Report of New Symptoms of Corynespora Leaf Fall Disease from the Non-traditional Rubber Growing Areas in Sri Lanka

Hevea brasiliensis (natural rubber) is one of the major economically important plantation crops in Sri Lanka. Traditionally, rubber was grown mainly in the wet zone of the country. Currently, the cultivation is being expanded to the intermediate and dry zone. These new rubber growing areas are known as non-traditional rubber growing areas. Corynespora leaf fall disease is regarded as the most destructive foliar disease and the disease has caused a major devastation in rubber industry resulting in a remarkable economic lass of Sri Lankan rubber industry. The causative agent is the fungus, Corynespora cassiicola. Characteristic symptom of the fungus on rubber leaves is the “railway track lesion”. Later more than ten different symptoms have been reported for this disease. The symptom varied based on the type of clone, maturity level and also on the environmental condition. Due to this reason, the disease identification has become complicated especially among the field staff. This study was carried out to report newly produced symptoms of this disease. Twelve different symptoms have been illustrated with the most characteristic symptom. In this study to collect Corynespora leaf disease samples from non-traditional rubber growing areas among the lesion and unusual symptoms was observed from Padiyathalawa. Then the fungus was isolated on to PDA. Later single spore isolation technique was employed to purify the culture. Koch’s postulates were proven. The fungus was identified as based on cultural and reproductive characteristics. The newly reported symptom is illustrated to aid disease diagnosis.Keywords: Hevea brasiliensis, Non-traditional areas, Symptoms, Corynespora cassiicola, Foliar diseas

Pathological characterization of Corynespora cassiicola isolates from traditional and non-traditional rubber growing areas

Rubber (Heveabrasiliensis) is one of the major economically important estate crops and generates the third largest export income of Sri Lanka. Rubber plantations established mostly in Wet Zone and certain regions in Intermediate Zone and the cultivated areas are known as traditional areas. However, presently rubber cultivation has been expanded to the dry zone of country and the cultivated areas are known as non-traditional areas. Corynesporacassiicola is the most destructive foliar pathogen of the rubber plant causing Corynespora leaf fall disease (CLFD) and the disease has caused a major devastation in rubber industry resulting in a remarkable economic loss. This study aimed to determine the variability of C. cassiicola isolates from traditional and non-traditional rubber growing areas using pathological factors. Ten isolates of C. cassiicola which had been isolated from diseased leaves of different clones grown in traditional (five isolates) and non-traditional (five isolates) areas were used for characterization. Variability in pathogenicity, temperature sensitivity, growth rate, conidia production, fungicide sensitivity to two fungicides; mancozeb and carbendazim and toxin production were examined. Data were statistically analyzed and the final analytical output revealed a statistically significant difference (p <0.05) between the isolates, but not between two geographical regions for all parameterstested except for toxin production and sensitivity to carbendazim. Though isolates of C.cassiicola show significant difference in pathological factors among isolates irrespective of geographical location, they do not behave differently in different climatic regions.Keywords: Corynesporacassiicola, CLFD, toxins, traditional and non-traditional areas

The Potential Use of Plant Growth-Promoting Rhizobacteria (PGPR) as Antagonists and Biocontrol Agents in Hevea Diseases

The rhizosphere related bacterial species have a significant capability in plant growth promotion, referred to as Plant Growth Promoting Rhizobacteria (PGPR). Several substances produced by antagonistic rhizobacteria have been related to pathogen control and indirect promotion of growth in many plants. The objectives of this study were to identify effective PGPR bacteria in rubber soils of Sri Lanka, determine the potential use as antagonists and biocontrol agents to control economically important Hevea diseases in nurseries, and identify effective novel inoculant formulations. Soil samples were collected from the root rhizozphere of cover crops in Hevea fields in Rubber Research Institute of Sri Lanka (RRISL), Dartonfield. Bacteria were isolated by TSA plate method. Isolated PGPR bacteria were subjected to test the enzyme secretion ability. Pectinase, Lipase, and Amylase secretion ability of PGPR were tested in specifically developed media. Auxin production of PGPR were measured using freshly prepared Salkowsky reagent. Phosphorous solubilizing ability of PGPR was tested using Pikovskaya (PVK) medium. Antagonistic ability of PGPR was tested against the Rigidoporous microporus, Fusarrium sp. and Pythium sp. causing white root disease and Patch Canker disease in rubber respectively. Paper disc diffusion method was used to test the antagonism of selected bacteria and the percentage growth inhibition was calculated. Plasmid DNA extraction of antagonistic PGPR was done following the alkaline SDS method. Gene sequencing of selected bacteria was further carried out. Among 13 selected PGPR bacterial species, 3 bacteria were showing significant antagonism towards R. microporus. (Percentage growth inhibition by 51.2%, 46.8% and 44.3% respectively) Fusarium sp. was controlled by 7 PGPR species. Pythium sp. was controlled by 2 PGPR (Percentage growth inhibition by 41.6% and 38.7% respectively) 5 types of combinations of bacteria were tested. Among 5 types, 2 types were highly effective in control of R. microporus (Percentage growth inhibition by 79.8% and 72.6% respectively). All 5 combinations were moderately effective in control Fusarium sp. and 1 type was highly effective in control of Pythium sp. (Percentage growth inhibition by 67.8%). PGPR can be used as effective biocontrol agents in control of Hevea pathogens. The combinations of PGPR are more effective than the individual bacterial species. This will be leading to improve the productivity of Hevea plantations in Sri Lanka. Keywords: Plant Growth Promoting Rhizobacteria, Antagonism, Hevea pathogen

Ability of the Native Antagonistic Fungi Against Rigidoporus microporus, to tolerate the Usage of Fungicides for White Root Disease in Rubber Growing Soil in Sri Lanka

Natural rubber, Hevea brasiliensis, is one of the major economically important crops in Sri Lanka. Diseases play a significant role to reduce the production of rubber cultivations. Among them White Root Disease is the most destructive root disease which has caused a huge economic loss to the country. Rigidoporus microporus is the causative pathogen of White Root Disease. In order to control the pathogen, Tebuconazole and Hexaconazole can be used as systemic fungicides. However, due to the toxicity of these chemicals, antagonistic fungi can be used as an environmental friendly solution to reduce the usage of chemicals. Hence an attempt was taken to find out the ability of native antagonistic fungi to tolerate the chemical fungicides. Soil samples were collected from different rubber growing soils in Sri Lanka and using dilution plate technique fungi were isolated. After preparing pure cultures antagonistic ability against R. microporus was tested using dual plate culture test. More than 60% inhibition showing fungi was selected and subjected to Poison Food Technique (PFT) test. 5, 10, 25, 50, 75 and 100 ppm concentrations of the two fungicides were used to test the ability of tolerance by the isolates. According to the results, twenty-three effective fungi isolates were selected. Fourteen isolated fungi were identified as Trichoderma spp., three as Aspergillus spp. and the rest were unidentified. Three Trichoderma isolates and one Aspergillus isolate showed the radial expansion even at 50 ppm concentration of tebuconazole. Five Trichoderma isolates showed the growth at 100 ppm concentration of the Hexaconazole. The radial expansion of R. microporus has been totally inhibited at 25 ppm of tebuconazole and 50 ppm of hexaconazole. Therefore these antagonistic fungi can be used even at the higher concentrations which are fatal to R. microporus.Keywords: White root disease, Tebuconazole, Hexaconazole, Native antagonistic fung

Potential of Making Fungal Toxins at Commercial Scale for Agarwood Inducement

Agarwood, a highly valuable resin formed in the stems and roots of certain tree species of the family Thymlaeaceae can be induced the formation in artificial manner by using different fungal species. The potential of fungal toxins using certain strains of Aspergillus niger and Fusarium solani to form agarwood in Gyrinops walla, which is a native species to Sri Lanka, was identified in 2016. However, the potential of extracting fungal toxins in large scale was not studied previously for the purpose of inducing agarwood formation at commercial scale. Therefore selected strains of A. niger and F. solani were grown on Potato Dextrose Agar (PDA) medium and three agar plugs of A. niger were inoculated in Czapek Dox Broth (CDB) and F. solani in Yeast Extract Glucose Broth (YEGB). A 50, 100, 150, 200, 250, 300 and 500 ml series was prepared for CDB and YEGB to identify the potential impacts of different volumes of toxins. Mycotoxins were extracted after 14 days for A. niger and after 10 days for F. solani based on the results of a previous study. The impacts of those toxins were detected by a bioassay using A. crassna and G. walla leaves. A comparison was made using diameters of lesions caused by toxins in A. crassna and G. walla leaves after 48 hour. According to the results, the effects of toxins of F. solani and A. niger were not significant on A. crassna. Further the damage caused by F. solani (F=1.02; P=0.428) and A. niger (F=1.41; P=0.238) grown in different volumes was not statistically significant. However, the effect of toxins of F. solani was significantly higher than that of A. niger (F=10.69; P=0.000) on G. walla. Damage caused by toxins formed in different volumes of F. solani was not significant (F=1.81; P=0.103) on the leaves of both species. However, the damages caused by toxins A. niger grown in 50 ml and 100 ml were significantly lower on G. walla leaves than the rest of volumes. Based on these results, 500 ml can be used as an effective volume to extract toxins from A. niger and F. solani for inducing agarwood formation at commercial scale.Keywords: Fusarium solani, Aspergillus niger, Agarwood resins, MycotoxinsAcknowledgement: Centre for Forestry and Environment of University of Sri Jayewardenepura and Sadaharitha Plantations Ltd, Colombo 03, Sri Lanka

Evaluation of the Antagonistic Effect of the Diethyl Ether Extracts of Local Plant Species on Rigidoporus microporus: the Causal Organism of White Root Disease of Rubber

With the objective of finding out the effectiveness of the diethyl ether extracts of local plant species on Rigidoporus microporus (Fr.) Overeem, the causal organism of white root disease of rubber, four different plant species were tested. The freshly uprooted rhizomes/ under-ground parts were ground into fine powder after surface sterilization and oven drying for 48 hours at 45o C. Afterwards the solvent extracts were obtained by shaking the powder in the solvent, centrifugation and rotary evaporation. In each of the extract, the particle concentration of the dried powder was kept constant. The presence of the white root-inhibiting compounds in the final extract was tested by Poison Food Technique (PFT), Soil Fungicide Screening Test (SFST) and colony growth in liquid medium. In each experiment, three concentrations of the botanicals i.e. 5%, 10% and 25% (V/V%) were tested in five replicates. Sterilised distilled water and diethyl ether were substituted for the extract in the control experiments. In each treatment, the percent inhibition of growth with respect to the control was calculated using a standard equation. Analysis of variance was carried out for the percentage inhibition-over the control and the means were compared. According to both PFT and SFST results, increasing the concentration from 5% to 25%, increased the inhibition percentage with all four species. For the concentration levels of 5% and 10%, the inhibitory effect of the four species was significantly different from each other. At those two concentrations, wild ginger (Curcuma xanthorrhiza) had the highest inhibition rate at both tests and its inhibitory effect was significantly higher than those of the other three species. However, at the 25% concentration level, wild ginger and galangale (Alpinia galanga) showed insignificant difference in the inhibitory action. Garlic (Allium cepa) followed by ginger (Zingiber officinale) showed the lowest inhibition rate compared to other species in all concentrations. The results of the liquid medium experiment did not show linearity with the results of the other two tests because, in this test method, a formation of rhizomorphs on the mycelial disc and a subsequent increase in the mycelial dry weight had been resulted as a defense reaction for the stress condition. The results of the study confirm the presence of effective compound/(s) especially in Galangale, Wild ginger and Ginger and suggest the potential of using diethyl ether extracts of these plant species for the further studies towards the identification of effective botanical compound/(s) against white root disease of rubber. Keywords: White root disease, Antagonistic plants, Growth inhibitio

Screening of Different Hevea brasiliensis Clones Based on the Reaction to Phytophthora Bark Rot

Disease conditions caused by Phytophthora species are common on rubber (Hevea brasiliensis) in all rubber-growing countries. Bark rot is the most economically damaging disease of the tapping panel of the rubber tree, leading to the cessation of tapping during the monsoon months in rubber plantations. To overcome this situation, the disease screening programs aiming disease resistant/tolerant clones are continually carried out. The current study attempted to evaluate the reaction of different clones of H. brasiliensis against the Phytophthora bark rot in order to recommend a suitable planting material for the industry. Forty clones of H. brasiliensis were screened at the bud wood nursery of Kuruwita substation of Rubber Research Institute of Sri Lanka during South West monsoon, 2014. Each clone consisted ten bud wood plants and in each bud wood plant, three branches were used for the inoculation. These plants of different clones were artificially inoculated with a standard spore suspension of Phytophthora meadii. This was prepared by scraping the sporangia on the surface of culture plates into sterile distilled water and the final concentration was adjusted to 1x104 spores per ml. At the inoculation, the bark of the selected branches was removed with a cork borer and the wood was exposed. A sterile absorbent cotton wool plug saturated with 2 ml of the zoospore suspension was placed on the exposed wood of the plant. The cotton wool plugs were taken in sterile Petri dishes to the experimental site and a freshly-prepared zoospore suspension was used. As the control, in each of the bud wood plant tested, one branch was inoculated with a cotton wool plug with sterile distill water. After the inoculation, the inoculated portions of the branch were sealed off with budding tape in such a manner to ensure aeration. Four weeks after the inoculation, the exposed lesions were traced on to tracing papers, after removing the bark and the area under the lesions were measured with a plannimeter. The area under the lesion was assumed to be inversely proportional to the resistance level of the relevant rubber clones towards the P. meadii infection. The area of the lesion resulted after each treatment was subjected to analysis of variance in SAS statistical software. The results of the trial carried out suggest the possibility of categorizing the screened clones into three clusters. The clones RRISL 222, RRISL 220, RRISL 208, RRISL 219, RRIM 600, RRIC 102, RRIM 717 and PB 235 are among the most resistant clones while the clones, PB 255, RRISL 206, RRISL 225, RRISL 201, RRIC 130, RRISL 211, RRISL 205 and RRIC 131 are among the most susceptible clones against Phytophthora bark rot. Therefore, it can be concluded that, the screened clones were showing differential reaction to the Phytophthora bark rot under bud wood nursery conditions.Keywords: Phytophthora bark rot, Hevea brasiliensis, Clonal screenin

In Vitro Evaluation of the Antagonistic Effect of Some Native Trichoderma Strains Against Economically Important Foliar Pathogens of Rubber

Rubber plants are subjected to serious fungal diseases and among them, the losses caused byfoliar diseases play an important role. The frequent use of chemical fungicides to controlcausative pathogens leads to environmental pollution, hazardous to human and may lead tothe development of new chemical resist pathogenic strains. Trichoderma species areubiquitous soil-borne Ascomycetes noted for their biocontrol capabilities against manyeconomically important plant pathogens. Hence, an attempt was made to investigate theantagonistic effect of some native Trichoderma isolates on the plant pathogenic fungi;Colletotrichum sp., Corynespora cassiicola, Phytophthora sp. and Drechslera heveae. Foliarpathogens were isolated from Rubber Research Institute, Dartonfield, Agalawaththa andidentified based on the symptoms, cultural and reproductive characteristics. FiveTrichoderma isolates isolated from different habitats in Sri Lanka were tested in vitro fortheir antagonistic effects against the four foliar pathogens. The results obtained from dualculture tests showed that all the five Trichoderma isolates effectively checked the growth ofthe four foliar pathogens. The test antagonists grew faster than the pathogen limiting theirgrowth. Trichoderma isolate A was the best antagonist against Drechslera heveae,Corynespora cassiicola and Colletotrichum sp. showing inhibition of 75.63%, 51.34% and74.46% respectively. Isolate B showed the best inhibition (70.99%) against Phytophthora sp.All antagonists showed their lowest inhibition against D. heveae. In conclusion, all the testedTrichoderma isolates showed antagonistic effect on the four foliar pathogens underinvestigation. Therefore, the fungal isolates under investigation can be used for furthergreenhouse and field studies to confirm the feasibility of using for the management of rubberfoliar pathogens.Keywords: Trichoderma spp., Drechslera heveae, Corynespora cassiicola, Colletotrichumspp., Phytopthora spp, Bio-control agent

Physiological Effect of Laccase, Secreted by Rigidoporus microporus on the Host Pathogen Interactions

Laccases (EC1.10.3.2) are multi-copper containing phenol oxidases that catalyse theoxidation of phenolic compounds and aromatic amines. They are widely distributed in higherplants and in many fungi. In fungi, they are involved in delignification, sporulation, pigmentproduction and plant pathogenesis and fruiting body formation. Rigidoporus microporus is awhite rot fungus efficient lignin degradation and causative agent of the most destructivewhite root disease. In this study, the production of laccase was detected qualitatively.Furthermore involvement of laccase in the host-pathogen interaction was also studied. Threeisolates of R. microporus were isolated from symptomatic Hevea brasiliensis (R1), Mucunabractiata (R2), and Artocarpus nobilis (R3). The level of laccase production was measuredqualitatively on solid media containing the colour indicator of guaiacol. 5 mm diameter ofmycelium discs from 7 day old cultures were innoculated into 2% PDA plates supplementedwith 0.2% glucose as a laccase degrading substrate with 0.02% Guaiacol (pH 5.5) andincubated at RT (28±20 C) under continuous dark conditions. After, an incubation period of 4days, the enzyme activity was examined by the decolorisation intensity. Morphology of thethree isolates was also evaluated on malt extract agar. Lignin degradation ability was studiedusing artificial inocula prepared by fresh root pieces of H. brasiliensis and incubated for fourmonths; thereafter they were splited longitudinally to observe the lignin degradation ability ofthe isolates. All the test isolates were laccase positive and R3 (A. nobilis) isolate producedhigher intensity showing comparatively larger diameter of the reddish brown halos indictinghigher level of laccase production. Moreover, the isolate R3 showed the formation ofrhizomorphs in vitro conditions while the other isolates did not show them. Formation ofrhizormorphs is associated with the laccase synthesis which has been reported to beresponsible for making polyphenolic glue that sticks the hyphae together. The degradation ofroots was observed to be comparatively faster by the isolate R3 leaving white pockets onroots by degrading cell wall component due to the removal of lignin in localized areas ofwood and observation can be attributed with the higher laccase enzyme. The root piecesinoculated with R1 and R2 did not show white pockets and a spongy nature until four monthincubation period. It has been revealed that the isolate R3 (A. nobilis) is a good laccaseproducer among the other isolates under investigation.Keywords: Laccase, Rhizormorphs, Rigidoporus microporu

Isolation of Rigidoporus microporus, the Cause of WRD of Rubber, from Some Forest Associated Plants

Natural rubber while promoting export earnings and livelihood of people supplementthousands of hectares to the forest cover. Over the past decades, the rubber yield hassignificantly increased due to the cultivation of high yielding clones. However, latexproduction still faces serious economic losses due to many biotic constraints which includesignificant losses caused by pathogenic fungi. Among them, White root disease (WRD) isvery destructive in rubber plantations of Sri Lanka and in many other rubber growingcountries. This disease has been identified as one of the major causes for the loss of plantsduring the first five years after planting resulting in low productivity levels. Inspite of the factthat disease management strategies have been clearly outlined by the Rubber ResearchInstitute, the disease incidence is showing an increasing trend. One of the main reasons forthis has been identified as the increment of the host range. In this situation expansion of thehost range of R.micrporus was undertaken. R. microporus was isolated from the symptomatichost plants such as Mucuna bractiacta, Camellia sinensis, Cinnamomum zeylanicum,Artocarpus nobilis, Alstonia macrophyll growing in and around rubber plantations. Some ofthe above crops such as C. sinensis, C. zeylanicum are intercrops recommended by the RRIwhile A. nobilis and A. macrophylla are important forest crop species grown in the vicinity ofrubber cultivations. M. bractiata is also one of the most commonly grown cover cropsrecommended for rubber plantations. The pathogen was isolated, on to Malt Extract Agarafter surface sterilizing in 70% ethanol for 3 minutes. Pure cultures were obtained and thecultural characteristics were recorded for the five isolates separately. For comparativepurpose isolate from H. brasiliensis was employed. Colonies showed white flattened cultures.Isolates from M. bractiacta, C. sinensis, C. zeylanicum, H. brsiliensis and A. nobilis showedpuffy aerial growth while A. macrophylla showed clear concentric zones which are prominentfrom the lower side of the colonies. Among the isolates investigated, the fastest growth ratewas observed in the isolate from C. sinensis showing 1.262cm/day while the lowest growthrate 1.205 cm/day was shown by the rubber isolate. The pathogenecity of the isolatesobtained from the above hosts were proven against the respective hosts by following theKoch’s postulates under green house conditions. The cross infection abilities of the isolateswere also assessed and all isolates infected Hevea brasiliensis showing cross infectionabilities. Pathogenic and genetic variability among the different isolates are beinginvestigated.The information will be valuable in the development of improved managementstrategies against white root disease of rubber