Control of gray mold disease of tomato caused by Botrytis cinerea using bacterial secondary metabolites

Gray mold disease is caused by Botrytis cinerea and it is the most significant and devastating disease affecting tomato cultivation worldwide. This disease was recently reported infecting tomato fruits in Cameroon Highland, Pahang. Biocontrol agents have been proven to be effective towards many phytopathogens. In this study, 43 bacterial isolates were screened against B. cinerea in-vitro. Five isolates with more than 65% inhibition of radial growth (PIRG) were selected for the subsequent experiments. Findings in dual culture assay revealed that BM11 and BC4 were the most effective biocontrol agent, with PIRG of 82.5% and 71.8%, respectively. BM11 and BC4 isolates were identified using the 16S rDNA gene. The identification results found that BM11 and BC4 were Pseudomonas protegens and Brevibacterium casei, respectively. Ten major bioactive compounds were successfully identified by Gas chromatography-mass spectrometry (GC-MS) from the culture filtrate of both biological control agent (BCA) isolates. These bioactive compounds may responsible for antifungal activity. In vivo study revealed that culture filtrate extract manages to suppress disease lesions on the treated tomato fruit from 0.86 -1.03 cM, 30 days after inoculation. Based on overall findings suggested that P. protegens and B. casei were the promising BCA in controlling gray mold disease of tomato fruit

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

Characterization and genetic diversity of Phytophthora palmivora from cocoa in Malaysia and its biological control using endophytic bacteria

Black pod disease of cocoa, caused by Phytophthora palmivora, is a serious problem to cocoa growers in Malaysia and worldwide. There are a few research efforts have been conducted to study the causal agent of black pod disease of cocoa in Malaysia. At present, there are no reports available on the determination of genetic diversity among P. palmivora populations in cocoa growing areas of Malaysia. Therefore, this study was initiated to isolate and identify Phytophthora species from main cocoa plantations infected by black pod disease in Malaysia using sequence analyses of the ITS rDNA, cytochrome c oxidase subunit 1, and translation elongation factor 1α regions. This study was also examined the genetic diversity within 56 P. palmivora isolates obtained from the different host, geographical regions and years in Malaysia using Inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. In addition, this study 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 the ability to reduce black pod rot disease were also investigated. A total of 36 Phytophthora isolates were obtained from different infected cocoa plantations from five states of Malaysia. Six Phytophthora isolates obtained from durian and available at the Department of Plant Protection, Universiti Putra Malaysia were also used in this study. Results of phylogenetic analyses of individual and combined dataset of the ITS rDNA, COX I and EF-1α confirmed that all Phytophthora isolates belonged to P. palmivora. The results also confirmed that the three examined regions could differentiate P. palmivora at the species level. P. palmivora isolates obtained from cocoa and durian were clustered into different subclades based on the three regions examined. The results of both markers indicated relatively high diversity among P. palmivora isolates. The complete separation was based on host and year of isolation. In total, 100 endophytic bacterial isolates were obtained from healthy cocoa tissues (leaves, branches and fruits) from seven states of Malaysia. The isolated bacteria were screened by dual culture assay and culture filtrate test for their antagonistic properties towards P. palmivora in vitro. The best two isolates Pseudomonas aeruginosa (AS1) and Chryseobacterium proteolyticum (AS2) with more than 80% inhibition of radial growth (PIRG) were selected for subsequent experiments. Bioactive volatile compounds from ethyl acetate crude extract were identified using gas chromatography-mass spectrometry (GC). The effectiveness of Pseudomonas aeruginosa and Chryseobacterium proteolyticum isolates in reducing black pod lesion was confirmed on detached cocoa pods. These results confirmed that these two bacterial isolates have potential to be used as bio-control agents against P. palmivora

Morphological and molecular characterisations of Phytophthora palmivora infecting cocoa in Pahang and Perak, Malaysia

Phytophthora spp. are one of the most destructive plant pathogens worldwide. Many economically important crop species such as cocoa, rubber, jackfruit, durian, taro, papaya, pepper, coconut, potato, citrus and forest trees are susceptible to various diseases caused by Phytophthora spp. Agronomists and plant pathologists have been aware of the economic importance of diseases caused by Phytophthora in Southeast Asia. However, there is a lack of published information on Phytophthora spp. in the tropics. Although many studies have been conducted over the past few decades to overcome diseases caused by Phytophthora spp., the general information is often not outlined with details while specific information of occurrence and economic impact are dispersed in many different publications in various languages. Black pod disease of cocoa caused by Phytophthora palmivora is a serious problem of economic importance to cocoa growers in Malaysia. It is also known to cause stem canker disease where their control in infected cocoa plantations is very difficult. This research was undertaken to study the cultural characteristics, reproduction, and pathogenicity as well as molecular characteristics of fourteen isolates of Phytophthora spp. randomly obtained from infected cocoa pods in two different states, Pahang and Perak in Peninsular Malaysia. The cultural characteristics of these isolates were assessed on four types of agar medium namely PDA , CA, VJA and CMA incubated at 25 ± 2°C showed various textures of colonies as fluffy with irregular margin on PDA, cottony with uniform margin on CA, slightly cottony with regular margin on VGA and flattish with regular margin on CMA. It did not show specific colony pattern on all four types of medium and each medium demonstrated its own morphological appearance. Growth rates of colonies were measured after three days of incubation on four types of agar media for one week and demonstrated that isolates grew significantly faster on CMA with 13.3mm/day, followed equally by VJA and CA with 10.0 and 10.2mm/day respectively, and the poorest growth was on PDA with 6.1mm/day. Dimensions of sporangia on CMA medium were measured averaging 43.48μm in length, 29.37 μm in width, 3.53 μm in pedicel length and 5.31 μm in size of exit pore. On culture media, the sporangia were of different shapes. Sporangia of isolates were all caducous. Zoospores were induced on four types of medium CA, VJA and CMA. Isolates on CA medium produced the largest number of zoospores than on VJA and CMA medium. Chlamydospores were produced on VJ broth ranging between 25.21 to 21.55 μm in diameter. Molecular identification using ITS region of rDNA confirmed that all isolates were Phytophthora palmivora. Eight isolates were selected randomly for pathogenicity tests on green healthy pods of KKM22 cocoa cultivar. The test results confirmed that isolates RB03 and RB04 from Pahang significantly more virulent with size of lesion 129mm and 117.75mm correspondingly compared to other isolates after five days of inoculation. Hence, complete brown to black lesions covered all inoculated after nine days

Detection and identification of cacao black pod causal agent by polymerase chain reaction (PCR)

Among the Phytophthora species that cause black pod of cacao, P. palmivora is the most virulent, posing a serious threat to cacao production in Asia. Correct identification of the species causing the black pod and understanding the virulence factors involved are important for developing sustainable disease management strategies. The present study had revealed molecular characterization of thirty isolates of P. palmivora randomly obtained from infected cocoa pods in different states in Malaysia. All 30 isolates of P. palmivora were subjected to molecular identification methods using ITS rDNA, the mitochondrial cox1 and elongation factor 1α genes. Phylogenetic tree for P. palmivora was constructed using the sequencing of ITS rDNA, mitochondrial cox1 and elongation factor 1α genes. Different Phytophthora spp. sequencing had been chosen from NCBI and Pythium undulatum as outgroup. Molecular identification of rDNA confirmed that all isolates were Phytophthora palmivora. Phylogenetic analysis based on rDNA sequencing of different genes were separated different species of Phytophthora to various clades. P. megakarya is considered the closest species with P. palmivora based on ITS rDNA, translation elongation factor 1alpha and Cox subunit I. The significant of these findings are also viewed in terms of the increasing importance of Phytophthora diseases in this region

Genetic variability of Rhizoctonia spp. isolated from different hosts and locations

Rhizoctonia species is well known as a destructive fungal pathogen in various agro-ecosystems globally. The current study attempted to investigate the genetic diversity among various anastomosis groups of Rhizoctonia species obtained from different locations and crops. Two types of markers were used to detect the genetic variability among isolates. The results showed that all the molecular markers were able to show reasonable polymorphisms. The clustering tree and PCA plot analysis supported the separation based on taxonomic groups more than the separation according to geographical origin and host. This indicates that there are other important factors that contribute to the genetic variation among Rhizoctonia isolates. The knowledge gathered in this study would be useful for developing crops that are resistant to Rhizoctonia diseases. This will assist in planning for the right crop rotation and proper disease management programs