We are investigating in this study the screening and selection of cocoa tree (Theobroma cacao L.) endophytic bacteria having antagonistic effects against the fungi Phytophthora spp. and to assess their inhibitory potential as biological control agents against black pod disease in Côte d'Ivoire. A total of One hundred and sixteen bacteria were isolated from symptomless root, leaf and stem tissues of two cocoa clones NA32 (susceptible) and P7 (resistant) young seedlings. These isolates restricted the radial growth of the mycelium of P. palmivora (strain BL7.11.2) and P. megakarya (strain 13P30.1) with percentages inhibition of 28.58 to 70.54% and 25.3 to 64.29% respectively. Four isolates, 48P, 60P, 23P and 18N were more effective in in vitro test and selected for in vivo confrontation test on leaf discs. Treatments based on two isolates S1 (48P) and S2 (18N) applied at the concentration 109 CFU/mL significantly (p < 0.05) reduced the black pod necrosis induced by the two Phytophthora strains on the leaf discs of three cocoa clones (NA32, PA150 and SCA6). In addition, simultaneous (C3PS1 and C3PS2) or separate (C3AVS1 and C3AVS2) inoculations of these two bacteria and Phytophthora, induced resistance to the clone NA32 and increased the intrinsic resistance of the clones PA150 and SCA6. This study showed that endophytic bacteria antagonistic to Phytophthora spp. can be an alternative solution in the biocontrol of black pod disease. In situ pod tests should be performed to confirm these preliminary results and propose effective bacterial inoculum.
The high incidence of black pod is a major constraint to cocoa production in West Africa, particularly in Côte d'Ivoire 1, 2. The control of this disease is therefore both a necessity and a priority 3, 4. Several integrated control approaches have been suggested to eradicate this disease. Copper and metalaxyl-based fungicides have been widely used 5. Other agronomic methods such as harvesting diseased pods and using partially resistant and/or tolerant varieties have been also applied 6, 7. However, none of them have given conclusive results. Biological control agents have been considered as an alternative approach to control various plant diseases 8. The use of endophytic bacteria as biological control agents against plant diseases has attracted considerable interest in scientific research 9. Their ability to colonize the host plant tissues has made them valuable and effective for sustainable agriculture and has provided a tool to improve crop yield compared to other biological agents 10, 11. As internal colonizers of the root system, endophytes can compete within the vascular system, inhibiting pathogens to obtain both nutrients and space for their proliferation. Many bacteria belonging to the genera Bacillus sp, Pseudomonas sp, Burkholderia sp and fungi such as Trichoderma spp and Aspergillus spp have effective antifungal activity to control Phytophthora diseases 12, 13, 14. The aim of this study was to screen and to select cocoa tree (Theobroma cacao L.) endophytic bacteria with antagonistic effects against Phytophthora spp. and to assess their potential as biological control agents against black pod disease in Côte d'Ivoire.
Two strains BL7.11.2 and 13P30.1 of Phytophthora palmivora and Phytophthora megakarya respectively isolated in the year 2000 and 2013 from naturally infected cocoa pods were used. These strains were stored in a mycobank at National Agronomic Research Center (CNRA)'s Phytopathology Laboratory in Bingerville (Côte d’Ivoire) and were periodically isolated once more from infected cocoa pods to maintain their virulence.
One hundred and sixteen (116) endophytic bacteria were isolated from young seedlings organs (roots, stems and leaves) of two clones (NA32 and P7 respectively susceptible and resistant to black pod) as described by Konaté et al. 15 and Rayda et al. 16.
Young symptomless leaves of three cocoa clones NA32, PA150 and SCA6 respectively susceptible, moderately resistant and resistant to black pod, were collected. The sampling was done from non-lignified twigs of ten trees per clone at the Divo research station (CNRA). A total of seven hundred and twenty (720) leaves were taken, at the rate of two hundred and forty (240) leaves per clone and twenty four (24) leaves per tree. The collected leaves were stored in moistened plastic bags and transported in a cooler.
2.2. MethodsIn vitro pairing assays were conducted on carrot extract agar against pathogens Phytophthora palmivora and P. megakarya. Bacteria were streaked in line dividing the agar plates into two equal parts 17. Two discs of mycelium calibrated to 5 mm in diameter from a five-day-old culture of each strain of Phytophthora grown on pea extract agar were placed apart on each side of the bacterial streaks (01 cm from the edge of the Petri dish). Control plates consisted of the mycelial discs without bacterial streaks. Plates were then incubated in darkness at 26 ± 2°C and were observed after twenty four hours. Three replicate plates were prepared for all bacteria and for each pathogen strain.
Growth of Phytophthora strains was recorded by measuring the diameter of the colonies each day using a ruler, as shown in Figure 1 and ended when one of the mycelium colonies covered the entire plate. The percentages inhibition (P.I.) exerted by endophytic bacteria on the radial growth of Phytophthora mycelium were estimated according to the following formula 18.
 |
Where P.I. (%) is the average percentage inhibition; Dn is the average diameter of mycelial colonies in the presence of bacteria and Dt is the average diameter of control plates mycelial colonies.
After thirty days of in vitro confrontation, seven bacteria (11P; 18N; 23P; 42P; 47P; 48P; 48P and 60P) having a percentage inhibition greater than or equal to 59 % with the two strains of Phytophthora were tested on wounded pods to see the survival and aggressiveness of the pathogen. Mycelium explants (05 mm in diameter) of each Phytophthora strain were collected along a line on the contact fronts of mycelium and bacterial colonies. The explants were inoculated in wounded pods at a rate of four pods per bacterial isolate. The pods were then placed in bins on foams soaked in distilled water and closed with black tarpaulins and were incubated at 26 ± 2°C for five days.
Fresh cultures of each bacterial isolate were performed in a pea extract broth. After forty eight (48) hours of incubation, the suspensions (109 CFU/mL) were diluted to a final concentration of 106 and 103 CFU/mL.
A suspension of zoospores of each strain was obtained from a sixteen-day-old culture on pea extract agar in Roux’s vials. After six days of incubation in darkness, the cultures were exposed to a twelve-hour photoperiod (an alternation of 12 h/12 h darkness and fluorescent light) for ten days. In order to obtain zoospores release, cultures of P. palmivora and P.megakarya were flooded with sterile distilled water at 4°C and exposed to incandescent light for forty five minutes. The zoospores suspension was then adjusted with a Malassez hemacytometer (SOVIREL, France) to 3 x 105 zoospores/mL 19.
The in vivo antagonist effect of endophytic bacteria against Phytophthora palmivora and P. megakarya was evaluated on leaf discs (15 mm in diameter). This test was conducted for each Phytophthora strain by a randomized four-factor split-plot experimental design. The first factor was the cocoa clone and three clones (NA32, PA150 and SCA6) which susceptibility to Phytophthora is known (susceptible, moderately resistant and resistant) were used. The second factor was the four bacterial isolates (60P; 48P; 18N and 23P) selected for good percentage inhibition. The third factor was the test of three concentrations (C1=103, C2=106, C3=109 UFC/mL) of each bacterial inoculum. Finally, the fourth factor was the variation of three application periods of the bacterial inoculum. First period named (AV) consisted in applying the bacterial inoculum one hour before the inoculum of Phytophthora spp. The second period (P) consisted in simultaneously applying the bacterial inoculum and that of Phytophthora sp. Finally, the third period (AP) consisted in applying the bacterial inoculum one hour after the Phytophthora spp. The leaf discs placed in bins on foam of (01cm) thickness soaked in distilled water were inoculated with aliquots of 10 μL of suspension of each bacterial fresh culture at different concentrations 103, 106 and 109 CFU/mL. Each leaf disc then received, according to the type of treatment, 10 μL of a suspension of a strain of Phytophthora calibrated at 3×105 zoospores/mL. Controls were inoculated only with zoospore suspensions of the Phytophthora strains used. A total of thirty six treatments at the rate of nine treatments per bacterial isolates were applied to the leaf discs of the three cocoa clones used. Ninety leaf discs per clone arranged in three lines of ten discs per type of treatment were inoculated with each bacterial suspension and distributed in four bins each constituting a repeat. After inoculation of the leaf discs, each bin was covered with a black tarpaulin and incubated at 26 ± 2°C for seven days. The results were read according to the Blaha scale 19, 20, whose black pod symptom score ranged from 0 to 5 (0: no symptoms; 1: point of penetration; 2: network points; 3: cross-linked task; 4: marbled task; 5: real task).
All data were analyzed using SAS® software 9.4 21. The P.I. were transformed at arcsinus√ to adjust them to the normal distribution 22 and then submitted with the data obtained in leaf discs tests to ANOVA according the PROC GLM program (Process of General Linear Model) followed by Student-Newman-Keuls test at the threshold α = 5 %.
Bacterial isolates of cocoa tree significantly inhibited mycelial growth of both Phytophthora strains in confrontation tests, as shown in Figure 2. The value of the P.I. of mycelium radial growth recorded after thirty days, varied significantly (p < 0.05) from 25.3 to 70.54 % depending on the bacterial isolates and the two fungal strains tested, as shown in Table 1. In fact, the isolates 48P; 18N; 42P; 60P; 47P; 47P and 23P were highly effective against P. palmivora with P.I. of 70.54 to 62.20% or diameters between 24.75 and 31.75 mm of mycelial growth. The isolates 42P; 47P; 60P; 18N; 22N; 66P and 63P were highly effective against P. megakarya with P.I. varying from 64.29 to 60.13% or diameters between 30 to 33.5 mm of mycelial growth. A total of four bacterial isolates 18N; 42P; 47P and 60P showed effective antagonistic activity against these two Phytophthora strains with P.I. above 60%.
The results of survival and virulence tests of Phytophthora after thirty days of in vitro confrontation test with the seven effective bacterial isolates (11P; 23P; 42P; 47P; 48P; 60P and 18N) were variously conclusive. Indeed, mycelial plugs collected from the plates containing isolates 18N; 23P; 48P and 60P did not induce any symptoms of black pod disease in the wounded pods during five days of incubation. However, the other three isolates (11P; 42P and 47P) did not reduce this aggressiveness.
The application of bacterial treatments significantly reduced (p < 0.05) the susceptibility to black pod disease of the three clones. Significant effects (p < 0.05) of factors clone and timing of bacterial inoculum application were noted. Similarly a significant interaction effect (p < 0.05) of clone and bacterial factors on the black pod disease symptoms was observed. Treatments based on bacteria S1 (48P), S2 (18P) and S4 (23P) applied to foliar discs significantly reduced black pod disease necrosis induced by both Phytophthora strains to the three clones (NA32; PA150 and SCA6). Leaf discs treated with these bacteria showed mostly no symptoms or simple penetration point (indices values 0 or 1), unlike the clone NA32 controls where necrosis network (indices values 2.8 and 3.0) were observed for Phytophthora palmivora and P. megakarya, as shown in Figure 3. Bacterial isolates S1 (48P) and S2 (18N) highly inhibited both Phytophthora strains. Furthermore, bacterial isolate S4 (23P) moderately inhibited both Phytophthora strains. Unlike the three bacterial isolates cited above, the isolate S3 (60P) showed no inhibitory effect towards the two Phytophthora strains. Because, treatments based on this bacteria were statistically close to the two controls of the clone NA32. Treatments at the concentration 109 CFU/ml of the two bacterial isolates S1 (48P) and S2 (18N) were highly effective. These bacterial isolates effectively reduced black pod necrosis on the foliar discs of the three clones, particularly the susceptible clone NA32, as shown in Figure 3.
3.2. DiscussionEndophytic bacteria isolated from the vegetative organs of cocoa tree exerted in vitro a high inhibitory activity on the mycelial growth of the both strains of Phytophthora. After thirty days of confrontation in vitro, an effective reduction of the mycelium diameter and an inhibition zone on both sides of bacterial streaks were observed. That demonstrate isolates which hindered the growth of Phytophthora produced inhibitory substances 23. Survival tests conducted on wounded pod confirmed the antagonistic effect of isolates 18N; 23P; 60P and 48P. However, isolates 42P; 47P and 11P were found to be ineffective. These isolates simply inhibited the growth of Phytophthora by competing for nutrients in the culture medium. Their antagonistic effects were simple fungistatic effects 3. In contrast, isolates 18N; 23P; 60P and 48P could have fungicide effects against Phytophthora palmivora and P. megakarya. Those effects would be more or less effective depending on the bacterial isolate and the strain of Phytophthora. Studies have shown the existence of Phytophthora antagonistic microorganisms in the cacao tree ecosystem 20, 23.
These isolates inhibited the radial growth of P. palmivora (BL7.11.2) and P. megakarya (13P30.1) mycelium on carrot extract agar plates with P.I. of 28.58 % to 70.54 % and 25.3 to 64.29 % respectively. Similar results were obtained by Akrofi et al. 18 who showed that epiphytic bacteria isolated from pods surface of three resistant cocoa clones (SCA6, T85/799 and IFC5), highly inhibited the growth of Phytophthora palmivora with P.I. of 69.7 and 65.8 %. Isolates S1 (48P) and S2 (18N) effectively reduced black pod symptomatic necrosis onto all three clones, particularly onto the susceptible clone NA32. This showed that these isolates induced resistance to the clone NA32 on the one hand, and increased the intrinsic resistance of the two clones (PA150 and SCA6) on the other hand. Kébé et al. 20 also isolated indigenous rhizobacteria from cocoa tree soils. These bacteria inhibited Phytophthora palmivora on leaf discs of two resistant cocoa clones (P7 and SCA6). According to Mejia et al. 24 the results of in vitro tests do not necessarily reflect those obtained in vivo or in field tests. Some microorganisms found to be highly antagonistic in vitro may be ineffective in in vivo or in field tests due to many ecological parameters.
The effectiveness of bacterial treatments applied for one hour on leaf discs before the introduction of the fungal inoculum proves that these endophytic bacteria, once installed, create a barrier against the penetration of pathogens in the stomata. This could explain the induction of resistance to the susceptible clone NA32. In addition, these isolates certainly produce antifungal substances that neutralize the development of zoospores on the foliar discs even when the inoculum of Phytophthora spp was applied one hour before the inoculum of the bacterial isolates was applied.
The cocoa tree naturally hosts endophytic bacteria that may have antagonistic effects against certain fungal pathogens such as Phytophthora palmivora and Phytophthora megakarya. In fact, these bacterial isolates significantly inhibited, in vitro, the mycelial growth of these two strains of Phytophthora and induced resistance against Phytophthora spp. to different cocoa clones. This resistance induction was more remarkable to the clone NA32, susceptible to black pod disease. This study therefore proves the antagonistic potential of cocoa endophytic bacteria and their possible use in biological management of black pod disease. These bacterial isolates may be tested in pod tests and field trials to confirm the stability of their antagonistic effects against Phytophthora spp.
We would like to thank Mr. Bakary Coulibaly, Research Technician and Mr. Touré Laurent, Research Assistant, at the Phytopathology laboratory, for the appreciable help provided during the leaf inoculation tests.
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Published with license by Science and Education Publishing, Copyright © 2020 OUATTARA Adama, COULIBALY Klotioloma, KONATE Ibrahim, KEBE Boubacar Ismaël, BEUGRE G.A. Maxwell, TIDOU Abiba Sanogo and Abdelkarim FILALI-MALTOUF
This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit
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| [1] | Ploetz R. The Impact of Diseases on Cacao Production: A Global Overview in: B.A. Bailey, L.W. Meinhardt (eds.), Cacao Diseases, Springer International Publishing Switzerland.p33-59. 2016. | ||
| In article | View Article | ||
| [2] | Coulibaly K., Aka R. A., Camara B., Kassin E., Kouakou K., Kébé B. I., Koffi N. K., Tahi M. G., Walet N. P., Guiraud S. B., Assi M. E., Kone B., N’Guessan K. F, Koné D.. Molecular Identification of Phytophthora palmivora in the Cocoa Tree Orchard of Côte d’Ivoire and Assessment of the Quantitative Component of Pathogenicity. Internationale journale of science, 7 (8). 7-15. August 2018. | ||
| In article | View Article | ||
| [3] | Mpika Joseph, Kebe Ismaël B., Druzhinina Irina S., Komon-Zélazowska Monika, Kubicek Christian P. & Aké Sevérin. Inhibition de Phytophthora palmivora, agent de pourriture brune des cabosses de cacaoyer en Côte d’Ivoire, par Trichoderma sp. Sciences & Nature, 6(1). 49-62. Mars 2009. | ||
| In article | View Article | ||
| [4] | Mbargaa J.B., Begoude B.A.D., Ambang Z., Meboma M., Kuate J., Schiffers B., Ewbankd W., Dedieu L., ten Hoopen G.M. A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya. Biological Control, 77. 15-22. June 2014. | ||
| In article | View Article | ||
| [5] | Pohe Jean, Pohe Sédjouény Steve Wilfrid, Okou Staelle Florence Famisso. Association oxyde de cuivre et metalaxyl dans la lutte contre la pourriture brune des cabosses de cacaoyer en Côte d’Ivoire. Journal of Animal &Plant Sciences, 16(3). 2362-2368. January 2013. | ||
| In article | |||
| [6] | Iwaro A.D., Thévenin J.-M., Butler D.R. and Eskes A.B. Usefulness of the detached pod test for assessment of cacao resistance to Phytophthora pod rot. European Journal of Plant Pathology, 113. 173-182. August 2005. | ||
| In article | View Article | ||
| [7] | Tahi, G.M., Kébé, B.I., N’Goran, A.K.N., Sangare´, A., Mondeil, F., Cilas, C., Eskes, A.B. Expected selection efficiency for resistance to cacao pod rot (Phytophthora palmivora) comparing lead disc inoculations with field observations. Euphytica 149, 35-44. November 2006. | ||
| In article | View Article | ||
| [8] | Tjamos, E.C., Tjamos, S.E. & Antoniou, P.P. Biological management of plant diseases: Highlights on research and application. Journal of Plant Pathology, 92(4). 17-21. May 2010. | ||
| In article | |||
| [9] | Soylu, S., Soylu, E.M., Kurt, S. & Ekici, O.K. Antagonistic potentials of rhizosphere-associated bacterial isolates against soil-borne diseases of tomato and pepper caused by Sclerotinia sclerotiorum and Rhizoctonia solani. Pakistan Journal of Biological Sciences, 8(1). 43-48. 2005. | ||
| In article | View Article | ||
| [10] | Nur Rashyeda Ramli, Maizatul Suriza Mohamed, Idris Abu Seman, Madihah Ahmad Zairun & Nasyaruddin MohamadThe Potential of Endophytic Bacteria as a Biological Control Agent for Ganoderma Disease in Oil Palm. Sains Malaysiana, 45(3). 401-409. 2016. | ||
| In article | |||
| [11] | Ouattara Adama, Coulibaly Klotioloma, Konate Ibrahim, Kebe Boubacar Ismaël, Tidou Abiba Sanogo, Filali-Maltouf Abdelkarim. Selection of Cocoa Tree (Theobroma cacao Linn) Endophytic Bacteria Solubilizing Tri-Calcium Phosphate, Isolated from Seedlings Grown on Soils of Six Producing Regions of Côte d’Ivoire. Advances in Microbiology, 9. 842-852. September 2019. | ||
| In article | View Article | ||
| [12] | Melnick Rachel L., Suárez Carmen, Bailey Bryan A., Backman Paul A. Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases. Biological Control, 57. 236-245. March 2011. | ||
| In article | View Article | ||
| [13] | Abdallah Aydi-Ben Rania, Marwa Hassine, Hayfa Jabnoun-Khiareddine, Hichem Ben Jannet & Mejda Daami-Remadi. Etude du pouvoir antifongique in vitro et in vivo des filtrats de culture et des extraits organiques des Aspergillus spp. contre Phytophthora sp. Microbiol. Hyg. Alim, 25(73).15-25. July 2013. | ||
| In article | |||
| [14] | Rika Fithri, N.B., Aris Tri, W. & Nurita, T.M. Control activity of potential antifungal-producing Burkholderia sp. in suppressing Ganoderma boninense growth in oil palm. Asian Journal of Agricultural Research, 8. 259-268. 2014. | ||
| In article | View Article | ||
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