Abstract: Background and Objective: The utilization of biological control agents using microorganisms is considered as one of the safest and most affordable strategies. This study was conducted to investigate the antagonistic potential of endophytic bacteria against Phytophthora palmivora causing black pod rot disease on cocoa in Indonesia. Materials and Methods: Endophytic bacteria were explored from healthy cocoa pods in Java, Sulawesi and Papua islands. Their antagonistic potential was screened using dual culture method. Bacterial isolates combating the growth of P. palmivora were grouped using rep-PCR technique (BOX1A, ERIC and REP primers). Their inhibition consistency was examined against P. palmivora using double layer technique. Histological assay on mycelial of pathogen was performed under SEM. The DNA of representative isolates was molecularly sequenced according to 16S rRNA and gyrB genes. The effectiveness of their antagonism under in vivo assay was observed on the P. palmivora-inoculated healthy cocoa pods. Results: The growth of P. palmivora was totally inhibited by 127 isolates. The clustering with rep-PCR assay revealed 12 groups of isolates which were independent on cacao clones, orchards and geographical origins. Several isolates showed the inhibition zone under double layer test. SEM viewed morphological abnormality as well as hyphal lysis, shrinking and wrinkling. The representative isolates were identified as members of Achromobacter, Alcaligenes, Bacillus, Burkholderia and Sphingobium genera. The optimum inhibition under in vivo experiment was exhibited by B. subtilis. Conclusion: The explored antagonists have possibility as alternative sustainable disease management strategy under appropriate formulation and application techniques as well as favourable environmental condition.
INTRODUCTION
Cacao (Theobroma cacao L.) is one of important commodities in Indonesia sharing about 15% of net export in the world1 and contributing around 19.50 and 15.43% of global production in 2010 and 2011, respectively (as the second leading country in cocoa bean production after Ivory Coast)2. Indonesia exported 521,300 t (equal to US$ 1.3 billion) of cocoa products in 2009 so that they occupied the third revenue in plantation sector following oil palm and rubber3. However, Indonesia was listed as the third cocoa producing country after Ivory Coast and Ghana with decreasing4 production from 740,513 t in 2012 to 659,800 t in 2017.
Black pod rot disease of cocoa caused by Phytophthora palmivora was considered as one of three main pests and diseases affecting cocoa production in Indonesia5. Many researchers have been conducted for management of black pod rot disease on cocoa in Indonesia, such as the use of antagonistic fungi under laboratory condition6,7, application of phosphonate through trunk injection8, liquid smoke of coconut shell9 and the combination of urea and lime10 as well as the screening on resistance cacao clones against P. palmivora11-15.
The utilization of biological control agents using microorganisms is interesting approach since it is considered as one of the safest and most affordable strategies16. Several species of bacterial endophytes have been isolated from various parts of cacao tree as well as other crops and their antagonistic potential as biological control agents against P. palmivora and other cacao pathogens has been examined17-25. However, there are a few reports associating with the study of potential indigenous antagonistic bacteria over P. palmivora. Therefore, we studied the antagonistic potential of indigenous bacteria towards P. palmivora causing black pod rot disease on cocoa in Indonesia, primarily their consistency in inhibiting the pathogen under in vitro and in vivo conditions, genetic diversity and interaction with pathogen under electron microscope as well as molecular identification.
MATERIALS AND METHODS
The present study including isolation, culture of isolates, in vitro and in vivo assays as well as molecular activities were carried out in Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta (Indonesia) during the year 2018-2019. Meanwhile, histological test and DNA sequencing were performed in The Integrated Research and Testing Laboratory of Universitas Gadjah Mada, Yogyakarta (Indonesia).
Exploration, isolation and preparation of bacterial and pathogen isolates: The potential antagonistic bacteria were explored from healthy cocoa pods of several cocoa producing areas in Indonesia, such as Java, Sulawesi and Papua islands. Samples were packed in dry paper, put into plastic bag to maintain the freshness during the transportation prior to isolation.
Endophytic bacteria were isolated using serial dilution method26. Healthy cocoa pods were peeled and cut into small pieces. Ten gram of pod pieces were put into Erlenmeyer containing 100 mL of phosphate buffer (pH 7) added with 10 μL of tween 20, shaken for 1 h and diluted up to 10–8 dilution. The suspension of 10–6 to 10–8 dilutions was spread on tryptone soya agar (TSA) medium and incubated for 48 h. Growing colonies were counted.
The isolates were purified as the single colony from tested serial dilution on TSA medium using streak isolation method and incubated for 48 h under temperature of 28°C. They were then selected according to its morphological characteristics such as shape (form, elevation and margin) as well as colony colour and kept were kept in slant TSA medium for further assay.
The WAT1 isolate of P. palmivora was one of the collection isolates from Kulon Progo, Yogyakarta (Indonesia) which had been morphologically identified based on its distinctive characters and molecularly detected using PCR with species-specific primers27. It was considered as high virulent isolate under virulence test.
Screening of potential antagonistic bacteria under dual culture assay: P. palmivora isolate was cultured on the centre of petri dish containing potato dextrose agar (PDA) medium and different bacterial isolates were streaked on the four margin sides of same plates. The hyphal growth of pathogen was observed and measured after 1 week incubation at room temperature. The radial of mycelia toward the streak inoculation sites (R2) and the mycelial of pathogen on control (streaked with sterile distilled water/SDW) (R1) were measured and percentage of inhibition was calculated using following formula25:
The bacterial isolates showing optimal inhibition (indicated with no hyphal growth of pathogen) were selected for further genetic diversity analysis.
DNA extraction: Genomic DNA of bacterial isolates were extracted following procedure of Joko et al.28 and Dwimartina et al.29 with a slight modification. Bacteria were cultured on TSA medium for 48 h at 28°C. Their colonies were swapped and suspended into 1.5 mL tubes containing a half volume of SDW. The solution was centrifuged at 5,000 rpm for 2 min and supernatant was discarded. As much of 500 μL of tris-EDTA (TE) buffer and then homogenized using vortex. The solution was added with 30 μL of 10% sodium dodecyl sulphate (SDS) and then incubated at 37°C for 1 h. The next step was addition of 80 μL 5M NaCl and 60 μL CTAB/NaCl as well as incubation at 65°C for 10 min (inverting the tube several times every 5 min). As much of 700 μL of chloroform isoamyl alcohol (CIAA) (24:1) was added, homogenized, centrifuged at 10,000 rpm for 10 min. Upper part of solution was transferred into new 1.5 mL tubes. Total of 600 μL phenol CIAA (PCIAA) (25:24:1) was added and centrifuged at 10,000 rpm for 10 min. Again, upper part of solution (about 500 μL) was transferred into new 1.5 mL tubes. DNA was then precipitated with about 300 μL isopropanol, incubated at -20°C for 1 h and centrifuged at 10,000 rpm for 10 min. As much of 300 μL of 70% ethanol was added, homogenized and then centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and the pellet was air-dried in the laminar air flow. The pellet was resuspended with 40 μL of TE buffer and kept under -20°C condition.
PCR assay using BOX, ERIC and REP primer sets: Primer sets of rep-PCR, namely BOX, ERIC and REP elements and their PCR condition were presented in Table 1. Each 25 μL of PCR reaction containing ddH2O, PCR ready mix (Bioline, London, UK), 100 μM of forward and reverse primers and DNA template was performed using PEQSTAR XS (VWR International Ltd., Lutterworth, Leicestershire, UK). PCR products were employed for electrophoresis in 2.5% agarose gel (added with 2 μL of Greensafe Premium staining solution (Nzytech, Lisboa, Portugal)) at 100 V for 45 min using electrophoresis device of Powerpac Basic (Bio-Rad, Hercules, CA, USA). The gel was visualized under Bio-Rad UV Transilluminator (Bio-Rad).
Phylogenetic analysis for rep-PCR assay: The band patterns were evaluated by recapitulating into 0-1 table (in which 0 for no appear band and 1 for appearing band). Dendrogram was constructed using NTSYS 2.10e program (Exeter Software, Setauket, New York, USA). For construction of dendrogram, the 0-1 table was set by putting the band arrangement in row and isolate number in column using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
| Table 1: | Primer sets used in this study |
aSequence of primer referred to previous researches, while PCR condition for 16S rRNA and gyrB were modified in this study | |
The table was saved in text format of Microsoft (Microsoft Corporation) and then analysed using Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm. Dendrogram was created and then saved in paint format of Microsoft (Microsoft Corporation).
Double layer test of potential antagonistic isolates: The isolates representing group or sub-group of genetic diversity were employed for using double-layer method of Gajbhiye et al.37 with a slight modification. One microliter of bacterial suspension was spread on PDA plates. Previously, its optical density (OD) was measured and adjusted to be 0.1 under wavelength of 600 nm using spectrophotometer GenesysTM 10S UV-VIS (Thermo Fisher Scientific, Waltham, MA, USA). The mycelial disc of P. palmivora isolates was cultured on same plates after the suspension dried. The growth of pathogen and inhibition zone was observed for 1 week. The percentage inhibition was calculated using above formulation25. The representative isolates showing optimal inhibition against pathogen were continued for identification with DNA sequencing using 16S rRNA and gyrase subunit B (gyrB) genes.
Observation of inhibition activity under scanning electron microscope (SEM): The histological analysis was conducted according to the method of Jung et al.38 and Mendez-Bravo et al.39. The plates of dual culture and double layer assays of P. palmivora and antagonistic bacterial isolates as well as untreated culture of pathogen were prepared for observation the antagonistic activity under scanning electron microscope (SEM) JSM-6510LA (JEOL Ltd., Akishima, Tokyo, Japan) at The Integrated Research and Testing Laboratory of Universitas Gadjah Mada, Yogyakarta (Indonesia).
DNA Sequencing using 16S rRNA and gyrB genes: Fragment of 16S rRNA and gyrB genes was amplified with corresponding universal primers and under PCR condition presented in Table 1. The 50 μL of PCR reaction containing ddH2O, PCR ready mix, 100 μM of forward and reverse primers and DNA template was performed using T100 Thermal Cycler (Bio-Rad). PCR products were analyzed by electrophoresis on 1% agarose gel (added with 2 μL of Greensafe Premium staining solution (Nzytech, Lisboa, Portugal) in TBE buffer at 70 V for 45 min using electrophoresis device of Powerpac Basic and then visualized under Bio-Rad UV transilluminator. The amplified products were sequenced using ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Phylogenetic analysis for identification of selected representative bacterial isolates: The consensus sequence was analysed using Mega 7.0 program40 and then treasured with BLAST program at NCBI (www.ncbi.nlm.nih.gov) to find sequence homology for identification of bacterial taxonomy. The phylogenetic tree was constructed under maximum likelihood method with 1000 replicates of bootstrap using Mega 7.0 program40. An outgroup species was included for comparison.
In vivo antagonism test of identified antagonistic bacterial isolates on cocoa pod: This assay was carried out following the method of Setyowati et al.41 on cocoa pod of the most susceptible clone (RCC71)12 collected from cocoa plantation of Segayung Unit, PT. Pagilaran Tbk (Batang, Central Java, Indonesia). Healthy cocoa pods were surface-sterilised using 70% alcohol and then rinsed with SDW. The pods were sprayed with suspension of 13 antagonistic bacterial isolates (108 CFU mL–1) (12 isolates from this study and one isolate from previous research of Setyowati et al.41 showing the highest inhibition level) prior to inoculation with high virulent isolate of P. palmivora. They were incubated for 3 days under room temperature and then the mycelial disc of pathogen was inoculated on wounded surface of the pods. The inoculated-pods were incubated at ambient temperature for a week. The uninoculated and bacterial untreated-pods was considered as negative control, while the inoculated ones with application of SDW was positive control. For comparison, the pods were sprayed with systemic fungicide (active ingredient of mefenoxam and mancozeb) (Ridomil Gold) (Syngenta International AG, Basel, Switzerland) on recommended dosage.
The lesion or necrotic area (cm2) was measured using transparent millimetre block and the disease severity was calculated using the following formula42:
| DS | = | Disease severity (%) |
| ni | = | Number of symptomatic pods on corresponding score |
| vi | = | Corresponding score of symptoms, i.e. |
| Score 0 | = | No symptom |
| Score 1 | = | Lesion or necrotic area between 0 and 20 cm2 |
| Score 2 | = | Lesion or necrotic area between 20 and 40 cm2 |
| Score 3 | = | Lesion or necrotic area between 40 and 60 cm2 |
| Score 4 | = | Lesion or necrotic area between 60 and 80 cm2 |
| score 5 | = | Lesion or necrotic area more than 80 cm2 |
| N | = | Total number of observed pods |
| V | = | Highest score |
RESULTS
Exploration and isolation of bacterial isolates: The explored isolates were various among serial dilutions, i.e., 1-673 colonies (3-474 colonies in average) with number of colony type 1-4 in range (average of 1-3) (Table 2). The colony types were differentiated according to shape (form, elevation and margin) and colour. It was found circular and irregular form with raised, convex and umbonate elevation, entire, undulate and lobate margin as well as white, cream and light brown colour.
Several plates from Batang (Central Java), Nganjuk (East Java), North Minahasa (North Sulawesi), Papua and Ciamis (West Java) showed the reducing colony number following the decline of serial dilutions from 10–6 to 10–8. However, such results were not consistently obtained from all cultures. Similarly, variation of colony type was not consistent among serial dilutions. Only some cultures from same geographical origins, excluding North Minahasa, revealed the decreasing colony type corresponding to diminishing serial dilutions.
| Table 2: | Bacterial isolates explored from healthy cocoa pods of cocoa growing areas of Indonesia |
Screening of potential indigenous antagonistic bacteria: From 362 screened isolates, the hyphal growth of P. palmivora was completely inhibited by 127 isolates (PI 100%), while around 2.5-38 mm (range of PI around 55.29-97.25%) of its hyphal growth was recorded in dual cultures with 72 bacterial isolates (Table 3). The remaining isolates were neglected since they could not grow under this antagonism assay, particularly those from North Sulawesi. The selected potential antagonistic bacterial isolates originated from West Java, Central Java, East Java and Papua (Table 4).
Genetic diversity of screened antagonistic bacteria using rep-PCR: There were 12 clusters of antagonistic bacteria within range of similarity index around 71-100% (Fig. 1). Those groups were independent on clone of cocoa, orchards and geographical origins. The group members varied from 1-37 isolates in which small groups consisted isolates from 1-3 geographical areas (i.e., group III, VI, VII, IX, XI and XII), while the remaining large clusters originated from 3-5 cocoa growing areas. According to their diversity within groups or sub-groups, a range of 1-14 isolates (total of 66 isolates) were selected from each cluster as representative isolates for double layer assay (Table 5).
Double layer test of potential antagonistic bacterial isolates: This second screening revealed that 46 isolates consistently showed maximum inhibition against P. palmivora with zero mycelial growth.
| Table 3: | In vitro screening of potential antagonistic bacteria isolates against Phytophthora palmivora using dual culture test |
aPercentage of inhibition (%) of antagonistic bacterial isolates against P. palmivora in which the mycelial growth of P. palmivora as control is about 85 mm | |
| Fig. 1: | Dendrogram of genetic diversity with rep-PCR elements clustering the potential antagonistic of endophytic bacteria after in vitro dual-culture test |
Most isolates did not express clear inhibition zone, while 19 isolates demonstrated consistent, quite consistent and inconsistent inhibition zone with range of diameter approximately 4.7-12.7 mm, 1.3-5.3 mm and 0.6-2.7 mm, respectively (Table 6).
A quite consistent inhibition was shown by other 5 isolates with colony diameter of P. palmivora about 0.7-2.0 mm (PI around 97.33-99.06% in range) and diameter of inconsistent inhibition zone about 0.6-13.3 mm in range. The growth of pathogen was also inconsistently inhibited by 4 isolates (colony diameter about 1.3-8.0 mm or PI around 78.88-98.27% in range) and even 11 isolates were the most inconsistent in hampering the growth of P. palmivora (colony diameter around 1.7-46.7 mm or PI about 37.73-97.73% in range).
These quite and inconsistent inhibiting isolates performed inconsistent inhibition zone with range of diameter approximately 0.6-13.3 mm. Meanwhile, there was no any inhibition zone which was exhibited by most of inconsistent and whole most inconsistent isolates.
The consistency of inhibition within bacterial isolates was independent on group. Only one-member clusters, such as cluster III, IX and XII were found consistently inhibiting the growth of P. palmivora with consistent inhibition zone, consistent inhibition without any inhibition zone and quite consistent inhibition with inconsistent inhibition zone, respectively.
Observation of inhibition activity under scanning electronic microscope (SEM): The scanning electron micrograph showed the morphological abnormality as well as shrinking and lysis indicating the damage of P. palmivora hyphae under confrontation with antagonistic bacterial isolates both on dual culture (Fig. 2a) and double layer tests (Fig. 2b).
| Table 4: | Selected isolates for further genetic diversity analysis using rep-PCR technique |
| Table 5: | Representative isolates corresponding to their diversity clusters for double layer test |
| Table 6: | Double layer test of selected potential antagonistic bacteria isolates against Phytophthora palmivora |
Percentage of inhibition (%) of antagonistic isolates against P. palmivora in which the mycelial growth of P. palmivora as control is about 75 mm, bTheir consistency in inhibiting mycelial growth is compared with the previous dual culture test and among three replications of double layer test, aTheir consistency in producing inhibition zone is compared among three replications of double layer test |
|
| Fig. 2(a-c): | (a) Scanning electron micrographs visualizing the antagonistic action of endophytic bacteria against P. palmivora under dual culture, (b) Double layer assays at 2,000X magnification and (c) Normal hyphal growth of P. palmivora without any lysis and bacterial cells adhering the mycelium showed by control at 3,000X magnification |
The lysis was extremely more severe and the adhering rod-shape bacteria were more abundant under dual culture than double layer tests. The intact and normal growing hyphae without any lysis was revealed on P. palmivora under control without any antagonist treatment (Fig. 2c).
Identification of selected representative isolates: Twelve isolates representing those with consistent inhibition, 4 geographical areas and ten clusters were proceeded to molecular identification. They were amplified with 16S rRNA and gyrB genes at approximately 1,475 bp and 940 bp, respectively (Table 7). All representative isolates were positively detected with 16S rRNA primers, while nine isolates were reacted with gyrB primers. Based on their DNA sequencing, they were identified as Achromobacter xylosoxydans, Alcaligenes faecalis subsp. faecalis, A. pakistanensis, Bacillus altitudinis, B. amyloliquefaciens, B. cereus, B. siamensis, B. subtilis, B. velezensis, Burkholderia cepacia, B. ptereochthonis and Sphingobium yanoikuyae with the percentage of identity around 81.12-100% (Fig. 3).
In vivo antagonism test of identified antagonistic bacterial isolates on cocoa pod: The initial disease symptom on bacterial-treated cocoa pods were recorded on 3rd day after inoculation with disease severity around 44-100% in range after a week incubation (Table 8). Meanwhile, fungicide treatment could delay the symptom appearance on the 5th day after inoculation and disease severity about 12% on the last incubation day. The lowest severity of disease was revealed by isolate number 1 corresponding to B. subtilis, whereas another four isolates (number 45, 99, 109 and 127 which were identified as Bacillus spp. and S. yanoikuyae) generated the highest one.
DISCUSSION
This study explored the bacterial on healthy cocoa pod collected from various cocoa clones and orchards in different geographical origins of Indonesia due to the common infection of P. palmivora on pod and little or no information of microorganisms for biological control from cocoa pod17. The resistance level of some cocoa clones in this experiment has been reported12,45. It was found that screened bacteria from those cocoa clones expressing antagonistic potential were around 5-17 isolates (Table 4). The availability of various microbes associating with cacao played important role in its resistance against pathogen12,25. However, current research did not elaborate the correlation of clonal resistance on cocoa with the number of screened antagonistic isolates. It may be investigated in further study.
None of antagonistic bacterial isolates from Sulawesi in this study was parallel to former findings17,22 and might be caused by high disease incidence in the field, i.e., 70-80%46. Future research is required to update novel prevalence of black pod rot disease in Indonesia and its correlation with the existence of beneficial endophytic microbes.
| Table 7: | Molecular identification of representative isolates corresponding consistent inhibition using 16S rRNA and gyrB genes |
| Fig. 3: | Phylogenetic tree constructed under maximum likelihood method with 1000 replicates of bootstrap using Mega 7.0 program for referring the representative potential antagonistic bacterial isolates to the closest bacterial strain at NCBI. Planctomycetes bacterium was considered as out group species |
| Table 8: | In vivo assay on inhibition of black pod rot disease on detached cocoa pod with the application of potential antagonistic bacteria |
Present study screened more bacterial isolates (362 isolates) from cocoa pods compared to previous works17,20,21,24,25,41,44 because of using serial dilution on common agar medium. This abundance finding was comparable to former investigation implementing same isolation technique and solid medium47,48. Higher dilutions of 10–3 to 10–5 could explore 114-511 colonies of epiphytic bacteria from healthy green cacao pods25. Then, the given technique could be recommended to isolate considerable useful indigenous microorganisms.
This experiment revealed the highest in vitro inhibition of antagonistic bacteria against P. palmivora among the previous investigations17,22-25,41,43,44,49-51 (Table 9). This suggested that the exploration of endophytic isolates from healthy pod might be considered as an essential screening stage of antagonistic bacteria for biological control.
Using rep-PCR assay, the present study could classify more than a hundred antagonistic bacterial isolates in short time and a few steps because its consistency and reliability in assessing the genetic diversity as well as specific region of targeted-PCR primers52. More distinct and more informative band profiles found in this investigation was also supported by the previous research on Anoxybacillus species53. It could be noticed that these primer sets were still relevant as rapid and appropriate tools for antagonistic screening.
This study might be considered as the first utilisation of rep-PCR methods in clustering the antagonistic endophytes from cocoa pods. The current findings of antagonistic bacteria reflected high degree genotypic diversity among them on healthy cocoa pods and they complied with former study54. It was assumed that those high genetic variability and independent clusters showed high adaptability of antagonistic bacteria to their environment and expressed the abundance of their hereditary capacity in the long-term evolution process.
Variation in consistency of inhibition performed by screened bacterial endophytes in this study indicated their dynamic antagonistic ability under different in vitro culture conditions. The consistent performance of inhibition under two cultural methods reflected the stability on antagonistic capability of the microbial endophytes under laboratory assay.
Similar antagonistic phenomena under SEM on phytopathogenic fungi were also reported55-61. However, current investigation did not find inhibition of zoospore production and sporangial breakdown since the in vitro antagonism assay on common artificial agar medium was more suitable for mycelial growth rather than the production of those asexual organs. The comprehensive observation is required in future to recognise the effect of antagonistic microbes on the development of reproduction features of pathogen.
Beyond the plant pathology, 16S rRNA and gyrB genes had been used for analysis of microbial community compositions62, recovering the results of long-established procedures63 and for comparative taxonomic analyses64. Hence, these housekeeping genes might be recommended for molecular identification of bacterial isolates using DNA sequencing approach.
| Table 9: | Comparison of documented inhibitory percentage on similar previous reports with this study |
aND means the species of antagonistic bacteria is not determined yet |
|
The dominance of Bacillus species as potential antagonistic endophytes against black pod rot pathogen on cocoa had been also documented18,65. The capability of those five genera of endophytic bacteria against fungal pathogens on cocoa and other crops had been recorded66-75. It convinced that they were potential as beneficial microorganisms for future strategy of sustainable crop disease management. Nevertheless, the scientific justification documenting antagonistic records of A. pakistanensis and B. ptereochthonis against phytopathogenic microorganisms could not be found. Their low identity percentages probably required more accurate and proper molecular identification technique using specific gene region.
The maximum and consistent inhibition of B. subiltis under in vivo test had been previously documented49,51,76-80. Surprisingly, inconsistent results of in vitro and in vivo assays using other antagonists in this experiment were parallel to previous reports41,81 but in disagreement with other works25,50. Such inconsistencies were possibly due to the dependency of in vitro test on interaction of competing microorganisms on rich-nutrient agar medium, the incubation under controlled conditions and the absence of host-plant tissue.
This fundamental study did not implement the antagonistic isolates under the field conditions as the estimated results could be reflected by the in vivo assay. Macagnan et al.17 presumed that variation of environmental conditions and competition amongst microflora of the pods could affect the effectiveness of biological control against cacao pathogens in the field and they suggested to investigate the population dynamics of these antagonists for minimizing the failure of field experiments. The dosage and composition of production medium for antagonist were also reported to affect the effectiveness of biological control in the field assays82. The advanced work is required to determine proper formulation to provide favourable environmental circumstances for optimal activity of microbes in the field.
CONCLUSION
Some endophytic bacteria had been successfully explored from healthy cocoa pods in Indonesia with antagonistic potential against P. palmivora causing black pod rod disease. They have possibility as alternative sustainable disease management strategy under appropriate formulation and application techniques as well as favourable environmental condition.
SIGNIFICANT STATEMENT
This study focused on exploration of bacterial isolates on healthy cocoa pods collected from various cocoa clones and orchards in different geographical origins of Indonesia, since the common infection of P. palmivora was found on pod. Comprehensively, this experiment screened the antagonistic endophytic bacteria through two in vitro assays, clustered them using rep-PCR technique and examined their in vivo inhibition on detached healthy cocoa pods. Furthermore, the representative isolates were then molecularly identified using DNA sequencing of 16S rRNA and gyrB genes. Such screening steps might be expected generates the most effective isolates as biological control agent of P. palmivora on cacao.
ACKNOWLEDGMENT
This research is part of doctoral program under the scholarship from Agency for Extension and Human Resources Development, Ministry of Agriculture, Republic of Indonesia. The grateful expression is also delivered to Universitas Gadjah Mada, Yogyakarta (Indonesia) for financial support of this experiment under RTA program 2019 with Contract No. 3226/UNI/DITLIT/DIT-LIT/LT/2019 and to PT. Pagilaran Tbk. as well as Indonesian Coffee and Cocoa Research Institute for providing the healthy cocoa pods.