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

Trunk Injection Method for Introducing Chemical Formulations in to Rubber Trees

Natural Rubber is an important commodity to the economy of Sri Lanka. This industry makesexport earnings, sustain the livelihood of over thousands of people, supplement thousands ofhectares to the forest cover and provide many other socio-economic and ecological benefits.The economically important diseases are regarded as a major constraint for achievingpotential productivity levels of rubber cultivations. Hence management of these diseasesprovides economic benefits to rubber growers. The most popular method of disease control ischemical control. There are various chemical application techniques and spray applications,soil drenching, dusting or broadcasting are popular methods. Based on the age, type of thedisease or the prevailing weather conditions, the application technology needs to be changed.These conventional technologies have many health and environmental drawbacks. Therefore,economical and environmental friendly chemical application techniques have becomeimportant. The present study was aimed at investigating the trunk injection method forintroducing chemical formulations into trees. Several types of potential injectors are availableand the chemjet injectors described by Guest et al., (1994) was used throughout theexperiments. These injectors [Chemjet Trading (Pvt) Ltd, Australia] contain 20ml of thesolution and have a working pressure of 1 – 1.5 bars facilitating the formulation into the treesthrough drilled holes. The injection process was tested at Dartonfield Estate, on the Heveabrasiliensis clone RRIC 121. The average girth at the tested plants was 89 cm. Two analysesviz. 45, 60 to a depth of 4 cm from the outer bark was done. The uptake rates weredetermined using 8% tebuconazole in aqueous solutions. Uptake of the fungicide was slightlyhigher in deep injected method compared to shallow injection. The two angles tested againstthe liquid uptake too were not significantly different from each other. No interaction wasobserved among the injection angle and the depth of injection on the liquid uptake. More than70% of the trees had uptaken the entire 20ml injected volume within 48 hrs of injection.Trunk diameter had no effect on the uptaking process. The results obtained proved thepossibility of injecting liquids / chemical solutions into rubber trees. Trunk injection requireslow volumes of chemicals resulting in reduced cost in chemical applications. Minimumwastage of chemicals could be achieved together with reducing the impact of hazardouschemicals on the environment. Compared to the currently practiced chemical applicationmethods into trees, this application technique has many advantages

Association of Forest-Origin Tree Species on the Development of Brown Root Disease in Sri Lankan Rubber Plantations

Brown root disease caused by Phellinus noxius is widely distributed in tropical and sub-tropical regions in Asia and becoming an important root affecting fungi in Sri Lankan rubber plantations. Most of the economically-important plantation and other crop species as well as forestry species have been reported to be affected by this disease. However, the significance of the brown root rot was recognized only recently in Sri Lanka with the expansion of rubber to non-traditional areas in Uva, Northern and Eastern provinces and with low adoption of the recommended land preparation measures. According to the literature, this disease is usually found in many plantations where crop established on cleared forest sites. The objectives of the current study was to investigate the local host species of the disease and to evaluate the role of those species in the development of brown root disease in rubber. Seventy brown root disease incidences were used to assess the host range and the disease transmitting pattern. A structured questionnaire was used to collect the background information. In association with the roots of the diseased tuber trees, decayed roots of twenty species of forestry origin which were kept in situ were identified, exhibiting the similar signs and symptoms of the disease. However, the pathogen isolates could be isolated from only five alternative species, as some of the roots were too decayed. They were Cereya arboria (kahata), Ginelina arboria (eth demata), Bridelia retusa (keta kela), Mangifera indica (mango) and Artocarpus heterophillus (jak). These isolates showed a variation in their morphological and physiological characteristics. The studies on the pathogenicity and the cross infection ability are in progress. It was observed that in all disease incidences, the initially diseased rubber tree had a root contact with a diseased roots of some other species. Moreover, at all the incidences, land had been previously either under the forest or abandoned for years with trees and shrubs of forest origin. Based on these facts, it can be concluded that the infection of brown root disease to rubber has a correlation with the presence of other tree species in the root contact. It can therefore be expected that the innoculum is present in native forests on infected roots or woody debris and rubber plants got infected when the roots made contact with the infected roots and other woody debris of cleared native forests.Keywords: Brown root disease, Rubber, Forest specie

Comparison of the Fungicide Sensitivity of the Two Fungal Species: Rigidoporus microporus and Phellinus noxius: the Causal Organisms of the Two Major Root Diseases of Rubber in Sri Lanka

White root disease caused by Rigidoporus microporus and the brown root disease, caused by Phellinus noxius are the two major root diseases of Sri Lankan rubber cultivations. In order to be used in a used in a collective approach towards both diseases, a comparison of the fungicide sensitivity of the two pathogens was intended in the study. In vitro screening of two potential fungicides namely: tebuconazole and hexaconazole was carried out using the poisoned food technique. One isolate from each pathogen was selected to have the highest pathogenicity level among the respective isolate collections. The experiment was carried out in a completely randomized design with four replicates in each treatment. A series of concentrations: 0.1,5,10,25,50 and 100 ppm, was used to identify the sensitivity levels of the respective fungicide towards the two pathogen species and sterilized distilled water was substituted for the fungicide in the control sets. After an incubation period of 4 days at room temperature, the percentage inhibition of the respective fungus over the control was observed in each culture plate. On tebuconazole, P. noxius showed no growth at any of the tested concentrations. However, R. microporus, exhibited a mycelial growth up to the 10 ppm concentration. Though no growth was observed at the concentration of 25 ppm, R. microporus could resume its growth when the fungal disk was transferred back to the growth medium. However, when exposed to concentrations 50 ppm or above, the fungus could not resume its growth in fungicide free medium. With hexaconazole, too, P. noxius showed no growth at any of the tested concentrations, while R. microporus grew up to the 10 ppm concentration. Though R. microporus exhibited no mycelial growth on the culture plate at the concentration of 25 ppm, a slight growth of the fungus was observed on inoculated discs after the incubation period. However, even at the 100 ppm concentration, R. microporus could resume its growth when transferred back onto fungicide free MEA. Therefore, it can be concluded that, when the two particular fungal isolates are considered, P. noxius shows high sensitivity than R. microporus towards the both tested fungicides.  Keywords: Rigidoporus microporus, Phellinus noxius, Fungicide sensitivity, Poisoned food techniqu

Variability of Pathogenicity of Different Isolates of Phellinus noxius: The Causal Organism of the Brown Root Disease of Rubber in Sri Lanka

Phellinus noxius is the pathogen causing brown root disease: an immerging disease condition in Sri Lanka rubber industry, especially in dry and intermediate regions. The possibility of the pathogen to be mutated into more virulent forms can be considered as one of the factors influencing the increased frequency of its occurrence in the country during the recent past especially in certain parts of the country. Considering the diversity of soi1 and climatic conditions in which the pathogen hasbeen reported and the large number of tree species it attacks, it is necessary to have an understanding whether there is any variation in pathogenicity within the population of the fungus. Ultimately, this heterogeneity in the pathogenicity of the pathogen population within the country would be helpful in the development of management strategies against the disease. The currentstudy has evaluated the pathogenicity of 24 Sri Lankan isolates of Phellinus noxius. A pot trial was carried out by artificial inoculation of three months-old rubber seedlings with an inoculated mixture (with respective fungal isolates grown on MEA) of rice bran and saw dust. Forty seedlings were inoculated by each isolate, and another forty seedlings were kept as control without inoculation. Starting after four months of inoculation, ten destructive samplings were done at two months intervals to observe the pathogenicity levels of different pathogen isolates against rubber seedlings. Based on the underground signs and symptoms, a pathogenicity score was given of each uprooted plant as; 0 (no infection), 1 (mycelial crust without root decay), 2 (mycelial crust with root decay) and 3 (plant death). The pathogenicity levels recorded as ranks were subjected to Kruskal–Wallis analysis and subsequently to the Wilcoxon rank-sum test, as the scores obtained for the different isolates were significant. A variation of pathogenicity was observed among the 24 Phellinus noxius isolates. As all the isolates had initiated to show a stabilised pathogenicity value at three and half months of the inoculation, a cluster analysis was performed for the mean score values ofpathogenicity rank at three and half months and the dendrogram showed that the test isolates were separated into two main clusters at the similarity level 0.8. Keywords: Brown root disease, Rubber, Pathogenicity of isolates, Clustogram

Variability of Trichoderma Population Isolated from Rubber Growing Soils, Showing Antagonistic Ability against Rigidoporus microporus, the Cause of White Root Disease of Rubber

Natural rubber (Hevea brasiliensis) is a very significant crop to the global economy. Loss of production of rubber plantations can be caused due to plant pathogens. Rigidoporus microporus, the causative agent of white root disease is abundant in rubber plantations of Sri Lanka. Since control of the disease by applying fungicide is expensive, fungal species can be used as a biological control agent against white root disease. Trichoderma isolates obtained from rubber growing soils were found antagonistic against R. microporus. An attempt was taken to isolate various antagonistic Trichoderma isolates from different rubber growing soils. Soil samples were collected from Kalutara, Rathnapura, Ampara and Vavuniya in Sri Lanka. Soil pH and moisture contents were measured using standard methods and fungi were isolated using the dilution plate technique. After preparing pure cultures, antagonistic ability against R. microporus was tested using dual plate culture test. More than 65% inhibition showing fungi were selected to observe the cultural and reproductive morphology. According to the results, the dual plate culture test indicated that five Trichoderma species were effective to show more than 65% inhibition against R. microporus. These included T. hamatum, T. koningii, T. spirale, T. ghanense and the teleomorph of T. harzianum (Hypocrea lixii) in DNA sequencing data after processing from Macrogen, Korea. Hypocrea lixii showed the maximum inhibition of 80% over the control in R. microporus followedby T. koningii. Trichoderma species were able to grow in rubber growing soils with a range of pH from 4.0 to 6.8. Hypocrea lixii were isolated from the dry zone where showed comparatively higher pH and lower moisture values than the wet zone. T. spirale and T. hamatum were observed in both dry and wet zones. The isolates produced irregular or circular colonies with filiform or entire types of margin showing maximum growth rate of 4.08 cm/day. Therefore, these antagonistic fungi show a potential to control R. microporus under field conditions too. Keywords: Rubber plantation, White root disease, Trichoderma s