Indian Journal of Agricultural Research

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Research Article

Indian Journal of Agricultural Research, volume 55 issue 6 (december 2021) : 727-732

​Control Activity and Antibiotic Gene Detection of Endophytic Bacteria in Suppressing Cocoa Black Pod Disease (Phytophthora palmivora Butl.)

Rian Arini1,3,*, Gusti Ayu Kade Sutariati2, Andi Khaeruni3, Teguh Wijayanto2, Novita Pramahsari Putri3, Tri Joko4
1Department of Agricultural Science, Post Graduate Program of Halu Oleo University, Kendari Southeast Sulawesi, Indonesia.
2Department of Agrotechnology, Faculty of Agriculture, University of Halu Oleo, Kendari, Southeast Sulawesi, Indonesia.
3Department of Plant Protection, Faculty of Agriculture, University of Halu Oleo, Kendari, Southeast Sulawesi, Indonesia.
4Department of Pest and Plant Disease, Faculty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia.
Cite article:- Arini Rian, Sutariati Kade Ayu Gusti, Khaeruni Andi, Wijayanto Teguh, Putri Pramahsari Novita, Joko Tri (2021). ​Control Activity and Antibiotic Gene Detection of Endophytic Bacteria in Suppressing Cocoa Black Pod Disease (Phytophthora palmivora Butl.) . Indian Journal of Agricultural Research. 55(6): 727-732. doi: 10.18805/IJARe.A-659.

ABSTRACT

Background: Black pod rot disease of cocoa caused by (Phytophthora palmivora Butl.) is one of the major diseases on cocoa plantations worldwide. Many attempts have been made to prevent or reduce the infection of pathogens, but they have not provided optimum results. This study aims to detect antibiotic genes in endophytic bacteria that can suppress cocoa black pod disease.

Methods: Eight endophytic bacteria were isolated from healthy cocoa pods and twigs that showed potentials in suppressing P. palmivora growth in vitro were used in suppressing of black pod rot disease in vivo tests. Antibiotic biosynthesis-related genes from eight endophytic bacterial isolates were confirmed by using PCR method, which includes phenazine-1-carboxylic (PCA), pyrrolnitrin (PRN), phenazine-1-carboxamide acid (PCN), pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DPAG/Phl).

Result: The endophytic bacteria, 4RSI, 5BR B3 and 2RW B2 isolates showed the highest disease suppressing index to black pod rot disease in vivo, i.e., 70.27%, 70.08% and 56.64%. The isolates 5BR B3 and 2RW B2 DNA yielded PCR product by using PCA primers (1400 bp), PRN primers (700 bp) and DAPG primers (1600 bp), while the 5RSI isolate yield PCR product using PRN primers only. Endophytic bacterial isolates 5BRB3 and 2RW B2 provided partial disease suppression to pod rot disease by inhibit pathogen growth and antibiotic compounds production.

INTRODUCTION

(Phytophthora palmivora Butl.) which causes pod rot is one of the important pathogens in cacao plants. This disease can reduce world cocoa production by 20-30% every year (Guest, 2007), it can even reduce the yield by 90% (Bowers et al., 2001). Various control techniques such as garden sanitation and use of resistant clones, as well as chemicals have been carried out, but this disease remains a major problem in cacao growing worldwide.
       
Endophytic bacterial is one of the biological agents that has the potential to be developed as a controlling agent for black pod rot disease in cocoa, which is effectively inhibits the development of pathogens and is safe for the environment and consumers (Marwan et al., 2011; Nguyen et al., 2016; Ramli et al., 2016). Endophytic bacteria live in plant tissue without causing disturbance and damage to plant tissue (Munif et al., 2013; Miliute et al., 2015; Kandel et al., 2017). The presence of endophytic bacteria in plant tissue can provide several benefits, one of which can play a role in plant pathogen biocontrol agents, such as: controlling blood diseases in banana plants (Marwan et al., 2011), inhibiting the growth of Fusarium oxysporum f. sp. vanillae causes stems rots of Vanilla (Suniti 2015) and Sclerotium sp. in peanut sprouts (Arios et al., 2014), suppressing the development of Xanthomonas axonopodis pv. glycines in soybean plants (Habazar et al., 2015), Ganoderma boninense in palm trees (Buana et al., 2014), Fusarium oxysporum and Meloidogyne incognita in pepper plants (Wiratno et al., 2019), as well as inhibiting the development of Oncobasidium theobromae causes of vascular streak dieback (VSD) in cocoa (Zubir et al., 2019).
       
Khaeruni et al., (2019a) reported that eight isolates of endophytic bacteria from healthy cacao plants had an inhibitory ability of >50% in vitro against 3 different P. palmivora isolates. A number of endophytic bacteria having the ability to inhibit the development of plant pathogens are reported to produce antimicrobial compounds such as pyrrolnitrin and phenazines (Kirner et al., 1998), 2,4-diecetylphloroglucinol (2,4-DAPG) and pyoluteorin (Subagio and Foster 2003). Genes related to the production of antimicrobial compounds from biocontrol agents can be detected by Polymerase Chain Reaction (PCR) techniques using gene specific primers that encodes phenazine-1-carboxylic acid, 2,4-DAPG, pyoluteorin, phenazine-1-c and pyrrolnitrin from the genomes of Pseudomonas and Burkholderia (Bakthavatchalu et al., 2013). With this background the study was taken up to determine the potential of endophytic bacteria from healthy cocoa plants as a biological controlling agents for control cocoa black pod rot disease in vivo and detect the presence of genes that encode antimicrobial compounds in these endophytic bacterial isolates.

MATERIALS AND METHODS

Source of endophytic bacterial isolates
 
The isolates used for the study include eight endophytic bacterial isolates from healthy cocoa pods and branches that had in vitro inhibition against P. palmivora >50%, namely: isolates 4RSI, 1RSI3, 2RWB2, 5BRB3, 5RSI, 3BTO1, 3BAE, 4RS (Khaeruni et al., 2019a), isolate P. palmivora KS01 collected from of Plant Protection Laboratory, Faculty of Agriculture, Halu Oleo University Kendari.
 
Fruit inoculation in living plants with endophytic bacteria
 
Inoculation of endophytic bacteria on fruit of cocoa in the tree was carried out at the Field Station of Plantation and Horticulture Office, Southeast Sulawesi Province, 8 endophytic bacteria were selected for the study that had inhibitory tests in vitro ≥50%. Selected endophytic bacteria were multiplied in (Trypticase Soy Agar) TSA medium and incubated at room temperature. After 2 days the bacteria were suspended in sterile distilled water until the cell density reached 108 CFU/mL. When treated in the field, bacterial suspension was mixed with 0.1% Tween 80 and sprayed on selected fruit with a relatively uniform age and size (length +10 cm). For each endophytic bacteria, spraying was carried out on three trees of five fruits per tree. A mixture of Tween 80 and sterile aquades was used as a control. After two weeks, the endophytic bacteria treated fruits were picked and then inoculated with P. palmivora in the laboratory.
 
Phytophthora palmivora inoculation on endophytic bacteria treated cocoa fruits
 
The P. palmivora isolate KS01, obtained from a single purified zoospora. The making of P. palmivora inoculum suspension was carried out in accordance with AVRDC Mycology (2000) method, The isolates were grown in V8 agar medium under continuous irradiation at 28°C for 4-5 days. Furthermore, isolates were divided into four, each part was transferred to a new petri dish and cut into 5 mm2 pieces. The pieces were immersed in water for one hour, discarded and flooded again and incubated under continuous irradiation for 24 hours. Then the isolates were transferred to a room at 4°C for 2 hours and 28°C for 1-2 hours. While the method of pathogen inoculation in fruit was carried out according to state by Tondok et al., (2012), harvested zoospores were diluted to 5 × 104 mL-1. Before use, zoospores were stored at 4°C for 3 days to germinate. Sterile circular filter paper 10 mm in diameter was dropped with 1 mL of zoospores, two papers were placed upside down on the surface of the cocoa pods inoculated by endophytic bacterial isolates.
       
Inoculated cocoa pods were then placed in plastic containers, five pods per container. Plastic containers were wrapped with thin plastic and then placed in wet tissue paper. The plastic container was then placed in an incubation rack arranged according to the experiments used. Disease severity was observed at two, four and six days after inoculation with a score: 1= x≤5; 2= 5<x≤15; 3= 15<x≤25; 4= 25<x≤35; 5= 35<x≤45 and 6= x>45, where x = range of symptoms (%) (Tondok et al., 2012).
       
The area under disease progress curve (AUDPC) value and disease Suppression Index (DSI) of each endophytic bacteria is calculated from the severity of the disease in cocoa pods. AUDPC was calculated by the formula proposed by Khaeruni et al., (2018) and DSI was calculated by the formula proposed by Wijayanto et al., (2017).
 
Antifungal encoding gene detection
 
(a) DNA extraction of endophytic antagonistic bacteria
 
Bacterial isolates were grown in the Yeast Peptone Agar (YPA) medium which was incubated for 24-48 hours. Furthermore, DNA extraction carried out according to the method of Schaad et al., (2001) with some modification. One loop of bacterial culture was suspended into 1000 µL sterile aquadest at Eppendorf tube 1.5 ml, then centrifuged at 10000 rpm for 10 minutes and supernatant removed. Next 500 mL TE buffer and 30 μL 10% SDS were added and carefully vortexed, then incubated at 37°C using water bath for an hour. After incubation, 100 μL 5 M NaCl and 10 μL CTAB/NaCl were added first to the above mixture followed by 750 μL Chloroform: Isoamyl-Alcohol, 24:1 (CIAA) and it was shaken until homogeneous for ± 2 minutes, continued with centrifugation at 14000 rpm for 5 minute. The top layer was then transferred to a new Eppendorf tube and added with 600 μL Phenol chloroform: isoamyl alcohol, 24:1 (PCIAA), shaken and centrifuged at 14000 rpm for 5 minutes.
       
As much as ±500 μL of the upper liquid phase was transferred to the new Eppendorf tube, then ± 500 μl of isopropanol was added and incubated at -20°C for an hour. After incubation, the DNA suspension was centrifuged at a speed of 14000 rpm for 5 minutes and then supernatant was carefully disposed. DNA pellets were washed by adding 1 mL of 70% ethanol gollowed by vortex and centrifuge at 14000 rpm for 5 minutes. Then, supernatant was removed and dried the pellet in the Laminar air flow cabinet for ±24 hours. DNA pellets were suspended in 20-40 μL TE buffer and stored in a refrigerator at -20°C.
 
(b)   Detection of antimicrobial encoding genes of endophytic antagonistic bacterial isolates
 
Detection of antimicrobial encoding genes in endophytic bacteria was carried out using PCR method. Specific primers that encoded the antibiotic compounds such as phenazine-1-carboxylic (PCA), pyrrolnitrin (PRN), phenazine-1-carboxamide (PCN), pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DPAG/Phl) genes were used according to the procedure of Bakthavatchalu et al., (2013) (Table 1). The PCR results of each genes were in electrophoreses with 1% agarose gel and visualized using Etidium Bromide (EtBr) via gel doc transilluminator.
 

Table 1: Primer and amplification of gene encoding antimicrobial genes in endophytic bacteria.


 
Statistical analysis
 
In the testing phase of P. palmivora inoculation on fruit that has been picked from the cacao tree, it was arranged based on a completely randomized design (CRD) with eight treatments of endophytic bacterial isolates plus control. Each treatment was repeated three times, bringing the total of the union units to 21 units. Variance analysis was carried out to determine the potential of endophytic bacteria in inhibiting the use of rotten cacao fruit in vivo. Based on the effect, proceeded with Duncan’s multiple range test (DMRT) at a 95% significance level.

RESULTS AND DISCUSSION

Disease severity, area under disease progress curve and disease suppressing index
 
All endophytic bacteria tested have the ability to inhibit the development of cocoa pod rot, this is evidenced by the disease severity which is always lower than the control (Table 2). At sixth DAI (day after inoculation), the endophytic bacterial isolates 2RWB2 and 4RSI treatments showed the lowest disease severity value of 25.55%, which is significantly different from control (70% disease severity). The suppression of disease progression by 2RWB2 and 4RSI endophytic bacteria was also evident from the lower AUDPC values compared to other treatments, recorded 11.12 and 11.11 units respectively (Table 2).
 

Table 2: Disease severity of cocoa fruit rot treated with endophytic bacteria.

  
 
The results of the analysis of the disease suppression index showed that eight endophytic bacterial isolates tested, 5 isolates were obtained, namely 4RSI, 5BRB3, 2RWB2; 3BAE and 1RSI3 which have disease suppression index> 50%, two of which endophytic bacteria 4RSI and 5BRB3 have disease suppression index above 70% (Table 3).
 

Table 3: AUDPC of fruit rot disease on cocoa treated with endophytic bacteria.

     
 
The PHZ1 and PHZ2 successfully amplified 1400 bp DNA fragment from 2RWB2, 5RSI and 5BRB3 isolates. The Prncf and Prncr were successfully amplified 700 bp size DNA fragment from the endophytic isolates of 2RW B2, 5RSI and 5BR B3, while the main pair of Prncf and Prncr successfully amplified DNA fragment of 700 bp in size from endophytic bacterial isolates of 2RW B2, 5RSI and 5BRB3. Succeeded in amplifying 1600 bp DNA fragment of DPAG/Phl gene in 2RW B2 and 5BRB3 isolates (Fig 1, 2 and 3).
 

Fig 1: PCR product of phenazine-1-carboxylic acid (PCA) gene amplification by PHZ1 and PHZ2 primers.


 

Fig 2: PCR product of pyrrolnitrin (PRN) gene amplification by Prncf and Prncr primers


 

Fig 3: PCR product of 2,4-diacetylphloroglucinol(DPAG/Phl) gene amplification by Phl2a and Phl2b primers.


 
The main pair of PhzH-up and PhzH-low and the main pair of PLTC1 and PLTC2 did not succeed in amplifying the DNA fragment of all tested isolates. These results indicate that endophytic bacterial isolates 2RW B2 and 5BRB3 contain three antimicrobial encoding genes namely pyrrolnitrin, phenazine-1-carboxamide and 2,4-diacetylphloroglucinol. Isolate 5RSI contain antimicrobial encoding genes such as pyrrolnitrin, while five other isolates contain none of the antimicrobial encoding genes that have tested (Table 4).
 

Table 4: Summary of PCR products of antibiotic genes from endophytic bacterial isolates.


 
Phytophthora palmivora is an important pathogen and can attack all parts of cacao plant (Azis et al., 2013), especially on immature fruits are the most detrimental (Rubiyo and Amaria 2013). Cocoa fruit inoculation in the field with endophytic bacteria followed by inoculation of P. palmivora in the laboratory gave different responses to the severity of the disease in cocoa. Endophytic bacterial inoculation in cocoa can inhibit the development of fruit rot disease, this is evidenced by the disease severity which is always lower at all times of observation in all the treatments of endophytic bacteria compared to control (Table 1).
       
At the end of the observation (sixth day after inoculation (DAI) the severity of the disease in the treatment of endophytic bacteria ranged from 25.55%-65.55%, while the control severity reached 70%. Endophytic bacterial treatment of 4RSI and 2RW B2 isolates showed the lowest disease severity of 25.55% and it was significantly different from control at 5% level. The lower disease severity in the treatment of endophytic bacteria 4RSI and 2RW B2 was recorded with followin by AUDPC values which were also relatively lower, respectively 11.11% and 11.22%, while the control reached 32.22%. Endophytic bacteria used in this study have the ability to inhibit the development of P. palmivora in vitro >50%. Of the 8 isolates tested, there were 5 isolates, namely: 4RSI, 5BRB3, 2RWB2, 1RSI3 and 3BAE, consistently showed an index of suppression to the development of P. palmivora in vivo >50%, even 4RSI3 and 2RWB2 isolates were able to suppress the development of cocoa black pod rot disease to 70.27% and 70.08% respectively (Table 2).
       
Previous researchers reported that the ability of endophytic bacteria as biological control agents against phytopathogens in cacao plants in both in vitro and in vivo. In vitro, endophytic bacteria LKM-UL is able to inhibit the development of P. palmivora from cacao plants (Hamzah et al., 2017), endophytic bacteria 2RWB2 and 5BPR1 in vitro are able to inhibit the development of Colletotrichum gloeosporioides causing anthracnose in cacao each by 69% and 62% (Khaeruni et al., 2019b). In vivo BT8 endophytic bacteria were able to inhibit the development of P. capsici inoculated on cacao leaves (Melnick et al., 2008) and LKM-BL endophytic bacteria at the age of 30 days after inoculation were able to inhibit the development of VSD (Vascular Streak Dieback) in the cacao nursery by 87.90% (Zubir et al., 2019).

The ability of endophytic bacteria to inhibit the development of pathogens and increase is related to various antagonistic mechanisms, including synthesizing antibiotic compounds, produce of hydrolytic enzymes, nutrient limitation and by priming plant defenses (Gao et al., 2010; Afzalab et al., 2019). The result of previous studies showed that some endophytic bacterial isolates used in this study, including isolates 5BRB3, 3BAE, were able to produce cellulase and protease hydrolytic enzymes (Khaeruni et al., 2019a), 2RWB2 isolate able tolyse the cell wall of C. gloesporioides, thought to be related its ability to cellulase produce the enzyme chitinase (Khaeruni et al., 2019b).
       
The results of antibiotic-encoding genes detection by PCR technique using specific primers showed that 2RWB2 and 5BRB3 DNA isolates are able to amplified PCA encoding genes of 1400 bp size, PRN encoding genes of 700 bp and DPAG encoding genes of 1600 bp, this indicating that both isolates have the potential to produce three antibiotic compounds namely phenazine-1-carboxylic acid, pyrrolnitrin and 2,4-diacetylphloroglucinol, whereas from the endophytic bacterium 5RSI isolate amplified PRN encoding gene size of 700 bp indicated the potential for producing pyrrolnitrin antibiotic compounds. DNA amplification measurements obtained from each antibiotic-coding gene primer were used in this study by accordance with the results of Bakthavatchalu et al., (2013). Pyrrolnitrin encoding gene was successfully amplified in three different isolates, this indicates that the pyrrolnitrin compound is one of the antibiotic compounds that play a role in controlling plant pathogens by antagonistic bacteria. Endophytic Bacteria, Burkholderia sp. which has antagonistic activity against G. anodermaboninense has the potential to produce pyrrolnitrin antibiotic compounds (Buana et al., 2014), Enterobacter agglomerans which have strong antagonistic activity against various types of pathogens detected to produce the enzyme chitinase and pyrrolnitrin compounds (Chernin et al., 1996). Pyrollnitrin is a chlorinated phenypyrrole antibiotic first isolated from Burkholderia pyrrocinia (Kloepper and Ryu, 2006). Pyrollnitrin is synthesized by four proteins encoded by 4 genes, prnA, prnB, prnC and prnD. The prnD gene is the final protein to form an active pyrrolnitrin compound. The prnD catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to the pyrrolnitrin form (Kirner et al., 1998).

CONCLUSION

It was concluded that the ability of endophytic bacterial isolates 2RW B2 and 5BR B3 to produce antibiotic compounds such as pyrrolnitrin, phenazine-1-carboxylic and 2,4-diacetylphloroglucinol suspected to be one of the antagonistic mechanisms play an important role in inhibiting P. palmivora infections in cocoa fruit, as evidenced by the value of 2,4-diacetylphloroglucinol. Disease Suppression Index (DSI) of the two isolates were quite high at 56.64% for 2RWB2 and 5BRB3 at 70.08%, witnessing the potential to develop as a biological control agent against cocoa pod rot. In addition, the 5RSI isolate also has the potential to be developed as a biological agent because it has a DSI of 70.27% cocoa pod rot disease, the highest of all the isolates tested, although no gene was detected for encoding the antibiotic compound tested, but perhaps the ability of the antagonist through other antibiotic encoding genes or another antagonistic mechanism.

ACKNOWLEDGEMENT

This research was funded by the Directorate of Research and Community Service of the Ministry of Research, Technology and Higher Education through the Competency Based Basic Research Grants in 2018, with Contract No: 479/UN29.20/PPM/2018 dated February 28, 2018.

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