Panama disease control agent for cavendish bananas and control method using same

Abstract

[Problem] To provide: a control agent having an antagonistic action on a pathogen Foc TR4 of Panama disease of Cavendish bananas; and a method for controlling the pathogen using the control agent. [Solution] The pathogen Foc TR4 of Panama disease of Cavendish bananas is controlled by using a mycelium fertilizer that is derived from a bacterium of the genus Bacillus, a heat-resistant actinomycete, or both and that is obtained by kneading 20-30 parts by weight of an organic substance having a carbon ratio of 15% or less and a water content of 25-30 parts by weight into a porous carrier having a pH of 7.5-9.5 and a particle size of 4-100 mesh.

Description

Control agent for Panama disease of Cavendish banana and control method using said agent

[0001] The present invention relates to providing an agent for controlling Panama disease of Cavendish bananas and a method for controlling the disease using the agent.

[0002] Global banana production is under serious threat from Panama disease. This disease, caused by the soil-borne fungus Fusarium oxysporum f.sp. cubense (Foc), wiped out the Caribbean banana industry based on the Gros Michel variety in the mid-20th century. The impact of Foc was overcome by switching to the resistant variety Cavendish, which now accounts for the largest share of bananas in the market.

[0003] In the 1990s, a new disease called Panama disease, caused by a new Fusarium fungus called Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), which is transmitted to the Cavendish banana plant, became widespread and remains a major threat to banana cultivation worldwide.

[0004] According to a May 2014 report by the Food and Agriculture Organization of the United Nations (FAO), entitled "Technical Manual Prevention and Diagnosis of Fusarium Wilt (Panama disease) of Bananas Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4)," "TR4 (VCG 01213) emerged in Malaysia and Indonesia in the early 1990s (Masdek et al., 2003; Nasdir, 2003) and spread throughout Southeast Asia and Australia within a decade, significantly impacting the livelihoods of thousands of workers and farmers in all affected countries."

[0005] JP 2015-105225 A JP 1-264987 A

[0006] Currently, there is an urgent need for effective control agents and methods that have antagonistic activity against Foc TR4, the pathogen of Panama disease.

[0007] In light of such circumstances, the inventors of the present application have conducted extensive research and found that mycelium fertilizer, which is a soil improvement material made from charcoal, has an effective antagonistic effect on Foc TR4.

[0008] "Mycelium fertilizer" is a soil improvement material containing beneficial microorganisms that are cultured and immobilized on a porous carrier such as coconut shell activated carbon, a type of charcoal (biochar) made from biological resources that is effective in activating organisms and improving the environment.

[0009] The invention of claim 1 relates to a mycelium fertilizer for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), the pathogen of Panama disease of Cavendish bananas, which is derived from Bacillus bacteria, thermotolerant actinomycetes, or both, and which comprises a porous carrier having a pH of 7.5 to 9.5 and a particle size of 4 to 100 mesh, and 20 to 30 parts by weight of an organic material having a carbon content of 15% or less and a moisture content of 25 to 30 parts by weight mixed therein.

[0010] The invention according to claim 2 relates to a method for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), the pathogen of Panama disease of Cavendish bananas, which comprises mixing the mycelium fertilizer with soil in which Cavendish bananas are grown.

[0011] The invention according to claim 3 relates to a method for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), the pathogen of Panama disease of Cavendish bananas, which comprises spraying the mycelium fertilizer on the planting soil or the base of Cavendish bananas at a rate of 0.5 kg to 2.5 kg per Cavendish banana plant.

[0012] According to the invention of claim 1, the mycelium fertilizer having an antagonistic effect against Foc TR4, the pathogen of Panama disease of Cavendish bananas, can suppress the onset and spread of Panama disease.

[0013] According to the invention of claim 2, by mixing the mycelium fertilizer having an antagonistic effect against Foc TR4, the pathogen of Panama disease of Cavendish bananas, into the soil where Cavendish bananas are planted, it is possible to effectively suppress the onset and spread of Panama disease.

[0014] According to the invention of claim 3, the onset and spread of Panama disease can be suppressed by applying the mycelium fertilizer having an antagonistic effect against Foc TR4, the pathogen of Panama disease of Cavendish bananas, to the planting soil or base of Cavendish bananas at a rate of 0.5 kg to 2.5 kg per Cavendish banana plant.

[0015] 1 shows external and internal symptoms of Panama disease-infected Cavendish bananas.

[0023] Figure 1 shows external and internal symptoms of Panama disease observed in a HyPro biochar (the same as HyPro, a brand name of mycelium fertilizer manufactured by King Coal Corporation) experiment in Ramanan, Calinan District, Davao City.

[0024] Figure 1 shows isolated Foc TR4 after 7 days of incubation.

[0025] Figure 2 shows bacteria isolated from HyPro biochar.

[0026] Figure 3 shows the effect of different concentrations of HyPro biochar pure culture on the growth of Foc TR4 after 7 days of incubation.

[0027] Figure 4 shows the effect of different rates of HyPro biochar on Cavendish banana seedlings infected with Foc TR4.

[0028] Figure 5 shows discoloration of vascular tissue in the rhizome of plants infected with Foc TR4.

[0029] Figure 6 shows (A) infection rate of Foc TR4, (B) control rate against Foc TR4, and (C) mean number of days until symptom onset by Foc TR4. 1 shows the soil treatment process using mycelium fertilizer mixed into the soil where Cavendish bananas are grown. 2 shows the average height of Cavendish banana plants 14 months after planting, to which either HyPro biochar or Hatake (a brand name of a biofertilizer manufactured by Hatake Harvest Philippines, Inc.) has been applied. 3 shows the trunk circumference of Cavendish banana plants 14 months after planting, to which either HyPro biochar or Hatake has been applied.

[0016] Below, we will explain in detail the laboratory experiments that the inventor conducted at a research center in Davao City and a research laboratory of a private plantation company, and then the experiments on seedlings that were conducted on Mindanao Island in the Philippines.

[0017] (Method for producing mycelium fertilizer) A preferred method for producing the mycelium fertilizer used in this experiment will be described.

[0018] First, 20 to 30 parts by weight of organic matter with a carbon content of 15% or less and a moisture content of 25 to 30 parts by weight is mixed with a porous carrier having a pH of 7.5 to 9.5 and a particle size of 4 to 100 mesh. Here, mycelium fertilizer produced using a porous carrier of 60 to 100 mesh can be preferably used when mixed with liquid fertilizer and injected into the roots of already planted Cavendish bananas or into the planting soil, and mycelium fertilizer produced using a porous carrier of 4 to 60 mesh can be preferably used when mixed with soil before planting Cavendish bananas.

[0019] As described above, the porous carrier has a pH range of 7.5 to 9.5 in order to ensure a suitable pH range for the growth of thermotolerant actinomycetes and thermotolerant bacteria (Bacillus bacteria) during the production and storage of the mycelium fertilizer. Limiting the pH range of the porous carrier also has the effect of inhibiting the proliferation of harmful bacteria such as filamentous fungi that do not grow well under alkaline conditions.

[0020] According to the technology disclosed in Japanese Patent Laid-Open No. 1-264987, a porous carrier such as coconut shell activated carbon, activated coke, or vermiculite is kneaded with 20 to 30 parts by weight of an organic material having a carbon content of 15% or less and a moisture content of 25 to 60%. The reason for limiting the organic material to a carbon content of 15% or less is based on the inventor's experimental findings that organic materials with a carbon content of more than 15% contain a high amount of fibrous cellulose, resulting in a high required amount of cellulase, which leads to the dominance of cellulase growth in actinomycetes, preventing the intended object of this invention from being achieved. The moisture content of the organic material is limited to 25 to 60% because the moisture in the fermentation system of the kneaded material is supplied solely from the organic material, and unless the moisture in the fermentation system is 25 to 60%, preferably 30 to 40%, a sufficient fermentation temperature for the thermotolerant actinomycetes cannot be obtained. Conversely, if the moisture content of the fermentation system exceeds 60% or is less than 25%, sufficient fermentation conditions for fermenting the thermotolerant actinomycetes cannot be obtained, which is undesirable.Furthermore, the reason for using 20 to 30 parts by weight of organic matter is that if the amount of organic matter is less than 20 parts by weight, the amount is too small, resulting in small growth of mycelia on each carrier, and the mycelia do not disperse uniformly on each porous carrier and adhere, while if the amount exceeds 30 parts by weight, the balance between the mycelia and the porous carrier is disrupted, resulting in an excess of mycelia or a high rate of undecomposed organic matter, which are undesirable in either case.

[0021] The organic matter used should not contain antibacterial substances. Chicken manure, etc., can be used as the organic matter, but in this case, the manure should be from chickens fed a diet that does not contain antibacterial substances. Antibacterial substances generally include antibiotics and synthetic antibacterial agents. Examples of antibiotics include aminoglycosides, cephims, tetracyclines, penicillins, and macrolides, and examples of antibacterial agents include quinolones, sulfonamides, thiamphenicols, and fluoroquinolones. However, the feed given to chickens does not contain these antibacterial substances. By using manure from chickens fed a diet that does not contain these antibacterial substances, such as antibiotics and synthetic antibacterial agents, the growth of thermotolerant actinomycetes and thermotolerant bacteria is promoted, and the mycelium can produce a total of 1.0 x 10 thermotolerant actinomycetes and thermotolerant bacteria per 1 g of mycelium fertilizer. ６It is possible to produce a mycelium fertilizer containing 1 / g or more of the lactic acid bacteria. Examples of chicken manure that can be used include, but are not limited to, Boris Brown (also known as Akatama chicken, scientific name Gallus gallus domesticus), and any domestic chicken. Examples of feed (antibacterial-free feed) for chickens include compound feed consisting of 50-60% grains, 10-20% vegetable oil cake, 5-10% animal feed, and cereals, such as Eten 17 (Nissin Marubeni Feed).

[0022] Next, the kneaded mixture is left to stand in an atmosphere capable of maintaining an internal temperature of 15° C. or higher. The reason for this is to shield the internal temperature from the outside air temperature and maintain a constant temperature to maintain uniformity of fermentation. If this temperature is below 15° C., the fermentation temperature will not rise sufficiently, which is not preferable.

[0023] After maintaining a constant temperature within the system and isolating it from the outside air, the temperature of the fermentation system is maintained at 55 to 80°C, preferably 60 to 70°C. If the fermentation temperature is below 55°C, in order to promote fermentation, air maintained at 15°C or higher within the system is fed (aeration) through an attached pipe or pump from the bottom of the fermentation tank for a certain period of time to maintain the fermentation temperature at 55 to 80°C. However, because the cellulose content is limited to organic matter with a carbon percentage of 15% or less and the moisture content is limited due to the large amount of porous carrier mixed in, it is practically impossible for the fermentation temperature to rise above 80°C.

[0024] Fermentation under these conditions for at least two days produces a mycelium fertilizer characterized by culturing and fermenting thermotolerant actinomycetes and thermotolerant bacteria (Bacillus bacteria) in a porous carrier at a pH of 7.5 to 9.5. The fermentation period is at least two days, preferably five days. The fermentation temperature is maintained at 55 to 80°C because, at temperatures below 55°C, thermotolerant actinomycetes and thermotolerant bacteria cannot be obtained in the desired proportion of mycelium colony, while temperatures above 80°C result in the growth of anaerobic bacteria, both of which are undesirable.

[0025] A suitable example of a mycelium fertilizer is HyPro Biochar (the same as HyPro, a trade name for a mycelium fertilizer manufactured by King Coal Co., Ltd.). HyPro Biochar is a mycelium fertilizer primarily composed of Bacillus bacteria, thermotolerant actinomycetes, or both.

[0026] As mentioned above, by using 20 to 30 parts by weight of organic matter with a carbon content of 15% or less, limiting the moisture content of this organic matter to 25 to 60%, and further by using 50 to 78 parts by weight of a porous carrier with a particle size of 4 to 100 mesh, the fermentation temperature will not exceed 80° C. Therefore, the fermentation temperature will not rise above 80° C. even without the so-called turning-over step, which is performed in the usual fermentation process, of pouring water over the fermented product to lower the fermentation temperature.

[0027] (Method for Identifying Foc TR4) The method for identifying the pathogen used in this experiment as Foc TR4 will be described below.

[0028] (Identification by Occurrence Status and Pathological Condition) From 2005 to 2007, ERDS (EAGRI Research and Development Services), a research institute accredited by the Philippine government, monitored the occurrence status of Panama disease at two Cavendish farms on Mindanao Island.

[0029] Figure 1 shows the external and internal symptoms of Panama disease on Cavendish plants, including yellowing of the older leaves on young plants (a) and older plants (b), division of the pseudostem (c), and dark brown discoloration of the vascular tissue in cross-sections of the pseudostem (d and e).

[0030] Figure 2 shows the external and internal symptoms of Panama disease observed in a hyprobiochar experiment in Ramanan, Calinan District, Davao City. Leaf yellowing begins on older leaves (A). Infected plants eventually die (B and C). At the end of the experiment, dark discoloration of vascular tissue was observed (D and E). The symptoms observed in this hyprobiochar experiment were similar to those of Foc TR4 mentioned in the paper.

[0031] Plants showing typical external and internal symptoms were regularly monitored, and the yellowing of leaves, dehiscence of rhizoids, and discoloration of the vascular tissue in the cross-section of rhizoids to a black-brown color were compared with the symptoms of TR4 mentioned in the paper, thereby identifying the pathogen used in this experiment as TR4.

[0032] The two papers referenced above are mainly the following: (1) Kristle Grace I. Aguilar-Hawod, et al., Genetic Diversity of Fusarium oxysporum f. sp. cubense Causing Panama Wilt of Banana in the Philippines (Published: 28 December 2019) (2) Agustin Molina, et al., CONFIRMATION OF TROPICAL RACE 4 OF Fusarium oxysporum f. sp. cubense INFECTING CAVENDISH BANANAS IN THE PHILIPPINES (Poster paper presented during the Centennial Meeting of the American Phytopathological Society, July 26-30, 2008, Minneapolis, Minnesota, USA)

[0033] (Isolation of Foc) In this experiment, we adopted the Foc isolation protocol described by Vicente et al. (2014). Infected ducts were cut into small pieces from the internal tissue. 3-6 mm long sections were taken from the distal end of the specimen. The cut tissues were then immersed in a plate containing 70% ethyl alcohol for approximately 1 minute, washed with three plates containing sterile distilled water (SDW), wiped dry with blotting paper, and evenly plated onto ¼-strength potato dextrose agar (PDA) or water agar (WA) on plates containing an antibacterial agent (1.2 mL streptomycin sulfate / 240 mL PDA).

[0034] (Mass Production of Foc TR4) Foc TR4 was mass-produced in pure culture on half-strength potato dextrose agar medium (PDA medium). Agar disks were obtained from the pure culture of Foc TR4 and inoculated into fresh PDA medium with a sterile needle (Figure 3).

[0035] Identification of Isolated Pathogens by Morphological Characteristics: Isolates cultured on potato dextrose agar (PDA) for 7 days showed considerable morphological variation. Colors ranged from pale purple, pale orange, to deep red, and some were unpigmented. The appearance of the hyphae varied from flat to aerial, flocculent to cotton-like, and round to irregular. Microconida were elliptical and kidney-shaped and usually unicellular, although some possessed a single septum arising from short, simple leaflets, resembling a pseudohead in the presentation of aerial hyphae. All strains produced numerous microfruiting bodies. Macroconida usually had three septa, were thin, linear to sickle-shaped, and had a distinct peduncle. Chlamydospores varied in morphology, with smooth and rough walls, and were found in monospores, paired spores, and conidiospores in the terminal and interstitial regions. These morphological features are consistent with those described by Leslie and Summerell, cited by Aguilar-Hawod et al. (2019), and identify the pathogen used in this study as Foc TR4.

[0036] (In vitro experiment on antagonistic effect of mycelium fertilizer on Foc TR4) This section describes in detail an experiment on the antagonistic effect of mycelium fertilizer on Foc TR4 conducted at the Davao City Research Center. Here, HyPro (manufactured by King Coal Corporation; the same applies hereinafter) was used as the mycelium fertilizer. (Objectives of the experiment) 1. Evaluation of the antagonistic effect of HyPro on Foc TR4 2. Determination of the most effective use rate of HyPro on Foc TR4

[0037] Experimental Design: The laboratory experiment was conducted using a completely randomized design. Four treatments were replicated three times, with three plates per replicate.

[0038] (Isolation of Foc) In this experiment, we adopted the Foc isolation protocol described by Vicente et al. (2014). Infected ducts were cut into small pieces from the internal tissue. 3-6 mm long sections were taken from the distal end of the specimen. The cut tissues were then immersed in a plate containing 70% ethyl alcohol for approximately 1 minute, washed with three plates containing sterile distilled water (SDW), wiped dry with blotting paper, and evenly plated onto ¼-strength potato dextrose agar (PDA) or water agar (WA) on plates containing an antibacterial agent (1.2 mL streptomycin sulfate / 240 mL PDA).

[0039] (Mass Production of Foc TR4) Foc TR4 was mass-produced in pure culture on half-strength potato dextrose agar medium (PDA medium). Agar disks were obtained from the pure culture of Foc TR4 and inoculated into fresh PDA medium with a sterile needle (Figure 3).

[0040] (Isolation and Mass Production of Bacteria from HyPro) HyPro was inoculated onto a nutrient agar plate. After 24 hours, the bacteria growing around the inoculated HyPro were transferred to another plate to obtain a pure culture of the target bacteria (Figure 4).

[0041] (Test for Antagonistic Effect of HyPro on Foc TR4) To evaluate the antagonistic effect of HyPro in vitro, the following concentrations of pure cultures of HyPro-derived bacteria were tested against Foc TR4. Treatment / Pure Culture Concentration (1) Foc TR4 (control) / - (2) HyPro + Foc TR4 / 5 ml pure culture (3) HyPro + Foc TR4 / 3 ml pure culture + 2 ml sterile distilled water (4) HyPro + Foc TR4 / 1 ml pure culture + 4 ml sterile distilled water. Different concentrations of pure cultures of HyPro-derived bacteria were evaluated using the disk diffusion method according to Kirby-Bauer (1940). The filter paper discs were immersed in various concentrations of pure cultures of Hypro-derived bacteria (5 ml pure culture, 3 ml pure culture + 2 ml sterile distilled water, or 1 ml pure culture + 4 ml sterile distilled water) for 2 minutes. Four of the immersed discs were then inoculated onto a plate 3 cm away from the Foc TR4 mycelium disc in the center. After incubation of the tested fungal strains at room temperature for 7 days, the diameters of the fungal growth on the control (Foc TR4) and treated plates were measured.

[0042] (Growth diameter and growth inhibition rate) The diameter of the cultured fungal colonies was measured in millimeters (mm) using a ruler to record the average of the two diameters, the longest and shortest, taken at right angles for each colony. Fungal growth was measured after 7 days of incubation, and the zone of inhibition was determined. The inhibition rate was calculated using the following formula (Alwathnani and Perveen; 2012):

[0043] where C is the average colony diameter on the control plate and T is the average colony diameter on the treated plate.

[0044] Statistical Analysis Data were analyzed using a completely randomized design analysis of variance (ANOVA), and differences in means between treatments were compared using Tukey's range test (THSD).

[0045] (Results and Discussion) The results of (1) growth diameter (mm) and (2) inhibition rate (%) of Foc TR4 after 7 days of culture at different concentrations of HyPro are shown in Table 1. In addition, the effect of different concentrations of pure culture solution of HyPro-derived bacteria on the growth of Foc TR4 after 7 days of culture is shown in Figure 5.

[0046]

[0047] The significant effect of the concentration of pure culture of Hypro-derived bacteria on the growth of Foc TR4 by analysis of variance (ANOVA) is shown in Table 2 .

[0048]

[0049] The results showed that treatment with 5 ml of pure culture of HyPro-derived bacteria significantly reduced growth (26.17 mm) after 7 days of incubation, with a growth inhibition rate of 54.09%. Concentrations of 3 ml and 1 ml of pure culture of HyPro-derived bacteria had similar effects on Foc TR4 growth (diameters ranging from 33.83 mm to 51.17 mm), with growth inhibition ranging from 10.23% to 40.65%. Meanwhile, the greatest growth was observed on the control plate, with an average diameter of 57.00 mm.

[0050] These results suggest that treatment with the highest level of pure culture medium from HyPro-derived bacteria significantly affected the growth of Foc TR4 in vitro. Furthermore, increasing the amount of pure culture medium from HyPro-derived bacteria inhibited the growth of Foc TR4.

[0051] This article describes the details and results of an in vitro bioassay test of Hyprobiochar (biochar) against Panama disease in Cavendish bananas, conducted by the private plantation company, Lapandy Agricultural & Development Corporation.

[0052] A bioassay study was conducted to evaluate the efficacy of HyPro Biochar (biochar) against the pathogen causing Fusarium wilt disease on Cavendish bananas. HyPro Biochar (biochar) was applied at concentrations of 1 to 5 gm / L and incubated at room temperature for 7 days. Growth inhibition rates of 51.34% to 63.33% were observed, comparable to those of commercially available fungicides. This demonstrated that HyPro Biochar (biochar) has the ability to inhibit the growth of the pathogen (Table 3).

[0053]

[0054] (Experiment 1 to evaluate the antagonistic effect of hyprobiochar against Foc TR4 in vivo) The following describes an experiment conducted at the ERDS Experimental Farm Station in Lamanan, Calinan District, Davao City, in which hyprobiochar was sprayed at the base of banana seedlings as a control agent against Foc TR4, and the biological effectiveness of hyprobiochar was evaluated.

[0055] (Experimental Environment) The experiment was conducted using a completely randomized design (CRD) with six treatment plots (T1-T6) replicated four times. Each replicate consisted of 10 sample plants, for a total of 40 plants per treatment. Sterilized potting soil (14W x 12L inches) was appropriately placed in the open field according to the experimental design, with a 12-inch spacing between pots and a 1.0 meter spacing between plots. The six treatment plots were as follows: T1: Control (untreated) T2: 50g of Hi-Pro biochar T3: 150g of Hi-Pro biochar T4: 250g of Hi-Pro biochar T5: 500g of Hi-Pro biochar T6: 1g of Hatake

[0056] Soil preparation and seeding: Soil medium was collected from the Foc TR4-infected area. The collected infected soil was placed in a 14W x 12L black plastic bag. After one day, the soil was seeded with 15 million Foc TR4 spores, and the pathogen was allowed to incubate for 25 days to ensure uniformity of the Foc TR4 inoculum in the potted medium.

[0057] (Treatment and Planting) Infested soil placed in large black plastic bags or pots (14 inches wide x 12 inches long) was treated with HyPro biochar and allowed to cure for 30 days in the treatment areas. Two-month-old tissue-cultured Cavendish banana seedlings were then planted. HyPro biochar was applied only once, with no further treatments given during plant growth. This trial began with a control treatment of HyPro biochar and Hatake. The treatments, application rates, and application methods were as follows: T1: control (untreated), T2: 50 g of HyPro biochar, T3: 150 g of HyPro biochar, T4: 250 g of HyPro biochar, T5: 500 g of HyPro biochar, and T6: 1 g of Hatake. For T2 to T5, the treatment was mixed into the soil prior to planting. For T6, the treatment was allowed to soak into the soil prior to planting. 50 g / hill of complete fertilizer was applied uniformly to the entire plant on the 14th and 45th days after planting.

[0058] Calibration of Foc TR4 and Application Timing: Cavendish banana "Grand Nine," a Foc TR4-susceptible cultivar, was used as the test plant. The Foc TR4 isolate was obtained from Lapanday Research Laboratory and mass-produced at the ERDS laboratory. A pure culture of Foc TR4 was mixed with 1 L of water to calibrate the volume of the Foc TR4 suspension. 1 ml of the suspension was collected and spores were counted under a microscope using a hemocytometer, achieving a standard inoculation dose of 75,000 spores / ml. Based on this calibration, 200 ml of the highly virulent Foc TR4 suspension was sprayed onto sterile garden soil to infect the soil. The inoculated Foc TR4 was allowed to incubate for 25 days before treatment with hyprobiochar. Foc TR4 was inoculated only once throughout the experiment, simulating a field situation considering that only the population of Foc TR4 present in the field infects the root system of the plants.

[0059] (Monitoring Method and Analysis) Plants were inspected every two weeks to monitor the occurrence of Foc TR4 infection. During the final week of reading, the lower part of the plant (corm) was cut off and examined to determine the condition of the vascular bundle and rhizome. The number of days from the appearance of the first symptoms to the appearance of yellow streaks on the old leaves was recorded. Analysis of variance (ANOVA) was performed using the statistical analysis software SPSS, and comparison of mean values ​​was determined using Duncan's multiple range test (DMRT). More details are as follows.

[0060] (a) Phytotoxicity: Phytotoxicity in the treated plots was monitored 30, 60, and 90 days after planting. Phytotoxicity in the form of spindly growth, chlorosis, leaf burn, leaf curl, and poor growth was evaluated using the standard rating scale below, relative to the untreated control. Scale 1: None (%); Scale 3: 1-10%; Scale 5: 11-20%; Scale 7: 21-30%; Scale 9: Greater than 30%; (b) Foc TR4 Infection Rate: Sample plants were observed and inspected every two weeks, and plants infected with Foc TR4 were recorded. Subsequently, monitoring was continued every two weeks for four months. (c) Days to Symptom Onset: Plants showing yellowing of older leaves were observed and recorded every two weeks. (d) Data Analysis Data were analyzed by analysis of variance (ANOVA) using the statistical software SPSS (Statistical Package for the Social Science). Differences between treatment means were determined at a 5% significance level using Duncan's Multiple Range Test (DMRT).

[0061] Results and Discussion: Symptoms of Panama disease caused by Foc TR4 were observed on plants 6 weeks after planting (Table 4). Infection occurred in all treatments, affecting 20% ​​of untreated plants. The two highest concentrations of HyPro biochar, 500g / plant and 250g / plant, showed the slowest onset of Panama disease in plants, with an infection rate of 2.5%. Meanwhile, 150g and 50g HyPro biochar helped plants resist Panama disease, with infection rates of 5% and 7.5%, respectively, significantly lower than the infection levels recorded in Hatake (18.5% infection rate) and untreated controls (20% infection rate). HyPro biochar provided long-term protection, with the 500g treatment showing no further infection even at 10 weeks, despite continued treatment. The 250g, 150g, and 50g high-pro biochar concentrations showed minimal infection by week 8, but by week 10, the latter two treatments showed reduced viability and resulted in a higher number of infected plants compared to the 500g and 250g high-pro biochar treatments. By week 12, infection symptoms progressed and became more severe the following week (week 14). However, despite the accumulation of Panama disease infestations, the highest high-pro biochar treatment, 500g, had the fewest infected plants. By week 16, the reduction in plant infection in the 500g high-pro biochar treatment indicated that control was indeed possible, as indicated by successful plant growth. Plants treated with 250g and 150g of Hi-Pro biochar per plant showed similar disease incidence due to Foc TR4, but infection was slightly more advanced at 50g / plant. Recovered plants showed signs of recovery, including shoot elongation and improved plant growth (Figure 6). In contrast, newly infected plants treated with Hatake began appearing at week 10, and infection incidence was significantly more severe, continuing to increase through week 16. Furthermore, the four Hi-Pro biochar treatments failed to provide a similar level of infection suppression at any stage of plant development.In the final week of evaluation (week 18), some infected plants were found to show discoloration of the vascular tissue of the rhizome (FIGS. 7(A) and (B)).

[0062] Panama disease infection rates: Only 15% of plants were infected in the 500g / plant high-pro biochar treatment, significantly lower than the other three high-pro biochar treatments (250g, 150g, and 50g) and Hatake (47.50%) (Figure 8A). Although infection rates increased in the 250g high-pro biochar treatments, this treatment was the best treatment for Foc TR4 control after the highest rate of 500g / plant. Fusarium disease infection progressed slowly in untreated plants, as plants remained asymptomatic for the first few weeks. However, by week 12, symptoms of yellowing of older leaves were evident, increasing through week 16, and by week 18, all plants were affected by Foc TR4. The results revealed that the effect of the highest rate of hyprobiochar, 500 g / plant, was very promising and provided excellent control of Foc TR4 on Cavendish banana, far superior to the other three rates of hyprobiochar and Hatake.

[0063]

[0064] (Control Effect of Foc TR4) This is explained based on Figure 8(B). The effect of the high-pro biochar in strengthening the root zone of the plant was confirmed by the absence of infected plants. Therefore, a healthy root system corresponds to a plant's ability to withstand disease attack. The most effective treatment for controlling Panama disease was 500 g / plant of high-pro biochar, which showed a high control rate of 85%. Furthermore, the effect of 250 g / plant of high-pro biochar, with a control rate of 76.67%, was comparable to the two lower rates of high-pro biochar, 50 g / plant and 150 g / plant, with a control rate of 70%. However, the latter two treatments showed better control than Hatake, which showed a control rate of 52.50%. Furthermore, it was confirmed that the highest rate of 500 g / plant of high-pro biochar had a more advantageous effect on controlling Foc TR4 than the other three high-pro biochar treatments.

[0065] (Average days to symptom onset) This is explained based on Figure 8(C). Visual observation of actual plant growth revealed that the onset of symptoms caused by Foc TR4 was not always pronounced, and that the plant's instinctive response to infection may not be synchronized, especially in untreated plants. Untreated plants were asymptomatic and did not immediately develop symptoms, but the plant's interior was already infected. However, in the case of the high-pro biochar treatments, the two lower concentrations of 50g / plant and 150g / plant were effective in delaying the onset of Panama disease infection, with an average of 105.60 days and 102.67 days, respectively. Although infection symptoms appeared earlier in plants treated with 250g and 500g of high-pro biochar, the rapid plant response to infection did not reduce the treatment effect. Furthermore, although yellowing symptoms were soon observed on older leaves, the disease did not progress rapidly, and only a few plants were affected. Additionally, the later onset of symptoms may be due to the control effect of the Bacillus strain contained in the hyprobiochar, which may have been able to promote plant growth and suppress the disease. Although the moss can also affect plants and delay infection, it cannot help them recover from the damage caused by Panama disease.

[0066] (Summary) The four high-pro biochar treatments failed to strongly suppress Foc TR4 infection, as did the Hatake fungus. However, the 500g / plant high-pro biochar treatment was the most effective treatment, with the lowest incidence of infected plants across all observations. By the final week, this treatment had a 15% infection rate, demonstrating its effectiveness against Foc TR4. This infection rate was significantly lower than the three lower rates of high-pro biochar (250g, 150g, and 50g) and Hatake, which showed 23.33%, 30%, and 47.5% infection rates, respectively, and discoloration of vascular tissue in rhizomes. Of the three lower rates of high-pro biochar, 250g / plant was the best treatment for Foc TR4 control, after the highest rate (500g / plant). The low-rate hi-pro biochar treatment delayed symptom onset the longest, at 105.60 and 102.67 days, but was not as effective at protecting plants from further infection as the two higher-rate hi-pro biochar treatments (500g / plant and 250g / plant). The 150g / plant and 50g / plant hi-pro biochar treatments slowed disease progression compared with the Hatake and untreated controls. The 500g / plant hi-pro biochar treatment provided the highest control of Foc TR4, at 85.00%, outperforming the three other hi-pro biochar treatments (250g, 150g, and 50g / plant). While the latter three treatments provided comparable control, their respective control rates of 76.67% and 70%, respectively, were significantly more favorable than the Hatake treatment at 1g / plant.

[0067] (Experiment 2 to Evaluate the Antagonistic Effect of Hyprobiochar on Foc TR4 in Vivo) The following describes an experiment conducted at Busia Farm in Tagakpan, Tugbok District, Davao City, aimed at determining the optimal amount of Hyprobiochar as a control agent against Foc TR4 and comparing it with Hatake, a common organic product, against Panama disease of Cavendish bananas.

[0068] (Experimental environment) The experiment was conducted using a randomized complete block design (RCBD), with six treatment plots (T1 to T6) replicated four times. Each replicate consisted of 10 sample plants, for a total of 40 plants per treatment plot. The distance between each plant was 2 x 2.5 meters, and the distance between each furrow was 2 meters. The planting holes were 12 inches deep and 12 inches wide. The six treatment plots were as follows: T1: Control (untreated) T2: Hypro biochar 0.5 kg / plant T3: Hypro biochar 1.0 kg / plant T4: Hypro biochar 1.75 kg / plant T5: Hypro biochar 2.5 kg / plant T6: Hatake 100 g / plant

[0069] (Soil preparation and sowing) Bracts infected with Foc TR4 were incised and planted on the soil surface of the experimental field, and the pathogen was cultured. The culture was continued for 7 days, and it was confirmed that the amount of Foc TR4 inoculated on the soil surface was uniform.

[0070] Before transplanting the seedlings, 0.5 kg (T2), 1.0 kg (T3), 1.75 kg (T4), and 2.5 kg (T5) of Hyprobiochar and 100 g (T6) of Hatake were added to the planting hole and mixed with the infected soil, and the infected soil was then treated for 30 days (Figure 9). The control agent was applied only once.

[0071] (Management) During the experimental period, except for fungicide application to the soil, routine management operations such as insecticide and pseudostem application, foliar fungicide application to control sigma moth, weeding, and irrigation were carried out uniformly according to standard methods.

[0072] (Monitoring Methods and Analysis) (a) Phytotoxicity. Phytotoxicity in the treated Cavendish bananas was monitored 30, 60, and 90 days after planting. Crop damage in the form of stunting, chlorosis, leaf burn, leaf curl, and poor growth was assessed using a standard rating scale relative to untreated controls. Scale 1: None (%) Crop Damage; Scale 3: 1-10 (%) Crop Damage; Scale 5: 11-20 (%) Crop Damage; Scale 7: 21-30 (%) Crop Damage; Scale 9: Greater than 30 (%) Crop Damage. (b) Infection Rate of Foc TR4. Sample plants were observed and inspected every two weeks, and the number of plants infected with Foc TR4 was recorded. Monitoring continued until the banana fruiting stage. (c) Data Analysis. Data were analyzed by analysis of variance (ANOVA) using the statistical software SPSS (Statistical Package for the Social Science). Differences between treatment means were determined at the 5% significance level using Duncan's multiple range test (DMRT).

[0073] (Results) Symptoms of Panama disease caused by Foc TR4 were observed three months after planting (Table 5). Infection occurred in T4 and T6 (hatake), with a 2.5% incidence rate for both. Meanwhile, by six months, the incidence rate in the untreated T1 plot was 7.5%. In the plot treated with low-concentration (0.5 kg / plant) high-pro biochar (T2), no Panama disease was observed for 12 months. However, statistically, T2 was comparable to T3, T5, and T6. One infected plant in T3 at five months recovered from Panama disease three months later. This demonstrates the ability of high-pro biochar to aid plant recovery. In T4, the incidence rate increased at 3, 7, 11, 12, 13, and 14 months. The untreated plot (T1) showed a very high incidence rate of 82.5% at 14 months. In the high concentration (2.5 kg / plant) high-pro biochar treatment area (T5), the incidence of Panama disease was 0% for 14 months.

[0074] Here, T1-CFP-UTC refers to a treatment area in which neither high-pro biochar nor moss was used in the planting soil (other conditions were the same as the other treatment areas).

[0075] (Growth of Cavendish Banana Plants) The average height of Cavendish bananas 14 months after planting was compared (Figure 10). The Hi-Pro biochar-treated plots (T1-T4) were taller than the untreated plot (T1) at 197.23 cm, with the Hi-Pro biochar 2.5 kg / plant treatment (T5) showing the highest height at 212.98 cm. Meanwhile, the Hatake 100 g / plant treatment (T6) showed the lowest height at 190.48 cm. The average trunk circumference of Cavendish bananas 14 months after planting was also compared (Figure 11). The average trunk circumferences of the untreated plot (T1) and the Hatake 100 g / plant treatment (T6) were 40.15 cm and 40.36 cm, respectively. In contrast, the Hi-Pro biochar-treated plots were 43.31 cm at T2, 43.14 cm at T3, 43.22 cm at T4, and 44.08 cm at T5.

[0076] (Cavendish Banana Harvest) Cavendish banana harvest data were compared 14 months after planting (Table 6). The average fruit weight per Cavendish banana plant was 14.50 kg (T1) and 17.53 kg (T6) in the untreated area and the 100g / plant treatment, respectively. In contrast, in the high-pro biochar treatments, the average fruit weight was 18.80 kg (T2), 18.12 kg (T3), 20.44 kg (T4), and 19.39 kg (T5). The Cavendish banana box stem ratio (BSR) was 1.07 and 1.30 in the untreated area (T1) and the 100g / plant treatment, respectively. In contrast, in the high-pro biochar treatments, the average fruit weight was 1.39 (T2), 1.34 (T3), 1.51 (T4), and 1.44 (T5).

[0077] Ave. No. of Hands: The average number of fruits per bunch. Ave. Bunch Weight: The average weight of fruit bunches harvested from one Cavendish banana plant. Ave. Fruit Weight: The average weight of fruit harvested from one Cavendish banana plant. BSR: Box Stem Ratio.

[0078] (Summary) Compared to the untreated plots, the four Hi-Pro biochar-treated plots were clearly able to prevent Foc TR4 infection. In particular, when Hi-Pro biochar was used at a high concentration of 2.5 kg per Cavendish banana plant, no Foc TR4 infection was confirmed for 14 months after planting. Therefore, this experiment demonstrated that the most effective amount of Hi-Pro biochar was 2.5 kg per plant. It was also found that the use of Hi-Pro biochar promoted Cavendish banana growth compared to when no Hi-Pro biochar was used or when fertilizer was used.

[0079] The control agent and control method of the present invention have an antagonistic effect against the pathogen of Panama disease of Cavendish bananas, and are therefore of great utility to Cavendish banana growers.

Claims

1. A mycelium fertilizer for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), the pathogen of Panama disease of Cavendish bananas, which is derived from Bacillus bacteria, thermotolerant actinomycetes, or both, and which is obtained by kneading 20 to 30 parts by weight of an organic matter having a carbon content of 15% or less and a moisture content of 25 to 30 parts by weight into a porous carrier having a pH of 7.5 to 9.5 and a particle size of 4 to 100 mesh.

2. A method for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), the pathogen of Panama disease of Cavendish bananas, which comprises mixing the mycelium fertilizer with soil in which Cavendish bananas are grown.

3. A method for controlling Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc TR4), which is a pathogen of Panama disease of Cavendish bananas, comprising spraying the mycelium fertilizer on the planting soil or at the base of Cavendish bananas at a rate of 0.5 kg to 2.5 kg per Cavendish banana plant.

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