Induction of Resistance in Abaca Against Wilt Disease Complex caused by Fusarium oxysporum f. sp. cubense (E. F. Smith) and Ralstonia solanacearum (Smith) Yabuuchi et. al. Using Chemical and Biological Elicitors Abstract Resistance in plants has been reported to be inducible using biological and or chemical elicitors. Fusarium wilt and bacterial wilt diseases are two major diseases hampering abaca production in the Philippines. This study was conducted to evaluate the efficacy of biological and chemical eliciors of resistance to control wilt disease complex in abaca caused by Fusarium oxysporum f. sp. ubense (FOC) and Ralstonia solanacearum. Suspected biological elicitors (Fomes and Xanthomonas oryzae pv. oryzae or Xoo) and known chemical elicitors (Asprin or Acetyl Salicylic acid and Boost) were sprayed to the abaca tissue cultured seedlings at 15 days interval together with water and Nordox (fungicide with bactericidal activity) as checks. The sprayed seedlings were challenged by simultaneous inoculation with FOC and Ralstonia solanacearum. The parameters used to compare the treatments were: percent infected plants, disease severity rating, percent area of vascular discoloration and number of remaining live plants.
Results show that Boost and Fomes resulted to an effective control of the wilt disease complex which were comparable or even better compared to Nordox, the fungicide check. Xoo and Acetyl Salicylic Acid on the other hand produced higher disease compared to the control. This study has demonstrated the potential of Fomes and Boost as biological and chemical elicitors of resistance, respectively in abaca which protected it from wilt disease complex. Keywords: Abaca, SAR, chemical elicitors, biological elicitors, Fusarium wilt, Bacterial wilt ————————————————- Student Researcher, VSU 2 Associate Professor, DPM, VSU and Research Adviser INTRODUCTION Abaca (Musa textilis Nee) is indigenous to the Philippines, and is one of the major dollar earners of our country. Abaca plant has many slender stalks, upright and pointed leaves. It is harvested for its strong but flexible fiber usually called as Manila hemp used for making twines, ropes, cordage, pulp and specialty papers, fiber crafts, textile and fabrics. It is also one of the few cash crops that can grow with relatively little input compared to the other crops (FIDA, 2007).
Abaca has a high market potential but its production has been limited due to the occurrence of diseases, such as bunchy top, bacterial wilt and fusarium wilt which have taken their toll on many abaca farms. This devastation caused by viruses, bacterial wilt and fusarium wilt in abaca aggravates the farmers’ production problems. Fusarium wilt disease is caused Fusarium oxysporum f. sp. cubense (E. F. Smith) Snyd. And Hans. which is also referred to as Agent Green. This fungus does so by colonizing the water-conducting vessels (xylem) of the abaca plant resulting for a blockage and breakdown of xylem.
Yellowing of the leaves, followed by drying and wilting and eventually plant death are the external symptoms of the disease. Most affected plants have discolored vascular bundles and eventually die before maturity thus, forcing the farmers to harvest the abaca plant prematurely. Another equally important disease is the bacterial wilt disease. Bacterial wilt is caused by the bacteria Ralstonia solanacearum (Smith) Yabuuchi et al. , 1995). The early symptoms of this disease include rusty brown streaks in the leaves which later turn blighted and water soaked and finally wilting of the whole plant.
The internal symptoms are the vascular discoloration, water soaking and finally the rotting of pseudo- stem. Fusarium wilt and bacterial wilt may occur singly or together as a complex in the field and had contributed much to the devastation of abaca in major growing areas in the Philippines. Host plant resistance is the most effective means of controlling wilt diseases. But the development of a resistant plant however takes time, effort and many resources. Resistance in plants however can be induced using chemical and biological elicitors.
Several studies have shown that certain chemicals can induce the resistance of plants and some of which are boost and salicylic acid. Boost is a novel synthetic chemical, benzo(1,2,3)thiadiazole-7-8-carbothioic acid S-methyl ester (BTH) was intially shown to induce acquired resistance in wheat (Gorlach, et al, 1996) and which is produced by Novartis and distributed in the Philippines by Syngenta. Aspirin or acetyl salicylic acid was also found to induce resistance in some plants (Gleason, 1999; Owen, 1996).
Salicylic acid (SA) has been reported to be elevated on plants that exhibit SAR for example in cucumber (Metraux, et al, 1990), tobacco (Malamy, et al, 1990), and Arabidopsis (Uknes, et al, 1993). There are evidences that suggest that SA plays an important role in the transduction pathway leading to SAR. Recent studies however indicate that SA is not likely to be the systematically transmitted signal (Vernooij, et al, 1994). ] There are also biotic elicitors that have been reported to elicit resistance in plants which includes chitin and chitosan.
Chitin is a safe material and was reported to induce resistance against soil-borne diseases ((Kuchitsu, et al, 1993). Moreover, chitin was reported to be used as soil fertilizer. Chitosan which can be extracted from chitin has anti-fungal activity against many plant pathogens (Herano, et al, 1990; El-mougy, et al, 2002). Moreover, chitosan is also reported to induce resistance against soil-borne fungi. Chitosan was also found to have anti-bacterial property and is capable of inducing resistance to the bacterial blight disease of rice cause Xanthomonas oryzae pv. oryzae (Modina, 2007).
Boost is a novel synthetic chemical, benzo(1,2,3)thiadiazole-7-8-carbothioic acid S-methyl ester (BTH) was intially shown to induce acquired resistance in wheat (Gorlach, et al, 1996) and which is produced by Novartis and distributed in the Philippines by Syngenta. Aspirin or acetyl salicylic acid was also found to induce resistance in some plants (Gleason, 1999; Owen, 1996). Salicylic acid (SA) has been reported to be elevated on plants that exhibit SAR for example in cucumber (Metraux, et al, 1990), tobacco (Malamy, et al, 1990), and Arabidopsis (Uknes, et al, 1993). There are evidences that suggest that SA lays an important role in the transduction pathway leading to SAR. Recent studies however indicate that SA is not likely to be the systematically transmitted signal (Vernooij, et al, 1994). ] This study therefore aimed to compare and contrast the efficacy of suspected elicitors of resistance in controlling the wilt disease complex in abaca. The efficacy of potential biological and chemical elicitors of systemic acquired resistance to control wilt disease complex of abaca caused by Fusarium oxysporum f. sp. cubense and Ralstonia solanacearum was also evaluated. METHODOLOGY Preparation and Sterilization of Culture Media
Potato dextrose agar (PDA) for the culture of FOC was prepared by dissolving 39 grams dehydrated PDA to 1 liter dH2O in a double boiler and cooked until the agar dissolves in H2O. It was dispensed to 250 ml Erlenmeyer Flasks and test tubes. Nutrient agar for the culture of Ralstonia solanacearum was prepared by dissolving 3g beef extract, 5 grams peptone and 17g agar to 1 liter dH2O in a double boiler and cooked until agar dissolves in H2O. It was dispensed to 250 Erlenmeyer Flasks and test tubes. The media were sterilized in a pressure cooker at 15 lbs/in2 pressure or 121°C temperature for 15 minutes.
Sterile media were allowed to cool. PDA and NA in tubes were slanted and stored in the refrigerator until use. Collection, Isolation and Culture of FOC Isolation plates were prepared by melting the stored PDA in flasks, pouring into sterile Petri plates and allowed to solidify. Fusarium wilt– infected abaca was collected from the National Abaca Research Center and it was brought to the Plant Disease Diagnostic Laboratory for isolation of the pathogen. Infected tissues from the advancing portion of the disease were disinfected with 1% sodium hypochlorite and were planted onto solidified PDA in plates (4 sections per plate).
After 2 or more days, mycelia that have radiated from the planted abaca tissue were cut aseptically using flamed sterilized flattened needle and were transferred to PDA slants where they were stored and maintained until use. Collection, Isolation and Culture of Ralstonia solanacearum Nutrient agar in flasks were melted and aseptically poured to Petri plates. The medium were allowed to solidify. The bacterial wilt pathogen, R. solanacaerum was isolated from a banana fruit infected with “Bugtok” disease. Infected tissues from the advancing portion of the disease were cut, disinfected with 1% hypochlorite and reused with sterile H20.
The sections were then placed in sterile water in a test tube and allowed to stand for 10 minutes to allow the bacteria to ooze out of the tissue. A loopful of the resulting bacterial suspension was streaked onto the solidified NA on plates and incubated. After 2 days individual colonies were picked with sterile wire loop and transferred to NA slants to purify. The cultures were incubated for 2 days and stored in the refrigerator for future use. Procurement and Preparation of Test Plants Four weeks old tissue cultured abaca plantlets were procured from National Abaca Research Center (NARC).
They were maintained in the vicinity of the Department of Pest Management and were given necessary care and maintenance such as watering, weeding, and fertilization. The plants were numbered and tagged according to the desired treatments. In numbering and tagging the plants, we gave them equal chances in belonging to a group. We did this through the drawn by lot method. Thus, we have used the technique randomization in giving names to the plants. Preparation and Application of Treatments The plants were sprayed with the different treatments (Fig. 1) at 15 days interval starting when the plants were 6 weeks old.
Treatments| Illustration| T0 (Water)| | T1 (Fomes at 100ppm)| | T2 (Attenuated Xanthomonas oryzae pv. oryzae)| | T3 (Aspirin or Acetyl Salicylic Acid at 100ppm)| | T4 (Boost at 100ppm)| | T5 (Nordox at 100ppm)| | Fig. 1. The different treatments used Fomes was prepared by oven drying (50? C for 3 days) and by pulverizing using mortar and pestle. After pulverizing, 0. 75g was obtained and was mixed with 750ml H20 to make a 100 ppm suspension. Xanthomonas oryzae pv. oryzae (Xoo) was prepared by transferring an isolate of Xoo (courtesy of PDDL at VSU) in NA slants 2 days before its scheduled time of application.
On the application time, 10ml sterile H20 was poured on the test tube slants and the bacteria were scraped using a wire loop without causing damage to the medium. Ten slants were used and the solution was diluted into a beaker to make 100 ml suspension. An approximately 650 ml H20 was added to have a total of 750 ml suspension. The suspension was heated for 5 minutes to attenuate the bacterium. Aspirin or Acetyl Salicylic Acid was prepared by making a stock solution. A tablet of aspirin was diluted to 1L H20 to make a 300 ppm suspension. Then, a volume of 37. 5ml was measured out from the stock and was added to 712. ml H20 to make a 100 ppm suspension. Boost was obtained from Syngenta Philippines. Two ml Boost was diluted and mixed to 1998 ml H20 to make a 2000 ppm suspension. A volume of 37. 5 ml was obtained out from the stock and was added to 712. 5 ml H20 to make a 100 ppm suspension. Nordox, copper-based fungicide with bactericidal activity was used as the positive check at the manufacturer’s recommended rate. It was prepared by weighing 1. 9g of Nordox and by mixing it thoroughly to 750 ml H2O. After preparing, the treatments were sprayed to the plants using a sprayit gun powered by an electric compressor starting from Water down to Nordox.
Each plant equally received 50ml of the said treatments (Fig. 2). The experiment was laid in a Completely Randomized Design. The six treatments were replicated 3 times and 5 plantlets constituted one replicate making a total of 90 plantlets needed for the experiment. Two consecutive spray applications of the treatments at 15 days interval were done before pathogen inoculation and sprayings were done until the last disease scoring. Fig. 2 Application of treatments to the plants Preparation of FOC Inoculum The pure cultures were first examined under the microscope to confirm the presence of real FOC pathogen (Fig. ) before they were mass cultured for inoculation. FOC that was used for inoculation to test plants was cultured and mass produced in corn meal-sand medium (CMS) which was prepared by mixing 1 part ground corn every 20 parts of sand. The mixture was placed in glass jars about 2/3 full and moistened and covered with aluminum foil then bound with rubber band at rim (Fig 4). They were sterilized at 15 lbs/in2 pressure and 121°C temperature for 1 hour. Pure culture of FOC from the PDA slant were aseptically seeded into the prepared CMS medium and incubated at room temperature until completely colonized by the fungus.
Fig 3. Photomicrograph of FOC macroconidia and microconidia Fig 4. Corn meal-sand medium with FOC Inoculation of FOC CMS cultures of FOC were first mixed thoroughly on a tray to assure even distribution of the fungal spores to the CMS medium before inoculation. FOC was inoculated into the soil of each potted plant by incorporating 1 tbsp of the inoculum near the base of the abaca plantlet and covering the inoculum with soil. Inoculation was done 2 days after the second spraying of the different treatments. Preparation of Ralstonia solanacearum, the bacterial wilt pathogen
Bacterial wilt pathogen was transferred to fresh NA slants. Two days after the isolation, each NA culture was mixed with 10ml dH2O and the pathogen was scraped using a wire loop without causing damage to the medium. Overall, 120ml of the bacterial suspension was used. One ml was injected to the stem of each plant. Bacterial Inoculation was done 12 days after FOC inoculation. Data Gathered The following data were gathered: A. Initial and Final Plant Height – This was gathered per plant basis before the application of treatments and inoculation of the pathogen and during the termination of the study.
B. Percent infection – (No. of plants showing foliage yellowing or wilting / Total No. of plants) x 100 starting 1 week after inoculation and weekly until most of the plants have died in any of the treatments. C. Visual Disease Severity Rating – This was gathered using a devised scale below: We gathered this data at 1 week interval until the termination of the study. Rating| Description| 0| No wilting| 1| Slight yellowing of lower leaves| 2| Yellowing of most leaves but no dried leaves yet| 3| Extensive yellowing and moderate wilting| 4| Extensive wilting but plant still alive| | Plant totally dead| D. Percent Area of Vascular Discoloration – This was taken during the termination of the study wherein all the plants were uprooted and the pseudostems were sliced longitudinally (Fig 5). Each plant was rated using the following scale (Borines et al. 2007) Rating | Description| 0-1| No discoloration| 1-2| Isolated points of vascular discoloration| 2. 1-3| Discoloration up to 1/3 of vascular tissue| 3. 1-4| Discoloration between 1/3 – 2/3 of vascular tissue| 4. 1-5| Discoloration greater than 2/3 of vascular tissue| 5. -6| Total discoloration of vascular tissue| Fig. 5 Slicing of pseudostem longitudinally E. No. of surviving plants per replicate per treatment. (Gathered at the termination of the experiment. ) Data gathered were analyzed using simple CRD with three replications. Treatments were compared using Duncan’s Multiple Range Tests (DMRT). 9) Disposal of Inoculated Plants After the experiment, the inoculated plants were exposed to direct sunlight and burned when dried. RESULTS and DISCUSSION Table 1 shows the percent infected plants at 14, 21, and 28 days after inoculation.
Based on the results, Boost produced significantly lower percentage infected plants compared to the control especially at 14 days after inoculation. At 21 and 28 days after inoculation, Boost still produced lower disease incidence although not statistically different from the control. Fomes, Boost and Nordox also showed lesser disease incidence values although not significantly different from the control. Table 1. Percent infected plants at 14, 21 and 28 days after inoculation Treatment| Percent Infected Plants| Days After Inoculation| 14 days| 21 days| 28 days| T0 (Control)| 73. 3 ab| 93. 33| 100| T1 (Fomes)| 66. 67 ab| 73. 33| 73. 33| T2 (Xoo)| 100. 00 a| 100. 00| 100| T3 (Aspirin)| 73. 33 ab| 100. 00| 100| T4 (Boost)| 26. 67 c| 60. 00| 80| T5 (Nordox)| 53. 33 bc| 66. 67| 83. 33| CV(%)| 26. 91| 26. 27| 21. 60| Means followed by a common letter are not significantly different at 5% DMRT. Another parameter used to measure the effect of the treatments was the mean disease severity rating per plant (Table 2). Based on this parameter, Boost, Nordox and Fomes have lower means of disease severity compared to the Control especially at 21 days after inoculation.
Xoo and Acetyl Salicylic Acid on the other hand have significantly higher means compared to Water. Thus the two treatments have even triggered the increase in wilt disease severity ratings of the plants. Table 2. Mean wilt severity rating per plant as affected by the different treatments Treatment| Mean Disease Severity Rating| Days After Inoculation| 14 days| 21 days| 28 days| T0 (Control)| 0. 93 c| 2. 47 ab| 3. 13 ab| T1 (Fomes)| 0. 80 c| 1. 53 bc| 2. 27 b| T2 (Xoo)| 2. 87 a| 3. 45 a| 3. 92 ab| T3 (Aspirin. )| 2. 07 b| 3. 20 a| 4. 33 a| T4 (Boost)| 0. 40 c | 1. 27 c| 2. 3 b| T5 (Nordox)| 0. 73 c| 1. 07 c| 2. 47 b| CV (%)| 27. 6| 25. 7| 27. 6| Means followed by a common letter are not significantly different at 5% DMRT. The percent area of vascular discoloration per plant is shown in Table 3. This was gathered upon termination of the study. All plants were uprooted and the pseudostems were sliced longitudinally. This actually shows the internal manifestations of disease and effects of the treatments to the plants. Based on statistical analysis, Boost still consistently showed lesser percent area of vascular discoloration compared to Water (Fig. ) which further confirm its effectiveness in controlling the wilt disease complex. Fomes and Nordox also showed much lower percent area of vascular discoloration compared to control, Xoo and Aspirin. Table No. 3 Percent Area of Vascular Discoloration Treatment| Percent area of vascular discoloration| No. of Live Plants Remaining| T0 (Control)| 55. 27 ab| 3. 00 ab| T1 (Fomes)| 38. 47 b | 3. 33 ab| T2 (Xoo)| 79. 27 a| 2. 00 bc| T3 (Aspirin)| 80. 27 a| 1. 33 c| T4 (Boost)| 37. 67 b| 4. 00 a| T5 (Nordox)| 38. 47 ab| 3. 33 ab| CV (%)| 31. 37| 52. 18|
Means followed by a common letter are not significantly different at 5% DMRT> Fig. 6 Internal Vascular Discoloration of Plants (left-right) Control, Fomes, Xoo, Salicylic Acid, Boost, Nordox The number of live plants remaining was also gathered during the termination of the study and shown in Table 3. Boost, had the highest average number of remaining live plants followed by Fomes and Nordox. Aspirin had the lowest no. of live plants or highest mortality which was even higher than that of water (Fig. 8d, p. 24). The height of abaca plants is shown in Fig. 7.
Based on the graph, the plants sprayed with Boost and Nordox have higher plant height compared to Conrol, Xoo, Fomes, and Aspirin. Among the parameters used to measure the effects of the different treatments, the mean disease severity rating on a per plant basis and mean percent area of vascular discoloration were able to differentiate more the effect of the different treatments and were considered more reliable compared to the rest of the parameters used. Fig. 7 Height of Abaca Plants at the start of the experiment, at 7, 14, 21 days and the final height before the termination of the study b c d ef Fig. 8. Group of abaca plants treated with a) water, b) Fomes, c) Xoo, d) Aspirin, e) Boost and f) Nordox. Boost is a commercially available elicitor of resistance in plants and it was found very effective in controlling the wilt disease complex in abaca. Boost, however is a little expensive, one spray cost P0. 65. Nordox is a commercial fungicide with bactericidal activity and it has also reduced the wilt disease incidence and severity. This is a little cheaper (P0. 45) compared to Boost. A cost-effective control for the wilt disease complex was Fomes.
It was able also to reduce disease incidence and severity. Although not as effective as Boost but, no cost has been incurred, except for the labor in gathering, drying and pulverizing. The use of Fomes as a biological elicitor of resistance in abaca to control wilt disease complex has been explored in this research. Most probably, being a fungus, the chitin in Fomes could be the elicitor of resistance. Kutchitsu et al (1993) reported that chitin induced resistance of plants. The active component of Fomes, that could elicit resistance in plants could be isolated in future research endeavor. CONCLUSION AND RECOMMENDATIONS
Boost (chemical elicitor) and Fomes (potential biological elicitor and Nordox (fungicide with bactericidal activity), were able to effectively control the fusarium wilt and bacterial wilt disease complex of abaca. Xoo and Aspirin were not able to control the disease but instead has aggravated the wilting of the plants. A follow-up study maybe conducted to re-evaluate the same elicitors for their effect on either fusarium wilt or bacterial wilt alone. Likewise, a study maybe conducted to further evaluate the potential of Fomes as biological elicitors of resistance to other diseases of abaca or to other plant diseases.
Furthermore, several other indigenously occurring biological elicitors of resistance may be evaluated against the wilt diseases of abaca. ACKNOWLEDGEMENT First and foremost, we would like to extend our gratitude to all the people behind the success of this study. We would like to thank our adviser, Dr. Lucia Borines for sharing to us her knowledge, skills effort and even her time though with a hectic schedule. To Ms. Christine Advincula, Ms. Victoria Palermo and Ms. Robleyn Piamonte, thank you so much for patiently guiding and helping us everytime we need assistance especially when our adviser is on travel.
Thank you also to Mr. George Valenzona, for the analysis of our data, and Mr. Marlon Tambis and Mr. Victor Calunangan for the computer assistance. In addition, our heartfelt thanks is extended to our loving parents who supported us in our financial needs and for enlightening our faith that we could be able to finish this research on time. No words indeed can express how much we owe you this success. REFERENCES 1) BASTASA, G. N. and A. A. BALIAD. 2005. Biological control of fusarium wilt of abaca (Fusarium oxysporum) with Trichoderma and Yeast. http://cropscience Philippines. blogspot. om/2006/032005-vol30-no-2-v30n02p01-02. html 2) BORINES, L. M. 2008. Efficacy of chemical activators of systemic acquired resistance for the control of bacterial blight and diseases of rice. Paper presented during the 2008 R&D Regional Conference held at Baybayon ni Agalon, Albuera Leyte, June 17-18, 2008. 3) BORINES, L. M. SALAMAT, E. E. CARDINES, R. B. 2007. Reaction of abaca (Musa textiles Nee) accessions and varieties to Fusarium wilt caused by Fusarium oxysporum f. sp. cubense. (E. F. Smith) Snyd. And Hanns. Annals of Tropical Research. Volume 29 (No. 1). 4) EL-MOUGY, N. S. , R. ABB-L-KAREEM and M. A.
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