Integrated management of eucalyptus bacterial wilt in Sumatra, Indonesia

Bacterial wilt is one of the most destructive plant diseases and responsible for great losses in eucalyptus plantations worldwide. Since the use of highly productive monoclonal stands became a common practice in forest companies, the industry have experienced severe bacterial wilt outbreaks. Thus, the present study aimed to provide alternative methods that can be incorporated in the management of bacterial wilt of eucalyptus in Sumatra, Indonesia. Pathogen identification with molecular markers, effect of plant propagation on the disease incidence, chemical sterilization of sand beds, antibacterial activity, and genetic resistance of eucalyptus clones were evaluated. Colonies obtained from diseased plants were morphologically indistinguishable, but specific primers 759F/760R and sequencing of 16S ribosomal RNA gene confirmed the pathogen identity as Ralstonia pseudosolanacearum. In-field evaluation showed that plants propagated via mini-cuttings had the highest disease incidence, 16.5%, while the tissue culture plants rooted in-vitro had the lowest disease incidence, 3%. Chemical sterilization of sand beds with NaOCl and H2O2 was effective on reducing bacterial cells by 97 and 42%, respectively. In-vitro assays confirmed the antibacterial activity of oxolinic acid and streptomycin by promoting a clear zone of 1.3 and 1.4 cm in diameter, respectively. The susceptibility to bacterial wilt varied among eucalyptus genotypes, and of the 21 clones tested eight were resistant, nine were moderate and four were susceptible. Therefore, an effective management of eucalyptus bacterial wilt can be achieved by combining all methods used in this study with best practices used in commercial nurseries and plantations.


Introduction
Eucalyptus is a genus with hundreds of woody and herbaceous species in the Myrtaceae family, mostly native to Australia, but have been cultivated worldwide.Some species are fast growing trees bringing characteristics of high pulp yield and high fiber quality, which makes this tree to form the basis of large commercial plantations for pulpwood production in several countries (Klein and Luna 2018;Turnbull 1999).Currently, the global area covered with eucalyptus plantations comprises more than 20 million hectares and are expanding rapidly (Booth 2013;Forrester and Smith 2012).Notably, tree improvement has mainly focused on development of superior clones targeting for more productive pulpwood plantations with fiber characteristics suiting a single end product.Thus, the use of highly productive monoclonal stands is a common practice present in forest companies globally (Rezende et al. 2014).This strategy may lead to a greater genetic vulnerability that poses a potential risk for the sustainability of commercial forest plantations due to the threat of biotic and abiotic agents (Wingfield et al. 2009).Amongst the biotic threats, bacterial wilt disease has proven to be of great concern to the forestry industry (Alfenas et al. 2006).
Bacterial wilt is considered as one the most destructive plant diseases and responsible for great losses in agriculture and forestry plantations worldwide (Alfenas et al. 2006;Denny 2006;Mansfield et al. 2012).The causal agent of bacterial wilt belong to the so-called Ralstonia solanacearum species complex, currently treated as a cluster of closely related bacteria (Fegan and Prior 2006) and most likely including many species yet to be described.Previously, classification of the isolates were based in race and biovar-race was determined by the host range and biovar by the ability to utilize or react with disaccharides and hexose alcohols (Hayward 1964(Hayward , 1991)).With the advancement of molecular tools, the classification was based on phylotyping (amplification of the 16S-23S rRNA internal transcribed spacer region), and also into a subdivision called sequence variants (sequevars), determined by the sequencing of endoglucanase (egl) gene.More recently, taxonomy of the R. solanacearum species complex has been resolved by analysis of genetic properties (Safni et al. 2014;Prior et al. 2016).The type strain of R. solanacearum included phylotype II, while phylotypes I and III were transferred to the new species R. pseudosolanacearum.All phylotype IV isolates were named as R. syzygii, and the species description included three new subspecies: subsp.syzygii, subsp.indonesiensis and subsp.celebesensis (Safni et al. 2014).
The first reports of bacterial wilt on eucalyptus occurred in China (Cao 1982), Brazil (Sudo et al. 1983), and Indonesia (Machmud 1985).Two Ralstonia species were found to be associated with bacterial wilt on eucalyptus: isolates obtained from diseased plants in South America represented R. solanacearum, while the isolates from Africa and Asia were R. pseudosolanacearum (Carstensen et al. 2017).These pathogens are soil and water borne, generally penetrate the host through the roots, colonize xylem vessels and promotes vascular dysfunction (Hayward 1991;Álvarez et al. 2010).Latent bacterial infections are commonly observed on eucalyptus cuttings in the nursery (Mafia et al. 2012), and asymptomatic plant material may contain large amount of bacterial populations (Swanson et al. 2007).Furthermore, based on field observations over many years, it has been suggested that the development of disease symptoms are also associated with stressful conditions arising from both biotic and abiotic factors that weaken the plant defense systems (Coutinho and Wingfield 2017).Since vegetative propagation of eucalyptus became a common practice, the forest industry have experienced severe bacterial wilt outbreaks (Alfenas et al. 2006).
Eucalyptus propagation via mini-cuttings is an alternative for rapid deployment of genetically improved material obtained from breeding programs and represents of one of the major drivers contributing for the increase in forest productivity (Assis 2011).Therefore, eucalyptus clonal nurseries with high propagation capacity have been built across Sumatra Island.Sand beds with drip irrigation system have allowed commercial nurseries to scale up sprouts production to supply plant material for the high demand of short-rotation eucalyptus plantations.Despite all benefits of clonal propagation via mini-cuttings, it has been demonstrated that Ralstonia inoculum can survive in the sand beds and consequently infect mother plants, and then spread via vegetative propagation process by harvesting infected cuttings from symptomless mother plants (Mafia et al. 2012).Besides significant economic losses that might arise from plant mortality in the nursery, those asymptomatic mini-cuttings may also face stressful conditions in the field and eventually express the disease symptoms (Coutinho and Wingfield 2017).Nevertheless, differences in the susceptibility of eucalyptus clones to Ralstonia species has been demonstrated previously (Wu and Liang 1988;Dianese et al. 1990;Gan et al. 2004;Wang et al. 2011;Wei et al. 2014;Fonseca et al. 2016), representing an additional alternative on managing bacterial wilt.Resistance to bacterial wilt is a highly desirable trait not always readily available in the breeding population but may be introduced via controlled crosses followed by screening for resistant genotypes.
It is noteworthy that no strategy is effective to manage plant diseases when applied individually (Lamichhane et al. 2017).Therefore, the objective of this study was to provide integrated management strategies for the control of bacterial wilt on eucalyptus in Indonesia by testing: 1) tools for accurate diagnosis; 2) the effect of plant propagation method on the disease incidence in the field; 3) chemical sterilization of sand used in mother beds in the nursery; 4) antimicrobials to inhibit bacterial growth; and 5) genetic resistance of eucalyptus clones.This study was developed in the province of Riau, Sumatra, Indonesia, and the experiments were conducted along the years of 2019 and 2020.

Isolation and molecular identification
Isolations were conducted from diseased plants collected in the nursery and field.Firstly, root and stem were washed with running water and detergent.Small pieces of discolored tissue were cut from the inner part and crushed with mortar and pestle.About 5-10 mL of sterile distilled water were added to the mortar containing the sample and stirred gently with the pestle.After 15 min, a total of 100 µL of the solution was streaked out onto semi-selective medium South Africa (SMSA) and incubated at 28 °C for 48 h (Elphinstone et al. 1996).Genomic DNA of reddish colonies was extracted using CTAB method (Doyle and Doyle 1987) with modifications.Molecular identification was conducted using a species-specific primer (759F/760R) developed by Opina et al. (1997) for Ralstonia solanacearum species complex.The amplification was made according to MyTaq DNA polymerase (Bioline, Germany) and the thermocycler settings were performed as described previously (Opina et al. 1997).Negative control reactions without template DNA were carried out simultaneously.Amplicons were visualized using 1% agarose gel electrophoresis.A second molecular identification method was performed through sequencing of 16S ribosomal RNA gene using primer 759F/760R.The amplification step included an initial denaturation step at 94 °C for 1 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 45 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min on an AB Veriti thermal cycler (Applied Biosystem, USA).After purification process, amplified DNA was sent for sequencing (IDT, Malaysia).Consensus sequences were aligned using Bioedit (Hall 1999) and subjected to NCBI BLAST search (http:// www.ncbi.nlm.nih.gov/ BLAST/) (Johnson et al. 2008).Blast result with the highest max score, query coverage, and percentage of identity was chosen as the identified species.

Effect of plant propagation on disease incidence
To understand the correlation between plant propagation method and bacterial wilt incidence in the field, eucalyptus plants of the same genetic material, ECL01, were propagated with four different methods: mini-cuttings, micro-cuttings (rooted in-vitro and rooted ex-vitro) and seedlings.The production of mini-cuttings was conducted by harvesting apical shoots from one-year-old mother plants; these minicuttings were rooted in mist chamber with RH at 80-85% for about 30 days, then kept in acclimatization house for another 30 days, and finally submitted to hardening process for another 20 days.The micro-cuttings were prepared in tissue culture laboratory according to the protocols of Le Roux and Van Staden (1991) for those plants rooted ex-vitro, after shoot multiplication the explants were kept in glass vials for about 28 days for elongation process and then transferred to rooting house in the nursery, and from that stage propagation followed the same protocol as described for mini-cuttings; for micro-cuttings rooted in-vitro, instead of transferring the explants from glass vials to the nursery, elongated shoots were transferred to glass vials containing root initiation medium for about 14 days (Le Roux and Van Staden 1991), and then transplanted to potting medium in the nursery-from that stage propagation followed the same protocol as described for mini-cuttings.Seedlings were propagated from seeds of open pollination obtained from a six-year-old tree of the same genotype as the micro and mini-cuttings (ECL01); seeds were sown in sand beds and after 14 days they were transferred to potting medium and kept for 28 days under acclimatization house, and then another 28 days for hardening process.A total of 800 plants were planted out in the field in a randomized block design consisting of 8 blocks with 4 treatments, and 25 plants per treatment.The selected area had a history of high eucalyptus mortality caused by bacterial wilt disease.Assessment of wilt symptoms were conducted at four months after planting.

Sand sterilization
The efficacy of chemical sterilization of the sand used in the nursery as growing medium for mother plants was tested with direct application of sodium hypochlorite (NaOCl) and hydrogen peroxide (H 2 O 2 ).PVC pipes of 554 cm 3 capacity were filled with sand collected from a commercial nursery located in the province of Riau, Sumatra, Indonesia.The trial consisted of three treatments: sodium hypochlorite, hydrogen peroxide, and sterilized water (control).A total of twelve PVC pipes per each treatment were arranged in a completely randomized design.Each pipe was inoculated with 100 mL of bacterial suspension adjusted spectrophotometrically (A540 = 1.0, approximately 1 × 10 9 CFU/mL) and incubated at 28 ± 2 °C for one week.After that, a solution of sodium hypochlorite at 0.5% was prepared and 140 mL was poured into the pipes, then 1 h after chemical treatment the sand was flushed with 200 mL of sterilized water for three times.For hydrogen peroxide, 30 mL of a solution adjusted to 3% was poured into the pipes.As a control, 150 mL of sterilized water was poured into the pipes.All pipes were incubated at 28 ± 2 °C.After four weeks, three samples were collected at 8, 18 and 28 cm depth, totaling 9 samples per pipe.From each sample, 10 g of the respective sand was added onto 100 mL of sterile distilled water and vortex for 5 min, then 100 µL originated from a serial dilution of 10 -6 was plated onto SMSA and incubated at 28 °C for 48 h.Colony counting was performed by placing the Petri dishes on a grid background and then colony forming unit (CFU) per gram of sand was calculated.

Antimicrobials
Antibacterial activity was tested in-vitro to evaluate the efficiency against Ralstonia pseudosolanacearum.Two antimicrobial chemical products, Starner 20 WP (Oxolinic acid) and Agrept 20 WP (Streptomycin) were tested in concentrations of 2 g/L and 1 g/L, respectively.Distilled water was used as negative control.A sensitivity test of Food Poison Technique (FPT) was performed to assess inhibition zone in-vitro.A bacterial suspension of isolate GRG2 was prepared and adjusted to 1 × 10 9 CFU/mL (A540 = 1.0).Then, 100 µL of bacterial suspension was added to 9 mL of NA medium and poured onto sterilized Petri dishes.Filter paper discs of 6 mm in diameter were soaked in the respective chemical solutions for about 10 min and then transferred to the center of Petri dishes containing solidified NA medium previously inoculated with bacterial inoculum.The inoculated plates were kept at 4 °C for 3 h to help on the chemical diffusion.Petri dishes were incubated at 28 °C for 72 h and the diameter of clear zone was measured on the second and fifth day after inoculation.

Genetic resistance
Twenty-one eucalyptus clones were tested for the resistance to Ralstonia pseudosolanacearum.Ninety-day-old cuttings were transferred to plastic bags of 2L capacity containing a mixture of cocopeat soil and rice husk 3:1 (v/v), and acclimatized for seven days inside the inoculation chamber.The inoculum was prepared with virulent colonies of the isolate GRG2 by selecting smooth, fluid colonies of white edges and red centers.Colonies were transferred to new Petri dishes with SMSA and incubated for 48 h at 28 °C.The inoculum was prepared by adjusting the bacterial suspension spectrophotometrically (A540 = 1.0, approximately 1 × 10 9 CFU/mL).For inoculation, 0.5 mL of the inoculum was deposited into the wound done with a sterile scalpel at the base of stem.A piece of damp cotton was placed below the stem cut.Afterwards, the wound and the cotton were covered with Parafilm® to prevent contamination by other pathogens and to maintain moisture.Plants were kept in a growth chamber at 28 ± 2 °C with a 12 h photoperiod and light intensity of 40 μmol/s/m 2 (Fig. 1).A total of twenty plants were used per each treatment.The trial was arranged in a completely randomized design.Plants of a susceptible clone ECL01 were inoculated with the same protocol and used as positive control while plants of the same clone inoculated with sterile distilled water served as negative control.Plants were evaluated daily for the presence of wilt symptoms.Final assessment was done at 30 days after inoculation by checking the percentage of wilting plants and presence of bacterial ooze (Fig. 1).To evaluate the presence of bacterial ooze, three fragments of stem tissue of every plant were removed from the stem base (3 cm above the inoculation point) and from the apical part (8-10 cm above inoculation point) and then deposited on a microscope slide containing a drop of water and examined under a light microscope at 100 × magnifications (Fig. 1).Data obtained from each plant was scored based on a rating scale developed specifically for bacterial wilt on eucalyptus in this study (Table 1).

Statistical analysis
Data from all experiments were statistically analyzed by using the agricolae package in R Project (De Mendiburu 2019).Analysis of variance (ANOVA) were conducted when experimental errors were normally distributed and homogeneity of variances observed.Then, Scott-Knott cluster test or Fisher's least significant difference (LSD) were used to evaluate the effects of plant propagation × disease incidence, chemicals × sand sterilization,

Disease symptoms and molecular verification
In the nursery, infections of mother plants were characterized by leaf necrosis, inner discoloration of roots and stem tissues, root necrosis, wilting and eventual plant death.In rooting house, infected cuttings showed symptoms of reddening of leaves and subsequent rot.In the field, disease symptoms were characterized by yellowing, ascending basal leaf loss, wilting, root necrosis, basal stem discoloration and consequently plant death.During diagnosis, bacterial ooze was observed streaming out of infected tissues (Fig. 2).After isolations in SMSA, a total of ten bacterial colonies with typical Ralstonia morphology obtained from field (GRG2, BAS4, SKB1, PEL4, KER1) and nursery (KCN2, KCN4, PCN2, PCN8, BCN3) were selected for molecular verification.These ten colonies had mucoid appearance and reddish color, in most cases with white edges and red center (Fig. 3).Among the isolated colonies, only one (GRG2) tested positive for Ralstonia complex using the specific primers 759/760R (Fig. 4).Sequence of 16S ribosomal RNA gene of the isolate confirmed as positive, GRG2, showed through BLASTn search with other entries in GenBank a nucleotide homology of 99.45% with R. psedosolanacearum (GenBank accession no.CP021762.1,query cover of 100%), thus confirming the pathogen identity as previously reported in other studies (Safni et al. 2014;Carstensen et al. 2017).

Effect of plant propagation on disease incidence
In-field evaluation of bacterial wilt incidence varied according to the plant propagation method used in the nursery (F = 8.31, p < 0.001), and no difference among the blocks was observed (F = 1.89, p = 0.068).Four months after planting, plants propagated via mini-cuttings showed a disease incidence of 16.5%, the highest incidence observed among all treatments.On the other hand, when plants were propagated in the tissue culture laboratory the disease incidence was significantly lower.Micro-cuttings rooted in-vitro  still able to reduce the bacterial cells by 42% in average when compared with the control (Fig. 6).

Antimicrobials
The two chemicals tested for antibacterial activity against Ralstonia pseudosolanacearum showed to be effective invitro (F = 12.3, p < 0.001).Streptomycin produced a clear zone of 1.3 cm in diameter at the fifth day of incubation.Same pattern was observed for oxolinic acid, which yielded on a clear zone of 1.4 cm at the fifth day of incubation, showing that both products were statistically different from the control for in-vitro assays (Table 2).

Genetic resistance
The susceptibility to bacterial wilt varied among eucalyptus clones (F = 14.60, p < 0.00001).The rating scale developed in the present study (Table 1) helped on giving proper disease scoring, which considered the wilt symptoms and exudation of bacterial cells from inner tissue, similar to the protocols of Fonseca et al. (2016).First wilt symptoms were observed showed the lowest disease incidence, 3.0%, while microcuttings rooted ex-vitro had a slightly higher disease incidence of 6.0%.Seedlings propagated from open pollinated seeds had a disease incidence of 9.5%, which is significantly lower than plants propagated via mini-cuttings (Fig. 5).

Sand sterilization
Chemical treatment used for sand sterilization in the nursery was effective on reducing bacterial cells in infested sand.However, based on ANOVA results, there was no significant difference on the chemical efficiency at different depths (F = 0.86, p < 0.424), so the Scott-Knott cluster test considered only the difference between chemicals (F = 70.8,p < 0.001).Sand treated with sodium hypochlorite (NaOCl) had an average of 0.39 CFU/g, while hydrogen peroxide (H 2 O 2 ) was 8.8 CFU/g, and sterilized water 15.22 CFU/g.Treatment with NaOCl was able to reduce the concentration of bacterial cells by 97%, in average, when compared with control treatment using only sterilized water (Fig. 6).Chemical treatment with H 2 O 2 was less effective than NaOCl, but sixteen days after inoculation on the susceptible clone ECL01 used as positive control.Of the 21 clones tested in this study, eight were resistant, nine were moderate, and four were susceptible (Table 3).None of the clones herein tested were highly susceptible.No symptoms and no bacterial exudation were observed on control plants inoculated with distilled water.The highest disease scoring was observed on the susceptible clone used as positive control as well as on clones ECL04 and ECL09.There was no clear correlation between eucalyptus species and resistance to bacterial wilt in the set of clones used in the present study.For instance, clones of E. pellita × E. robusta had different levels of resistance and were classified as susceptible (ECL04), moderate (ECL08 and ECL06), or resistant (ECL10 and ECL05).

Discussion
Since the first report of bacterial wilt on eucalyptus in Indonesia (Machmud 1985), the disease has been associated with great losses in commercial stands of Sumatra (Alfenas 1993;Old et al. 2003, Siregar et al. 2021), suggesting that alternative disease control strategies are required to manage the problem.Besides the well-known preventative methods to ensure disease free planting material as well as proper silviculture to reduce plant stress in forestry plantations, the present study provided alternative control measures to enhance the integrated management of bacterial wilt on eucalyptus.It was demonstrated that alternative plant propagation method through tissue culture, chemical sterilization of sand with sodium hypochlorite and hydrogen peroxide, plant treatment with antibacterial chemicals such as streptomycin and oxolinic acid and selection of resistant genotypes suitable for field deployment, were all able to mitigate the disease impact.
Effective control measures depend on proper identification of disease-causing agent, making diagnosis crucial for plant disease management.An inaccurate diagnosis may lead to the use of less effective control measures and consequently further plant losses.Bacterial colonies are often obtained from symptomatic or asymptomatic plants, but it has been demonstrated that in certain cases the exudate produced in infected eucalyptus plants may contain other endophytic bacteria (Coutinho and Wingfield 2017).Identification of bacterial colonies belonging to the Ralstonia solanacearum species complex has been facilitated with the development of specific molecular markers (Opina et al. 1997), allowing pathogen detection even from latent bacterial infections commonly observed on eucalyptus cuttings (Alfenas et al. 2006).In this study, specific primers 759F/760R helped to differentiate morphologically indistinguishable colonies, since colony identification based solely on morphology may be mistaken.Also, sequencing of 16S ribosomal RNA gene revealed that isolates obtained from diseased eucalyptus plants in Sumatra belong to R. pseudosolanacearum, same as previously reported (Safni et al. 2014;Carstensen et al. 2017).Molecular verification prior to trial establishment was particularly important in the present study due to the fact that bacterial colonies obtained from eucalyptus plants could be endophytes and not the disease causal agent, compromising experimental results.As previously discussed, members of Ralstonia solanacearum species complex can spread via vegetative propagation process by harvesting infected cuttings from symptomless mother plants (Alfenas et al. 2006).Infected shoots from several eucalyptus clones resulted in a significant decrease in rooting rate and consequent reduction of nursery productivity (Mafia et al. 2012).For that reason, water, sand and tools used in the nursery are expected to be sterilized prior to planting of mother plants in order to avoid potential infections with Ralstonia inoculum.Pathogen free water can be obtained through sand filtration system combined with chlorination process (Ferreira et al. 2012) and sterilization of tools can be done by means of thermal treatment at 85-90 °C for at least 30 s (Mafia et al. 2012).However, an issue experienced by nursery managers (author's personal information) is related to operationalization of steam sterilization currently used in most forestry clonal nurseries.Hence the need for an alternative sterilization method.Our findings show that chemical treatment with sodium hypochlorite and hydrogen peroxide were able to reduce the concentration of bacterial cells in the sand by 97% and 42%, respectively, when compared with the control.Thus, chemical sterilization becomes an important and effective alternative method to eradicate inoculum from the sand.Another advantage of using chemical treatment is that the sand can be placed directly into the beds and then treated, helping on the operationalization of large nurseries.It is noteworthy that flushing with sterile water must always happen after chemical treatment to avoid phytotoxicity, especially when the sand is treated with sodium hypochlorite.
Obviously, treatment of vascular diseases is considered very difficult.Instead, preventative measures to avoid the interaction between plant and pathogen should be considered as the major strategy for disease management.Preventative measures have been widely studied to avoid bacterial inoculum in forestry nurseries (Alfenas et al. 2009).Nevertheless, when Ralstonia is somehow established in the nursery, antibacterial chemicals becomes an important tool to assist on plant treatment.Oxolinic acid has been used commercially in Israel since 1998 for the suppression of Erwinia amylovora, a bacterial disease in pear orchards (Shtienberg et al. 2001).Moreover, antibacterial efficacy of streptomycin has already been demonstrated against R. solanacearum in solanaceous crops (Verma et al. 2014).Although the use of antibacterial chemicals have been successfully applied, it is important to note that a reasonable use of pesticide must be considered when treating the plants in order to avoid pathogen resistance (Manulis et al. 2003;Kleitman et al. 2005).
Deployment of genetically improved plant material through vegetative propagation, especially via mini-cuttings, is an important strategy to increase forestry productivity, but also brings great concerns about potential bacterial wilt outbreaks (Alfenas et al. 2006).In our study, when a susceptible eucalyptus clone was submitted to different propagation methods and challenged to bacterial wilt infections under field conditions, plants propagated via mini-cuttings showed the highest disease incidence amongst all treatments.On the other hand, micro-cuttings propagated in the tissue culture laboratory showed a significantly lower disease incidence.Plant propagation in tissue culture is based on aseptic techniques that offers various advantages over the conventional methods and has a special role on supplying disease free planting material (Srivastava et al. 1999).Micro-cuttings do not require preparation of mother plants to supply propagation material; instead, clean explants are obtained from shoot multiplication in aseptic tissue culture laboratory and then later transferred to the nursery.This process minimizes the chances of spreading the disease from nursery to the field, since the planting material is supposedly disease free.As expected, bacterial wilt incidence on micro-cuttings in the field was not absent, but significantly lower.These infections may have occurred in the nursery after shoot multiplication in the tissue culture laboratory, suggesting that water, potting media or tools used in nursery facilities were not free from bacterial inoculum.Moreover, the site where the trial was established had a history of high bacterial wilt incidence and field inoculum may have contributed to the symptoms observed in micro-cuttings.
Based on the research conducted over the past 30 years, it has been demonstrated that there are sources of resistance against bacterial wilt on eucalyptus, and that the selection for disease resistance is indeed possible (Wu and Liang 1988;Dianese et al. 1990;Gan et al. 2004;Wang et al. 2011;Wei et al. 2014;Fonseca et al. 2016).Screening for disease resistance is particularly difficult for the pathosystem Rasltonia × Eucalyptus due to inconsistent reproducibility of symptoms even under controlled conditions, however, efficient inoculation methods have been studied previously and provided reliable protocols (Wei et al. 2014;Fonseca et al. 2016).In our study, the inoculation protocol was successfully implemented and helped to properly screen eucalyptus genotypes for resistance to bacterial wilt.Eucalyptus clones varied in their susceptibility to infection by R. pseudosolanacearum, and most of the clones herein tested were classified as resistant, including E. pellita clones.Eucalyptus pellita is amongst the most planted eucalyptus species in Sumatra, by means of pure species or in interspecific hybrid crosses.Despite observations of previous studies have pointed out a higher susceptibility of E. pellita clones to bacterial wilt (Wu and Liang 1988;Old et al. 2003), our study showed no clear evidence that this species was more susceptible than others.Different levels of resistance were observed in our set of clones, which included pure species and hybrids.Of the eight resistant clones, three were pure E. pellita.Thus, all these resistant materials can be further deployed in plantations, although other silviculture problems would then also have to be addressed.To validate the results of artificial inoculations, additional evaluation under field conditions should be considered prior to the commercial deployment (Sniezko 2006), especially when stressful conditions in the field are important contributing factors for the expression of disease symptoms (Coutinho and Wingfield 2017).
The most important principles and practices for integrated disease management relies on prevention and suppression of the pathogen, followed by monitoring and decision making, and finally control with the use of pesticides (Steiro et al. 2020).These concepts are well aligned with the management of bacterial wilt of eucalyptus.Therefore, to achieve a satisfactory control is deemed important to combine control methods documented in this study with the current good practices adopted in forestry nurseries and plantations in Sumatra, Indonesia.

Fig. 1
Fig. 1 Inoculation method of Ralstonia pseudosolanacearum on eucalyptus.A -Inoculation done at the base of the stem; B -Incubation at 28 °C, RH 70-80%, and 12 h photoperiod; C -Wilt symptoms at 4 weeks after inoculation; G -Comparison between susceptible and resistant clones; E -Xylem colonization; F -Bacterial ooze observed under a light microscope at 100 × magnifications

Fig. 2
Fig. 2 Symptoms of bacterial wilt caused by Ralstonia pseudosolanacearum on eucalyptus in the province of Riau, Sumatra, Indonesia.A and C -Wilt symptoms in the field; B -Red arrows indicating bacteria exuding from freshly cut stem; D -Bacterial ooze streaming out of infected tissues of one-year-old mother plant; E -Wilt symptoms in the nursery; F -Xylem discoloration

Fig. 6 Fig. 5
Fig. 6 Chemical sterilization of sand beds.A and B -Methodology used to test chemical treatment at different depths in nursery beds; C -Efficiency of chemical sterilization.Different lower case letters above bars indicate statistically different means based on Fisher's least significant difference test (p < 0.001)

Table 1
Rating scale to assess the resistance of eucalyptus plants against Ralstonia pseudosolanacearum

Table 3
Resistance of eucalyptus clones to bacterial wilt caused by Ralstonia pseudosolanacearum1 Eucalyptus clone ECL01 2 Means followed by the same letter within the column do not differ statistically from each other by Scott-Knott cluster test (p < 0.001)

Table 2
Efficacy of chemical treatment on the control of Ralstonia psedosolanacearum on eucalyptus