Antagonistic Activities of Epiphytic Bacteria on Black Pod Disease of Cocoa

A study was conducted to determine the antagonistic activity of epiphytic bacteria isolated from cocoa pod surface on Phytophthora palmivora (Butler) Butler, the causal agent of black pod disease of cocoa. Out of 233 isolates of epiphytic bacteria obtained from non symptomatic and diseased cocoa pods, only were found to have antagonistic activities against the fungal pathogen when screened by the dual-culture method. Three isolates, (LKM/B/1, LKM/B/35, LKM/B/76c) were identified as Pseudomonas putida Biotype A, 2 isolates (LKM/B/5 and LKM/B/62b) were identified as Pseudomonas aeruginosa and one isolate each was identified as Pseudomonas spinosa (LKM/B/2), Burkholderia glidioli (LKM/B/4) and Burkholderia sp (LKM/B/6). Identification of the epiphytic bacteria was done using the BIOLOG® Identification System. Percentage inhibition of the radial growth (PIRG) of P. palmivora by the epiphytic bacteria ranged from 66.0% to 82.1%. All isolates of epiphytic bacteria showed optimum antagonistic activities at 300 C. pH has no influence on the antagonistic activities of the epiphytic bacteria. Detached pod studies showed that pods treated with the epiphytic bacteria retarded the growth of black pod lesion up to 12 days after inoculation. Isolate Burkholderia glidioli (LKM/B/4), Pseudomonas aeruginosa (LKM/B/5) and Pseudomonas putida Biotype A (LKM/B/76c) produced volatile substances that affect the growth of P. palmivora, while isolates LKM/B/1 and LKM/B/35 of Pseudomonas putida Biotype A and isolate LKM/B62b of Pseudomonas aeruginosa did not show the production of volatile substances. Isolate LKM/B/76c also produced diffusible metabolites that could significantly inhibit the growth of P. palmivora compared to other epiphytic bacteria especially isolate LKM/B/35 which showed good antagonistic through dual culture method. Microscopic observations of P. palmivora at the periphery of the inhibition zone indicated that all isolates of epiphytic bacteria inhibited P. palmivora by the process of cell wall degradation and growth retardation

Effect of climate variables on cocoa black pod incidence in Sabah using ARIMAX model

Cocoa black pod disease is one of the major diseases affecting the cocoa production in Malaysia and also around the world. Studies have shown that the climate variables have influenced the cocoa black pod disease incidence and it is important to quantify the black pod disease variation due to the effect of climate variables. Application of time series analysis especially auto-regressive moving average (ARIMA) model has been widely used in economics study and can be used to quantify the effect of climate variables on black pod incidence to forecast the right time to control the incidence. However, ARIMA model does not capture some turning points in cocoa black pod incidence. In order to improve forecasting performance, other explanatory variables such as climate variables should be included into ARIMA model as ARIMAX model. Therefore, this paper is to study the effect of climate variables on the cocoa black pod disease incidence using ARIMAX model. The findings of the study showed ARIMAX model using MA(1) and relative humidity at lag 7 days, RHt − 7 gave better R square value compared to ARIMA model using MA(1) which could be used to forecast the black pod incidence to assist the farmers determine timely application of fungicide spraying and culture practices to control the black pod incidence

Isolation, identification and characterization of endophytic bacteria antagonistic to Phytophthora palmivora causing black pod of cocoa in Malaysia

Black pod rot is the most significant factor limiting production of cocoa (Theobroma cacao) in Malaysia with average annual losses of above 30%. This work was carried out to isolate, characterize and screen bacterial endophytes from cocoa plants for their biological control activities. Their mechanisms of action as well as abilities to reduce black pod rot disease were also investigated. In total, 103 endophytic bacterial isolates were obtained from healthy cocoa tissues (leaves, branches and fruits) from seven states of Malaysia in 2016 and screened for their antagonism against P. palmivora in vitro. The best two isolates AS1 and AS2 with more than 80% inhibition of radial growth (PIRG) were selected for subsequent experiments. Sequence analysis of the 16S rRNA region indicated that these two isolates belonged to Pseudomonas aeruginosa (AS1) and Chryseobacterium proteolyticum (AS2). Bioactive volatile compounds were identified using gas chromatography-mass spectrometry (GCMS). Major compounds present in P. aeruginosa extract were identified as Eicosane (9.11%), Hexatriacontane (6.87%), Tetratetracontane (5.17%), trans-2-Decenoic acid (17.04%) and 1-Phenanthrenecarboxylic acid, 1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl) (3.60%). In C. proteolyticum extract, major compounds were identified as Eicosane (11.29%), Tetratetracontane (10.82%), Heneicosane (10.78%), Hexatriacontane (9.04%) and Phenol, 2,4-bis(1,1-dimethylethyl) (5.92%). Effectiveness of P. aeruginosa and C. proteolyticum in reducing black pod lesion was confirmed on detached cocoa pods with 100% inhibition for both isolates. These results indicated that these two bacterial isolates have potential to be used as bio-control agents against P. palmivora

ISOLATION, IDENTIFICATION, PATHOGENICITY AND SENSITIVITY OF RHIZOCTONIA SPP. TO PHENAZINE-1-CARBOXYLIC ACID (PCA)-PRODUCING PSEUDOMONAS SPP.

Rhizoctonia root rot and bare patch is the most important disease of direct-seeded wheat and barley in the Inland Pacific Northwest. Major gaps remain in understanding the epidemiology of this disease and the biology and ecology of Rhizoctonia solani AG-8 and R. oryzae, the causal agents. In these studies, a collection of 498 isolates of R. solani, AG-I-like binucleate Rhizoctonia sp., and R. oryzae groups was assembled from fields throughout the Inland Pacific Northwest. Isolates were identified by PCR with primers specific to internal transcribed spacers of the nuclear ribosomal DNA and/or by DNA sequence analysis of the ITS regions. The results revealed the geographic distribution of Rhizoctoniaisolates in cereal-based production systems. R. solaniAG-8 and R. oryzae groups II and III (but not group I) caused severe root rot on wheat. R. solaniAG-2-1 caused only mild root rot and the other groups showed trace discoloration of the roots. In contrast, R. solaniAG-2-1 caused severe damping-off of canola and killed seedlings in greenhouse assays. Distinctive morphological characteristics were described for isolates of R. solaniAG-8, AG-2-1, and AG-10, AG-I-like binucleate Rhizoctonia, and R. oryzae groups I, II, III. These results demonstrated for the first time that colony morphology and amplification by specific PCR primers are predictive of the identity of an isolate of Rhizoctonia on wheat and canola.The distribution of fluorescent Pseudomonas spp. capable of producing the antibiotic phenazine-1-carboxylic acid (PCA) was shown to overlap closely with that of R. solaniAG-8 but not that of R. oryzae AG-2-1 and to exhibit a highly significant inverse correlation with annual precipitation. Sensitivity of R. solaniAG-8 and AG-2-1 to PCA was not correlated with exposure in nature or with virulence, indicating that tolerance of these pathogens to the antibiotic does not develop in nature.Representative isolates from each of the four major phylogenetic groups of Phz+ pseudomonads were shown to control root rot of wheat caused by R. solanias well or better than did the model PCA producer P. fluorescens 2-79

Grouping Tolerant Level of Cocoa Genotypes Against Pod Rot Disease in Malaysia

Cocoa pod rot disease (CPRD), caused by Phytophthora palmivora, is the main disease that caused major losses in Malaysia. It is important for screening the cocoa genotypes available in Malaysia for their tolerance level against the CPRD. This paper has an objective to select the potential genotypes tolerant to P. palmivora by grouping the cocoa genotypes available in Malaysia based on four tolerant levels such as tolerant, moderately tolerant, moderately susceptible and susceptible. The experiment was carried out at the laboratory of Plant Pathology at the Cocoa Development and Research Centre Jengka, Pahang using the detachedpod test. Isolate of P. palmivora was obtained from a naturally infected cocoa pod in cocoa field at the Cocoa Research and Development Centre Tawau, Sabah,Malaysia then inoculated by a single point on the ridges of pod to 40 mature unripe pods of each tested genotypes. Fifty genotypes were tested in this study.The assessed disease severity was the rate of lesion area development from 1 to 7 days after inoculation and the proportion of pod area infected by CPRD. Thedisease severity was significantly different among tested genotypes showing tolerance variability against CPRD. Four nonlinear models consisted of Monomolecularmodel, Exponential model, Logistic model and Gompertz model were used to fit the proportion pod infection area curve. The best fitted Gompertz model was used in calculated the area under disease progress curve (AUDPC). The variability of both disease severity variables was used to group the genotypes into four tolerant levels using the k-means clustering method with 10 genotypes in group I (torelant), 14 genotypes in group II (moderately tolerant), 13 genotypes in group III (moderately susceptible) and 13 genotypes in group IV(susceptible). Six genotypes in group I, namely MCBC 13, PBC 221, BAL 209, KKM 19, QH 1176 and KKM 22 were identified to have lower disease severity values compared to control tolerant genotype PBC 123 that could be suggested to the farmers to be planted in the field.&nbsp

Cocoa pod husks as precursors for biosynthesis of carbon dots as potential bioimaging tool

Recent zero-dimensional carbon dots (CDs) have dominated the world of nanomaterials due to their ease of synthesis and nature of their precursors. The aim of this study is to synthesize, characterize and evaluate cytotoxicity of CDs from agricultural waste CPH for its potential use in the bioimaging field. The TEM analysis and particle size distribution curve revealed that the particles had a diameter of 10-30 nm, sphere-shaped and exhibited lattice fringes with a d-spacing of 0.196 nm. XRD analysis revealed a broad peak at 2Ɵ = 20.71°, indicating the existence of carbon. FTIR confirmed the presence of multiple functional chemical groups on the surface of the CPH CDs consist of C=O, N–H, C–N, and C–O–C. Due to the electronic transition’s characteristic of CDs, the UV-Vis absorption spectrum revealed two distinct peaks at 235 and 293 nm. PL spectra of CPH CDs revealed a red shift in the emission peak from 400 to 410 nm as the excitation wavelength increased from 320 to 380 nm. We used brine shrimp and human colon adenocarcinoma cells (Caco2) in vitro to determine the cytotoxicity of CPH CDs. In terms of brine shrimp assay, we found that 0.001 mg/ml showed lower lethality percentage with 57.93 ± 9.77 %. The cytotoxicity of CPH CDs was assessed in vitro using the MTT assay and Caco2 cell line's viability decreased with increasing concentration (IC50 = 155 ug/ml). Due to their favorable properties and low cytotoxicity, CPH CDs have the potential to be used as bioimaging tools

Phylogenetic analysis and genetic diversity of Phytophthora palmivora causing black pod disease of cocoa in Malaysia

Black pod, caused by Phytophthora spp., occurs worldwide and is a major problem for cocoa farmers in Malaysia. Limited studies addressed causal agents of black pod disease of cocoa in Malaysia as well as their genetic diversity. Therefore, this study was initiated to isolate and identify Phytophthora from the main cocoa plantations infected by black pod in Malaysia using sequence analyses of the ITS rDNA, EF-1α, and COX1 gene regions. A total of 36 Phytophthora isolates were obtained from infected cocoa plantations from five states of Malaysia in 2016 and 14 isolates in 2013. Six Phytophthora isolates obtained from the durian crop, “the king of fruit”, in 2013 were also used in this study. Results of phylogenetic analyses of the combined dataset of the ITS rDNA, COX1, and EF-1α confirmed that all Phytophthora isolates belonged to P. palmivora. P. palmivora isolates obtained from cocoa and durian clustered into different subclades based on the three regions examined. The study also examined the genetic diversity within a population of 56 P. palmivora isolates using random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. The results of both markers indicated relatively high diversity among P. palmivora isolates. The complete separation was based on host and year of isolation. The study suggests that one species of Phytophthora, namely, P. palmivora, is responsible for black pod of cocoa in Malaysia. However, the relatively high genetic diversity and separation of isolates into different clades may suggest that P. palmivora has been introduced into Malaysia via different sources