DOI: https://doi.org/10.56669/WPMN7356
ABSTRACT
Crown rot disease (CRD) of papaya is considered one of the most important diseases affecting papaya in Malaysia. Various management methods such as chemical, biocontrol and farm practices were evaluated and failed to control the disease effectively. Hence a new strategy to induce the plant’s resistance to manage this disease was developed. One hundred and thirty-nine plant growth-promoting rhizobacteria (PGPR) were isolated and screened for systemic resistance induction on papaya seedlings. Thirty-one of the bacterial isolates displayed an induction of induced systemic resistance (ISR) with disease control scores ranging from 20–100%. Eight isolates had recorded 100% disease suppression. All the 8 isolates were further tested on hotspot and after one crop cycle of 24 months, seven isolates showed 100% control against CRD. One of the best performing isolates in terms of disease control was MIB 106 which was further selected for up-scaling in commercial farm. The isolate recorded 95% resistance after one crop cycle. After the completion of the up-scaling, this technology was thoroughly evaluated for its commercial potential by Technology Commercialization and Business Centre (TCO), MARDI based on the criteria such as proof of concept, novelty of innovation, technology competitiveness and potential market. Once the technology potential had been determined, it was protected by intellectual property rights, launched, and commercialized. MARDI has approved the usage of the technology following thorough review by technology user based on testimonials who provided feedback on the outcomes and issues of the technology.
Keywords: Crown Rot Disease, papaya, Induced Systemic Resistance, plant growth-promoting rhizobacteria
INTRODUCTION
Papaya is one of the significant fruits grown in Malaysia. In 2021, the area cultivated to papaya is approximately 2,822 ha (DOSM, 2022). The variety Sekaki is the most widely grown, with Eksotika, Hawaiian Solo, Subang, and Batu Arang being grown on a smaller scale. The disease known as papaya bacterial dieback, or also known as crown rot disease of papaya, was first reported to infected papaya crop in Malaysia in 2003. Another incidence was then reported in Perak in October 2004. By the end of 2006, the disease had spread to five other states on the west coast of Peninsular Malaysia. The causal agent of the disease was initially identified as Erwinia papayae (Makhatr et al., 2008). Later it was re-classified as Erwinia mallotivora (Mat Amin et al., 2010).
Since the disease appearance was first noted, it has destroyed an estimated 800 hectares of cultivated papaya, affected 1 million trees nationwide resulting in a loss of 200,000 ton. Malaysia's export of papaya has then decreased from RM200 million in 2004 to RM28 million in 2012.
The mode of action of the pathogen is through invading and colonizing all the papaya plant parts, including the shoot, leaf, bark, and fruit. Early symptoms of CRD on papaya include yellowing and necrosis along the leaf margins, followed by wet spots on the stalk base, crowns, and midribs of the leaves. Following the development of necrotic and water-soaked patches on stems, the pathogen then spread into the internal tissues. At infection sites, dark and oily spots have occasionally been observed (Mat Amin et al., 2011). The plant part that produces fruit, will completely rot as the infection on the crown spread over time. At the final infection stage, the water-soaked leaf stalk ultimately caused the infected part to die. Once the pathogen entered the plant, there is currently no effective means to cure the disease.
The issues faced by the papaya industry in Malaysia, notably caused by the CRD, had been addressed with concerted efforts centered on short-, mid-, and long-term strategies.
i) Development of ISR technology to control crown rot disease of papaya
Induced systemic resistance (ISR) technology to control the CRD of papaya was developed by bioprospection and manipulation of ISR inducing PGPR from papaya cultivated soil. The study involved isolation and screening for potential PGPR strains selection and testing of the ISR seedlings on an open field (hotspot) for papaya CRD. The detail explanation on the technology development process is given below:
- Isolation of PGPR
The soil from healthy papaya plants were sampled from three locations viz Serdang, Selangor and Lunas and Keladi in Kedah by using selective media for Bacillus and Pseudomonas. The bacilli were isolated using mannitol egg yolk polymyxin agar (MEYP) (Atlas, 1997) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA) (Atlas, 1997) and the pseudomonads were isolated using Pseudomonas F agar (Atlas, 1997) and King's media B (Atlas, 1997).
A total of 139 samples of bacilli and pseudomonad were isolated from roots of papaya in three locations namely Serdang, Keladi and Lunas. This is shown in Table 1. From the samples collected, nine bacilli were isolated from Serdang, 42 from Keladi and 36 from Lunas. Out of 52 pseudomonads isolated, 20 samples were from Serdang, 19 samples were from Keladi and 13 were from Lunas.
- Screening of ISR inducing PGPR
The Eksotika papaya seeds were soaked overnight in a container containing distilled water. Floating seeds were discarded as they were considered non-viable. The submerged seeds were soaked into a container containing PGPR suspension for 2 hours. For control treatment, the seeds were not soaked in PGPR suspension. Both the PGPR treated, and control seeds were then sown into germination tray filled with peat moss. Two weeks after sowing, each young seedling with 3-4 leaves stage was transferred into a 15 x 23 cm perforated polybag containing soil mixture of 2 parts of topsoil, 1 part of organic matter and 1 part of sand and kept under 50% shade in the nursery.
When the seedlings were two and six weeks old, the treatments were treated with the PGPR bacterial suspension (1mL/seedling with 1:50 dilution in water), while the seedlings in the control treatment were applied with sterile distilled water only. Each treatment was replicated 5 times. The seedlings were maintained for another two weeks (8 weeks after transferred to polybag) before being challenged with the papaya crown rot pathogen, E. mallotivora. This experiment was conducted under glasshouse conditions in MARDI Serdang.
The pathogen was grown in a nutrient broth with agitation for 48 hours at room temperature. The seedlings in the treated and control treatments were pricked (20 pricks/seedling) with a sterile needle on the stem close to the crown. Ten mL of the E. mallotivora suspension was sprayed at the pricked area using a hand-held sprayer. The seedlings sprayed with the pathogen were then covered with a plastic bag for two weeks. The disease symptom development and plant survival rate were observed at 4, 7 and 14 days after challenging with the pathogen.
Four days after inoculation with E. mallotivora, the first symptom of bacterial crown rot was observed. The seedling's shoot and stems developed watery lesions and gradually became necrotic, which are symptoms of papaya crown rot disease. Generally, these symptoms were developed in all treatments including control. After being inoculated with the pathogen for 14 days, the survival rates of the seedlings were recorded. Out of the thirty-one isolates tested, at least one plant survived from the disease. Eight of the treatments, which were inoculated with isolates MIB 10, MIB 13, MIB 43, MIB 58, MIB 100, MIB 106, MIB 113, and MIB 132, recorded 100% healing and survival. All plants in the control treatment were infected with the disease (Figure 1).
- Evaluation of ISR seedlings for disease resistance in hotspot
All the eight ISR positive PGPR isolates namely, (with 100% suppression against crown rot disease), MIB 10, MIB 13, MIB 43, MIB 58, MIB 100, MIB 106, MIB 113, and MIB 132 were used to produce ISR seedlings as mentioned above. Control (non-ISR) seedlings were also produced without PGPR inoculation as mentioned in the method above. The non-ISR seedlings were used to compare with the ISR inoculated seedlings for disease resistance. After the seedlings were transferred, a routine application of ISR inoculant was conducted once a month throughout the crop cycle. Control plants were also prepared and planted for comparison with ISR plants to evaluate the effects of disease infection. Fertilizer application and pest control were done according to standard practice (Chan et al., 1991). Good Agriculture Practice was employed throughout the crop cycle. Disease infection and plant death caused by the crown rot disease were recorded.
Generally, all the ISR seedlings showed more vigorous vegetative growth as compared to the seedlings in the control treatment at nursery stage. Both the ISR and control seedlings performance against papaya CRD infection were evaluated under open field in MARDI Serdang which was considered as a hotspot (where the disease is prevalent). After the completion of one crop cycle (24 months) evaluation at the hotspot and experiencing second rainy seasons (3 months), all the control plants were infected with the disease and eventually died (Table 2). Almost all the ISR plants survived until 24 months and were not infected with crown rot disease, except the treatment inoculated with MIB 13 isolate. The isolate MIB 13 recorded only 87.25% resistance against papaya crown rot disease. All the control plants were succumbed to crown rot disease within 11 months after being transferred to the field.
The ISR plants showed better vegetative growth, yield and no significant changes in fruit quality compared to plants in the control plot. The quality of the fruits produced by the ISR plants had Total Soluble Solid readings between 13-14% and red intense colour flesh.
ii) Pre-commercialisation
The ISR technology was pre-commercialized by up-scaling on a commercial farm. After being sown at MARDI Serdang, a total of 2,850 ISR Eksotika I papaya seedlings were sent to the commercial farm in Ampang Tinggi, Negeri Sembilan. The site was regarded as a hotspot because the previous papaya crop was completely afflicted and died from crown rot disease. After the seedlings were transplanted to the field, a routine application of ISR inoculants were applied once a month throughout the crop cycle. Fertilizer application and management of insect pests and diseases were done according to standard practice (Chan et al., 1991). Good Agriculture Practice was employed during the whole crop cycle. Disease infection and number of plants died caused by the crown rot disease were recorded.
Three months after the seedlings were planted in the field, the first incidence of crown rot disease infection was observed, affecting 85 papaya plants. The infection was contributed by the uncontrolled weed growth surrounding the root region of the papaya plants. Thirteen months later, the disease spread to another 61 plants. No further crown rot disease infection was observed until the project was completed. Only 5% of the papaya plants had crown rot disease at the end of the crop cycle. It was found that the fruits produced in this experiment were of similar quality to the fruits produced from the control plot, suggesting that the ISR treatment had no effect on the fruit quality.
iii) Evaluation process of the ISR technology and approval for commercialization
The Technology Commercialization and Business Centre (TCO), MARDI, will thoroughly assess each technology produced by its researchers for its commercial potential based on criteria such as proof of concept, novelty of innovation, technology competitiveness, and market potential (Ganisan Krishnen, et al., 2016). Positive feedback on the ISR technology has been obtained through field evaluations and up-scaling activities to prepare for use by the end users or stakeholders. Once the technology has been transferred to the end users or stakeholders, the TCO will then evaluate the impact of the technology. Corrective action will be recommended after an analysis of feedback regarding the technology's shortcomings and performance is completed (Ganisan Krishnen, et al, 2016).
- Perceptions and testimonies of papaya farmers about ISR technology
The ISR technology was developed as a preventive measure against the papaya crown rot disease. Plants that have been infected, especially those with severe infections cannot be cured with this method. However, many papaya growers used Dieback Buster (trade name of the ISR bacterial inoculants) when their papaya plants were infected with crown rot disease expecting that this product will cure their plants from the disease.Since this product is recommended for disease prevention, the best results to control the disease can only be achieved by applying Dieback Buster during seed sowing, booster application during nursery stage and continuous monthly application in the field for up to two years of production (Ganisan Krishnen and Mohamad Roff Mohd. Noor, 2021).
In general, farmers that used Dieback Buster and ISR technology as a preventive tool to manage the disease reported positive testimony on the effectiveness in controlling the crown rot disease and increasing production. To sustain high papaya production at their farms and produce high income, these farmers are willing to continuously adopt ISR technology (Krishnen and Mohd Noor, 2021).
- Intellectual property rights protection
A patent application was made with the Intellectual Property Corporation of Malaysia (MyIPO) to protect the ISR technology for inducing resistance against the papaya crown rot disease. The submitted patent is anticipated to protect the process of inducing systemic resistance in papaya at seedling stage in the nursery and in the field. The application for patent granting is now being reviewed by the patent agency.
The ISR technology has been sold by MARDI to Arif Efektif Sdn. Bhd., a Malaysian company. Arif Efektif Sdn. Bhd is currently the legal owner of the technology. No one is allowed to produce the inoculum without the company's prior written permission. Nonetheless, farmers can use Dieback Buster, which they purchase in the market, to grow their own ISR seedlings. The Dieback Buster and Adjustable Volume Liquid Gun are available for purchase from any All Cosmos Industries Sdn. Bhd. dealers in Malaysia.
- Technology launching and commercialization
For commercial purposes, the bacterial inoculants that induced systemic resistance were marketed under the brand name "Dieback Buster". The bacterial inoculants both induce resistance and effectively combat crown rot disease. To ensure that every papaya plant in the field receives the same amount of Dieback Buster spray volume, an equipment was designed. The equipment that was developed was known as Adjustable Volume Liquid Gun. When compared to manual application utilizing containers in the past, this equipment facilitates and expedites the application of Dieback Buster in the field.
On August 1, 2017, at the Exotic Star Sdn. Bhd. farm in Dengkil, Selangor, the Deputy Minister of Agriculture and Agro-based Industry Malaysia launched the Dieback Buster and Adjustable Volume Liquid Gun package technology. Numerous national newspapers reported on this new technology, which helped inform Malaysia's papaya growers and other interested parties.
On October 7, 2018, Arif Efektif Sdn. Bhd., a subsidiary company of All Cosmos Industries Sdn. Bhd. (a public listed company with BioNexus status), commercialized the ISR technology in Malaysia. Now, the company has successfully produced the inoculants in large volume and marketing the Dieback Buster. Since the product was introduced to the market in April 2019, about 393 ha of cultivated papaya have benefited from this technology. In order to effectively, introduce the technology to extension agents and farmers, Arif Efektif Sdn. Bhd. together with MARDI started a number of initiatives, including seminars, hands-on training sessions, and presentations in various scientific and technical forums. The company also uses social media and print media to promote the product through its dealers.
As Malaysian organizations who firmly support the Sustainable Development Goals, MARDI, Arif Efektif Sdn. Bhd. and All Cosmos Industries Sdn. Bhd. are happy to provide this technology so that papaya growers around the world, especially those in the Asia Pacific region, can benefit from it. It is possible to transfer the papaya ISR technology to other nations through correspondence with either MARDI, Arif Efektif Sdn. Bhd. or All Cosmos Industries Sdn. Bhd. Any correspondence regarding the technology transfer to foreign countries can be sent to aniadila@mardi.gov.my (MARDI) or wan_azha@allcosmos.com (Arif Efektif Sdn. Bhd.).
CONCLUSION
Malaysian papaya industry was pushed to the brink of collapse by the emergence of an alien invasive papaya crown rot disease. The introduction of this disease has devastated the production and exports of papaya. To address this issue, MARDI had developed an ISR technology to combat the deadly papaya disease. This technology was developed by bioprospecting of systemic inducing PGPR. Eight PGPR isolates with the highest disease control capacity (100%), were used to produce ISR seedlings, and evaluated them in an open field (hotspot). The results showed complete control against the papaya crown rot disease. The isolate MIB 106, was selected for activities involving upscaling. Additionally, this method was revalidated for the production of organic papaya in MARDI and for conventional production in three commercial farms which use chemical inputs. Both organic and inorganic papaya production, benefit greatly from this technology. ISR technology was protected through the Intellectual Property Corporation of Malaysia (MyIPO) patent filing process.
The ISR inoculant containing ISR inducing bacteria isolate MIB 106 was given the brand name DIEBACK BUSTER and the ISR inoculant application equipment as Adjustable Volume Liquid Gun. Both these technologies were launched in one of the farms where the validation was conducted. These package technologies were commercialized to Arif Efektif Sdn. Bhd., a subsidiary of All Cosmos Industries Sdn. Bhd. The products have been available in the market since April 2019. About 393 ha of cultivated papaya had benefitted from this technology. Various seminars and trainings were conducted to disseminates the knowledge and technology transfer of this technology to stakeholders, farmers, and extension agents. Farmers who used this method to prevent papaya crown rot disease gave positive testimonies and indicated their interest to continue using it. Currently, this is the only way to grow papaya while minimizing losses from bacterial crown rot disease.
The only hope for reviving Malaysia's flagging papaya industry and increasing both production and its exports is ISR technology. It is envisaged that when papaya can be effectively produced with the disease papaya crown rot eradicated, Malaysia would one day regain its position as the world's leading papaya exporter.
REFERENCES
Atlas Ronald, M. and Parks Lawrence, C. (1997). Handbook of Microbiological Media. Second edition. Boca Raton, CRC Press Inc., 1706 pages.
Chan Y.K., P. Raveendranathan, R. Zahar and P. F. Lam. (1991). Business proposal for the cultivation of papaya (cv. Eksotika Malaysia). Business Proposal Series No. 8. 83 pp. Ministry of Agriculture Malaysia, Kuala Lumpur.
DOSM. (2022). Selected Agricultural Indicators, Malaysia 2022. Department of Statistics Malaysia. Retrieved from https://www.dosm.gov.my/
Ganisan Krishnen and Mohamad Roff Mohd Noor. (2021) Success Story on Induced Systemic Resistance – A New Hope for Malaysian Papaya Industry. Asia Pacific Association of Agricultural Research Institutions, Bangkok, Thailand. xiv+63p
Ganisan Krishnen, Mohamad Roff Mohd. Noor, Alicia Jack, and Sharif Haron. (2016) Research, Development and Commercialization of Biofertilizer and Biopesticide in Malaysia. In Agriculturally Important Microorganisms: Commercialization and Regulatory , Eds. H. Bahadur Singh, B. K. Sarma and C. Keswani, pp. 149-166, Singapore: Springer Nature. doi 10.1007/978-981-10-2576-1
Ignatius, C. (2022). Malaysia needs “Big Innovation Purpose” to stay ahead. Business Today, 1 March 2022 . https://www.businesstoday.com.my/2022/03/01/malaysia-needs-big- innovation-purpose-to-stay-ahead/
Maktar, N.H., Kamis, S., Mohd Yusof, F.Z. and Hussain, N.H. (2008). Erwinia papayae causing papaya dieback in Malaysia. Plant Pathology, 57: 774; https//doi: 10.1111/j.1365- 3059.2008.01877.x
Mat Amin, N., Bunawan, H., Redzuan, R.A. and Jaganath, I.B. (2010). Erwinia mallotivora sp., a new pathogen of papaya (Carica papaya) in Peninsular Malaysia. Int. J. Mol. Sci. December 24;.12(1):39-45; https://doi.org/10.3390/ijms12010039
Norain Ismail, Mohd Jailani Mohd Nor and Safiah Sidek. (2015) A framework for a successful research products commercialization: A case of Malaysian Academic Researchers. Procedia-Social and Behavioral Sciences 195: 283-292. doi: 10.1016/j.sbspro.2015.06.163
Research, Development, and Commercialization of Induced Systemic Resistance Technology for Controlling the Crown Rot Disease of Papaya in Malaysia
DOI: https://doi.org/10.56669/WPMN7356
ABSTRACT
Crown rot disease (CRD) of papaya is considered one of the most important diseases affecting papaya in Malaysia. Various management methods such as chemical, biocontrol and farm practices were evaluated and failed to control the disease effectively. Hence a new strategy to induce the plant’s resistance to manage this disease was developed. One hundred and thirty-nine plant growth-promoting rhizobacteria (PGPR) were isolated and screened for systemic resistance induction on papaya seedlings. Thirty-one of the bacterial isolates displayed an induction of induced systemic resistance (ISR) with disease control scores ranging from 20–100%. Eight isolates had recorded 100% disease suppression. All the 8 isolates were further tested on hotspot and after one crop cycle of 24 months, seven isolates showed 100% control against CRD. One of the best performing isolates in terms of disease control was MIB 106 which was further selected for up-scaling in commercial farm. The isolate recorded 95% resistance after one crop cycle. After the completion of the up-scaling, this technology was thoroughly evaluated for its commercial potential by Technology Commercialization and Business Centre (TCO), MARDI based on the criteria such as proof of concept, novelty of innovation, technology competitiveness and potential market. Once the technology potential had been determined, it was protected by intellectual property rights, launched, and commercialized. MARDI has approved the usage of the technology following thorough review by technology user based on testimonials who provided feedback on the outcomes and issues of the technology.
Keywords: Crown Rot Disease, papaya, Induced Systemic Resistance, plant growth-promoting rhizobacteria
INTRODUCTION
Papaya is one of the significant fruits grown in Malaysia. In 2021, the area cultivated to papaya is approximately 2,822 ha (DOSM, 2022). The variety Sekaki is the most widely grown, with Eksotika, Hawaiian Solo, Subang, and Batu Arang being grown on a smaller scale. The disease known as papaya bacterial dieback, or also known as crown rot disease of papaya, was first reported to infected papaya crop in Malaysia in 2003. Another incidence was then reported in Perak in October 2004. By the end of 2006, the disease had spread to five other states on the west coast of Peninsular Malaysia. The causal agent of the disease was initially identified as Erwinia papayae (Makhatr et al., 2008). Later it was re-classified as Erwinia mallotivora (Mat Amin et al., 2010).
Since the disease appearance was first noted, it has destroyed an estimated 800 hectares of cultivated papaya, affected 1 million trees nationwide resulting in a loss of 200,000 ton. Malaysia's export of papaya has then decreased from RM200 million in 2004 to RM28 million in 2012.
The mode of action of the pathogen is through invading and colonizing all the papaya plant parts, including the shoot, leaf, bark, and fruit. Early symptoms of CRD on papaya include yellowing and necrosis along the leaf margins, followed by wet spots on the stalk base, crowns, and midribs of the leaves. Following the development of necrotic and water-soaked patches on stems, the pathogen then spread into the internal tissues. At infection sites, dark and oily spots have occasionally been observed (Mat Amin et al., 2011). The plant part that produces fruit, will completely rot as the infection on the crown spread over time. At the final infection stage, the water-soaked leaf stalk ultimately caused the infected part to die. Once the pathogen entered the plant, there is currently no effective means to cure the disease.
The issues faced by the papaya industry in Malaysia, notably caused by the CRD, had been addressed with concerted efforts centered on short-, mid-, and long-term strategies.
i) Development of ISR technology to control crown rot disease of papaya
Induced systemic resistance (ISR) technology to control the CRD of papaya was developed by bioprospection and manipulation of ISR inducing PGPR from papaya cultivated soil. The study involved isolation and screening for potential PGPR strains selection and testing of the ISR seedlings on an open field (hotspot) for papaya CRD. The detail explanation on the technology development process is given below:
The soil from healthy papaya plants were sampled from three locations viz Serdang, Selangor and Lunas and Keladi in Kedah by using selective media for Bacillus and Pseudomonas. The bacilli were isolated using mannitol egg yolk polymyxin agar (MEYP) (Atlas, 1997) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA) (Atlas, 1997) and the pseudomonads were isolated using Pseudomonas F agar (Atlas, 1997) and King's media B (Atlas, 1997).
A total of 139 samples of bacilli and pseudomonad were isolated from roots of papaya in three locations namely Serdang, Keladi and Lunas. This is shown in Table 1. From the samples collected, nine bacilli were isolated from Serdang, 42 from Keladi and 36 from Lunas. Out of 52 pseudomonads isolated, 20 samples were from Serdang, 19 samples were from Keladi and 13 were from Lunas.
The Eksotika papaya seeds were soaked overnight in a container containing distilled water. Floating seeds were discarded as they were considered non-viable. The submerged seeds were soaked into a container containing PGPR suspension for 2 hours. For control treatment, the seeds were not soaked in PGPR suspension. Both the PGPR treated, and control seeds were then sown into germination tray filled with peat moss. Two weeks after sowing, each young seedling with 3-4 leaves stage was transferred into a 15 x 23 cm perforated polybag containing soil mixture of 2 parts of topsoil, 1 part of organic matter and 1 part of sand and kept under 50% shade in the nursery.
When the seedlings were two and six weeks old, the treatments were treated with the PGPR bacterial suspension (1mL/seedling with 1:50 dilution in water), while the seedlings in the control treatment were applied with sterile distilled water only. Each treatment was replicated 5 times. The seedlings were maintained for another two weeks (8 weeks after transferred to polybag) before being challenged with the papaya crown rot pathogen, E. mallotivora. This experiment was conducted under glasshouse conditions in MARDI Serdang.
The pathogen was grown in a nutrient broth with agitation for 48 hours at room temperature. The seedlings in the treated and control treatments were pricked (20 pricks/seedling) with a sterile needle on the stem close to the crown. Ten mL of the E. mallotivora suspension was sprayed at the pricked area using a hand-held sprayer. The seedlings sprayed with the pathogen were then covered with a plastic bag for two weeks. The disease symptom development and plant survival rate were observed at 4, 7 and 14 days after challenging with the pathogen.
Four days after inoculation with E. mallotivora, the first symptom of bacterial crown rot was observed. The seedling's shoot and stems developed watery lesions and gradually became necrotic, which are symptoms of papaya crown rot disease. Generally, these symptoms were developed in all treatments including control. After being inoculated with the pathogen for 14 days, the survival rates of the seedlings were recorded. Out of the thirty-one isolates tested, at least one plant survived from the disease. Eight of the treatments, which were inoculated with isolates MIB 10, MIB 13, MIB 43, MIB 58, MIB 100, MIB 106, MIB 113, and MIB 132, recorded 100% healing and survival. All plants in the control treatment were infected with the disease (Figure 1).
All the eight ISR positive PGPR isolates namely, (with 100% suppression against crown rot disease), MIB 10, MIB 13, MIB 43, MIB 58, MIB 100, MIB 106, MIB 113, and MIB 132 were used to produce ISR seedlings as mentioned above. Control (non-ISR) seedlings were also produced without PGPR inoculation as mentioned in the method above. The non-ISR seedlings were used to compare with the ISR inoculated seedlings for disease resistance. After the seedlings were transferred, a routine application of ISR inoculant was conducted once a month throughout the crop cycle. Control plants were also prepared and planted for comparison with ISR plants to evaluate the effects of disease infection. Fertilizer application and pest control were done according to standard practice (Chan et al., 1991). Good Agriculture Practice was employed throughout the crop cycle. Disease infection and plant death caused by the crown rot disease were recorded.
Generally, all the ISR seedlings showed more vigorous vegetative growth as compared to the seedlings in the control treatment at nursery stage. Both the ISR and control seedlings performance against papaya CRD infection were evaluated under open field in MARDI Serdang which was considered as a hotspot (where the disease is prevalent). After the completion of one crop cycle (24 months) evaluation at the hotspot and experiencing second rainy seasons (3 months), all the control plants were infected with the disease and eventually died (Table 2). Almost all the ISR plants survived until 24 months and were not infected with crown rot disease, except the treatment inoculated with MIB 13 isolate. The isolate MIB 13 recorded only 87.25% resistance against papaya crown rot disease. All the control plants were succumbed to crown rot disease within 11 months after being transferred to the field.
The ISR plants showed better vegetative growth, yield and no significant changes in fruit quality compared to plants in the control plot. The quality of the fruits produced by the ISR plants had Total Soluble Solid readings between 13-14% and red intense colour flesh.
ii) Pre-commercialisation
The ISR technology was pre-commercialized by up-scaling on a commercial farm. After being sown at MARDI Serdang, a total of 2,850 ISR Eksotika I papaya seedlings were sent to the commercial farm in Ampang Tinggi, Negeri Sembilan. The site was regarded as a hotspot because the previous papaya crop was completely afflicted and died from crown rot disease. After the seedlings were transplanted to the field, a routine application of ISR inoculants were applied once a month throughout the crop cycle. Fertilizer application and management of insect pests and diseases were done according to standard practice (Chan et al., 1991). Good Agriculture Practice was employed during the whole crop cycle. Disease infection and number of plants died caused by the crown rot disease were recorded.
Three months after the seedlings were planted in the field, the first incidence of crown rot disease infection was observed, affecting 85 papaya plants. The infection was contributed by the uncontrolled weed growth surrounding the root region of the papaya plants. Thirteen months later, the disease spread to another 61 plants. No further crown rot disease infection was observed until the project was completed. Only 5% of the papaya plants had crown rot disease at the end of the crop cycle. It was found that the fruits produced in this experiment were of similar quality to the fruits produced from the control plot, suggesting that the ISR treatment had no effect on the fruit quality.
iii) Evaluation process of the ISR technology and approval for commercialization
The Technology Commercialization and Business Centre (TCO), MARDI, will thoroughly assess each technology produced by its researchers for its commercial potential based on criteria such as proof of concept, novelty of innovation, technology competitiveness, and market potential (Ganisan Krishnen, et al., 2016). Positive feedback on the ISR technology has been obtained through field evaluations and up-scaling activities to prepare for use by the end users or stakeholders. Once the technology has been transferred to the end users or stakeholders, the TCO will then evaluate the impact of the technology. Corrective action will be recommended after an analysis of feedback regarding the technology's shortcomings and performance is completed (Ganisan Krishnen, et al, 2016).
The ISR technology was developed as a preventive measure against the papaya crown rot disease. Plants that have been infected, especially those with severe infections cannot be cured with this method. However, many papaya growers used Dieback Buster (trade name of the ISR bacterial inoculants) when their papaya plants were infected with crown rot disease expecting that this product will cure their plants from the disease.Since this product is recommended for disease prevention, the best results to control the disease can only be achieved by applying Dieback Buster during seed sowing, booster application during nursery stage and continuous monthly application in the field for up to two years of production (Ganisan Krishnen and Mohamad Roff Mohd. Noor, 2021).
In general, farmers that used Dieback Buster and ISR technology as a preventive tool to manage the disease reported positive testimony on the effectiveness in controlling the crown rot disease and increasing production. To sustain high papaya production at their farms and produce high income, these farmers are willing to continuously adopt ISR technology (Krishnen and Mohd Noor, 2021).
A patent application was made with the Intellectual Property Corporation of Malaysia (MyIPO) to protect the ISR technology for inducing resistance against the papaya crown rot disease. The submitted patent is anticipated to protect the process of inducing systemic resistance in papaya at seedling stage in the nursery and in the field. The application for patent granting is now being reviewed by the patent agency.
The ISR technology has been sold by MARDI to Arif Efektif Sdn. Bhd., a Malaysian company. Arif Efektif Sdn. Bhd is currently the legal owner of the technology. No one is allowed to produce the inoculum without the company's prior written permission. Nonetheless, farmers can use Dieback Buster, which they purchase in the market, to grow their own ISR seedlings. The Dieback Buster and Adjustable Volume Liquid Gun are available for purchase from any All Cosmos Industries Sdn. Bhd. dealers in Malaysia.
For commercial purposes, the bacterial inoculants that induced systemic resistance were marketed under the brand name "Dieback Buster". The bacterial inoculants both induce resistance and effectively combat crown rot disease. To ensure that every papaya plant in the field receives the same amount of Dieback Buster spray volume, an equipment was designed. The equipment that was developed was known as Adjustable Volume Liquid Gun. When compared to manual application utilizing containers in the past, this equipment facilitates and expedites the application of Dieback Buster in the field.
On August 1, 2017, at the Exotic Star Sdn. Bhd. farm in Dengkil, Selangor, the Deputy Minister of Agriculture and Agro-based Industry Malaysia launched the Dieback Buster and Adjustable Volume Liquid Gun package technology. Numerous national newspapers reported on this new technology, which helped inform Malaysia's papaya growers and other interested parties.
On October 7, 2018, Arif Efektif Sdn. Bhd., a subsidiary company of All Cosmos Industries Sdn. Bhd. (a public listed company with BioNexus status), commercialized the ISR technology in Malaysia. Now, the company has successfully produced the inoculants in large volume and marketing the Dieback Buster. Since the product was introduced to the market in April 2019, about 393 ha of cultivated papaya have benefited from this technology. In order to effectively, introduce the technology to extension agents and farmers, Arif Efektif Sdn. Bhd. together with MARDI started a number of initiatives, including seminars, hands-on training sessions, and presentations in various scientific and technical forums. The company also uses social media and print media to promote the product through its dealers.
As Malaysian organizations who firmly support the Sustainable Development Goals, MARDI, Arif Efektif Sdn. Bhd. and All Cosmos Industries Sdn. Bhd. are happy to provide this technology so that papaya growers around the world, especially those in the Asia Pacific region, can benefit from it. It is possible to transfer the papaya ISR technology to other nations through correspondence with either MARDI, Arif Efektif Sdn. Bhd. or All Cosmos Industries Sdn. Bhd. Any correspondence regarding the technology transfer to foreign countries can be sent to aniadila@mardi.gov.my (MARDI) or wan_azha@allcosmos.com (Arif Efektif Sdn. Bhd.).
CONCLUSION
Malaysian papaya industry was pushed to the brink of collapse by the emergence of an alien invasive papaya crown rot disease. The introduction of this disease has devastated the production and exports of papaya. To address this issue, MARDI had developed an ISR technology to combat the deadly papaya disease. This technology was developed by bioprospecting of systemic inducing PGPR. Eight PGPR isolates with the highest disease control capacity (100%), were used to produce ISR seedlings, and evaluated them in an open field (hotspot). The results showed complete control against the papaya crown rot disease. The isolate MIB 106, was selected for activities involving upscaling. Additionally, this method was revalidated for the production of organic papaya in MARDI and for conventional production in three commercial farms which use chemical inputs. Both organic and inorganic papaya production, benefit greatly from this technology. ISR technology was protected through the Intellectual Property Corporation of Malaysia (MyIPO) patent filing process.
The ISR inoculant containing ISR inducing bacteria isolate MIB 106 was given the brand name DIEBACK BUSTER and the ISR inoculant application equipment as Adjustable Volume Liquid Gun. Both these technologies were launched in one of the farms where the validation was conducted. These package technologies were commercialized to Arif Efektif Sdn. Bhd., a subsidiary of All Cosmos Industries Sdn. Bhd. The products have been available in the market since April 2019. About 393 ha of cultivated papaya had benefitted from this technology. Various seminars and trainings were conducted to disseminates the knowledge and technology transfer of this technology to stakeholders, farmers, and extension agents. Farmers who used this method to prevent papaya crown rot disease gave positive testimonies and indicated their interest to continue using it. Currently, this is the only way to grow papaya while minimizing losses from bacterial crown rot disease.
The only hope for reviving Malaysia's flagging papaya industry and increasing both production and its exports is ISR technology. It is envisaged that when papaya can be effectively produced with the disease papaya crown rot eradicated, Malaysia would one day regain its position as the world's leading papaya exporter.
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