves (4 days after inoculation) as indicated by an arrow

Fig. 4(a-b):

Pathogenicity assay on clove seedlings, (a) Healthy clove seedlings without inoculation (control treatment) and (b) Clove seedlings with Ralstonia syzygii subsp. syzygii inoculation at 35 days after inoculation

Hypersensitivity reactions and pathogenicity of Ralstonia syzygii subsp. syzygii: The isolate of R. syzygii subsp. syzygii showed hypersensitivity reactions to tobacco leaves. Areas of necrosis on the leaves were formed 4 days after the pathogen was inoculated (Fig. 3). Pathogenicity assay of R. syzygii subsp. syzygii performed on one-year-old clove seedlings showed that clove plants inoculated using a suspension of R. syzygii subsp. syzygii showed symptoms of Sumatra disease in the form of yellowing of the leaves starting from the bones and edges of the leaves and then drying and scorched brown in color (Fig. 4a). Meanwhile, uninoculated clove plants did not develop symptoms until 35 days after inoculation (DAI) (Fig. 4b). Detection of isolates of R. syzygii subsp. syzygii using PCR showed the formation of a single band of single DNA bands at 378 bp.

Transmission of Sumatra disease in clove plant seedlings: The control plants showed no symptoms (Fig. 5a), while symptoms of Sumatra disease occurred in the shoot titration, root immersion, root watering and root wounding treatments (Fig. 5b-e). The clove plants inoculated using the titration method was marked by a change in leaf color to yellowish starting from the leaves that were positioned close to the inoculation point (Fig. 5b). Meanwhile, the treatment with root immersion (Fig. 5c), root watering (Fig. 5d) and root wounding (Fig. 5e) experienced symptoms of Sumatra disease which was indicated by drying of the leaves starting from the bottom as a sign of R. syzygii subsp. syzygii infection.

Within 70 days after inoculation (DAI), all leaves and stems dried out and turned brown in color with plant shoots showing symptoms like scorched. Furthermore, some of the dried leaves fell and some of the leaves remained on the stems and twigs of the plant.

Incubation period, disease severity and AUDPC of Sumatra disease: Treatment using different inoculation methods showed differences in symptoms, incubation period, disease severity and AUDPC of Sumatra disease in clove seedlings in the greenhouse. The inoculation treatment by root immersion, root watering and root wounding had a faster incubation period of disease of 12 days after inoculation compared to the titration treatment which had a longer incubation period of 17 days after inoculation (Table 2).

The percentage of the disease severity in all treatments was directly proportional to the AUDPC value. The highest disease severity and AUDPC value occurred in the inoculation treatment using the root wounding method, namely 37.04% and 201.85, while the lowest disease severity and AUDPC value occurred in the titration treatment, namely 16.05 and 85.81%. All treatments had a percentage of disease severity and AUDPC values that were significantly different from the control treatments (Table 2). Based on this, it can be seen that the inoculation method affects the disease severity and AUDPC.

Fig. 5(a-e):

Symptoms of Sumatra disease in clove seedlings in the greenhouse, (a) Healthy plant without inoculation, (b) Shoot titration inoculation method, (c) Root immersion inoculation method, (d) Root watering inoculation method and (e) Root wounding inoculation method

Fig. 6:

Visualization of DNA amplicons from plant seedling samples using specific primers UGMRss-F/UGMRss-R PCR products were analyzed using 1% agarose gel electrophoresis in 1x TBE buffer (Lane 1-5: Leaf samples-control, shoot titration, root immersion, root watering, root wounding, Lane 6-10: Stem samples-control, shoot titration, root immersion, root watering, root wounding, Lane 11-15: Control, shoot titration, root immersion, root watering, root wounding and M: 100 bp DNA Marker)

Confirmation of Sumatra disease symptoms in clove seedlings using PCR: Detection of R. syzygii subsp. syzygii in plants with symptoms of Sumatra disease was carried out on all parts of the plant including leaves, stems and roots of each treatment. Detection using PCR showed that the leaves, stems and roots of clove seedlings treated with shoot titration, root immersion, root watering and root wounding showed the formation of DNA bands at 378 bp (Fig. 6), while the control treatment without R. syzygii subsp. syzygii did not show any formation of DNA bands that matched the target. This indicated that R. syzygii subsp. syzygii could colonize all parts of the clove plant from root to leaf and vice versa.

DISCUSSION

Symptoms shown on two-year-old clove plants in the replanting area are typical symptoms of Sumatra disease. In general, the external symptoms of Sumatra disease are infected leaves that turn yellow and fall or suddenly wither, then quickly turn brown and remain attached to stems and branches as if scorched by fire. Infected branches change color from green to reddish brown and progressively die off starting from the tip of the plant¹⁵.

Some of the outbreaks of the Sumatra disease of clove population that occurred in West Sumatra Province caused population deaths reaching 70% of the total area of 15,000 ha during 1975-1985. The death of this population also included plants that were replanted in the same area (replanting)³. This showed that the soil and clove plant roots in the remaining and decayed areas could still be hosts of R. syzygii subsp. syzygii. Based on the results of this study, as a pathogen belonging to the RSSC group, R. syzygii subsp. syzygii could be transmitted through the soil. Furthermore, R. solanacearum and R. pseudosolanacearum can also survive in the soil as saprophytic bacteria¹⁶.

Based on Choudhary et al.¹⁷ R. solanacearum is a pathogen that can survive in aquatic habitats and irrigation water contaminated by diseased plant tissues as a source of inoculum. According to Navitasari et al.¹⁸ when the plant dies, R. solanacearum changes its life cycle to become a saprophyte in soil or other environments and can survive until it finds a new host. When there are no host plants, R. solanacearum can survive in the natural environment, but its population is influenced by biotic and abiotic factors. Several ways for R. solanacearum to survive outside the host plant and in unfavorable environmental conditions include in the form of Viable but Non-Culturable Bacteria (VBNC), starved cells, PC type and biofilms.

Under conditions of status as VBNC bacteria, bacterial metabolic activity continues, but cannot be cultured on solid media that is generally used and forms dormant cells. Ralstonia solanacearum can survive in soil as the VBNC form in less than one month at 4°C. In the condition of being starved cells, bacteria survive using population management and do not grow, but can still be cultured. Besides that, R. solanacearum can form biofilm-like structures when in contact with biotic and abiotic surfaces.

Ralstonia syzygii subsp. syzygii exhibits a hypersensitive reaction in tobacco plants. Tobacco leaf cell membranes that come into contact with pathogenic bacteria will experience destruction so that the leaves become dry and necrotic¹⁹. This shows that bacterial isolates injected into leaf cells have the potential act as plant pathogens. The ability of R. syzygii subsp. syzygii to show a hypersensitive reaction on tobacco leaves because this pathogen has a Type-III Hrp secretion system which has an important role in the pathogenesis process²⁰. In the pathogenicity assay, R. syzygii subsp. syzygii in this study showed a longer emergence of symptoms compared to the study by Danaatmadja et al.²¹ and Trianom et al.²² due to differences in the age of clove seedlings and the density of bacterial cells used.

The transmission of Sumatra disease experiment in clove seedlings showed that there were differences in the location of the initial symptoms between the titration inoculation method and other methods. This was presumably because the development of Sumatra disease symptoms corresponds to the entry point of the bacterial colony R. syzygii subsp. syzygii. Generally, R. solanacearum enters the host plant tissue through wounds at the ends of the roots and the point where the roots emerge. It then gradually infiltrates the xylem vessels and spreads via the vascular system to the aerial parts of the plant (stems and leaves). Wilting symptoms are typically brought on by the xylem tissues significant bacterial colonization and the excessive production of exopolysaccharide (EPS), which obstructs the passage of water and nutrients²³.

The presence of pathogens in the xylem tissue can cause embolism and reduce the conductivity of the vascular tissue. In plants with wilting symptoms, the association between pathogens, vascular tissue embolism and symptom development is closely related and the formation of bubbles triggers a defensive response of the plant, resulting in occlusion of the vessels due to gel production and tylosis²⁴. In general, the main factor that causes symptoms to appear in infected plants is the result of blockade in the xylem tissue due to bacterial biofilm activity as well as due to tylose or sap that is actively produced by plants.

Ralstonia syzygii subsp. syzygii, which was inoculated from the roots, could be detected on the roots, stems, the tops of the clove plant seedlings. Research conducted by McElrone et al.²⁵ on one of the xylem inhibiting bacteria, Xylella fastidiosa, indicates that the movement of pathogens occurs from the base of the plant to the top of the Parthenocissus quinquefolia plant. The petioles lying at the base are colonized first and eventually limit all movement of water into the leaf. In addition, cutting off the leaf stalks on vines with symptoms of scorch indicates a blockage by bacterial colonies in the vessels on the leaves.

The same thing happened to the treatment by inoculation using the shoot titration method, which showed that R. syzygii subsp. syzygii can be detected up to the roots of the clove plant. Based on research by Saponari et al.²⁶ inoculation of X. fastidiosa using the stem prick method showed that at 4 weeks after inoculation, X. fastidiosa was detected in the roots of the Olea europaea. According to Petit et al.²⁷ the presence of a high diffusion signal factor (DSF) can induce bacterial synthesis and produce polygalacturonase, which can degrade cell walls and endo-1,4-b-glucanase, which can enlarge the pores between xylem vessels, allowing the transfer of bacteria between vascular tissues. The presence of these enzymes is thought to play an important role in the distribution of bacterial mass from the leaf shoots as entry points during inoculation, to all parts of the plant including stems and roots²⁸.

CONCLUSION

Ralstonia syzygii subsp. syzygii can be detected in symptomatic plants, as well as in the rhizosphere soil and debris of infected plant roots. Artificial inoculation through root wounding was able to cause Sumatra disease symptoms in clove seedlings. This indicated that the transmission of Sumatra disease can occur through the soil.

SIGNIFICANCE STATEMENT

Ralstonia syzygii subsp. syzygii is known to be detectable molecularly using PCR techniques on soil and debris from infected plant roots that have been logged for 2 years. In addition, artificial inoculation using root immersion, root watering and root wounding methods was able to show the appearance of Sumatra disease symptoms on clove seedlings. This can be the basis for realizing that the transmission of Sumatra disease can occur through the soil. Knowledge about the transmission of Sumatra disease can be the basis for making decisions related to the management of Sumatra disease in the field so that it is more effective and able to prevent transmission of Sumatra disease in a wider area.

ACKNOWLEDGMENTS

This study is financially supported by the Ministry of Education, Culture, Research and Technology, Republic of Indonesia under the Tesis Magister Research Program with contract number 3231/UN1/DITLIT/Dit-Lit/PT.01.03/2023. This manuscript is part of an MSc thesis by BAI under the supervision of TJ and Sy.