Instant biofumigation using natural products from papaya seed waste for sustainable management of soil-borne plant pathogens

Progress report for SW20-911

Project Type: Research and Education
Funds awarded in 2020: $349,995.00
Projected End Date: 05/31/2023
Grant Recipient: University of Hawaii at Manoa, College of Tropical Ag & Human Resources (CTAHR)
Region: Western
State: Hawaii
Principal Investigator:
Dr. Wei Wen Su
University of Hawaii at Manoa, College of Tropical Ag & Human Resources (CTAHR)
Co-Investigators:
Dr. Stuart Nakamoto Nakamoto
U. of Hawaii Manoa, Human Nutrition, Food, and Animal Sciences
Dr. Koon-Hui Wang
University of Hawaii
Dr. Tao Yan
Dept. of Civil & Environ. Engineering, University of Hawaii at M
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Project Information

Abstract:

Soil-borne diseases caused by fungi and nematodes bring about serious damage to many crops of agricultural importance. Conventional soil fumigation for controlling soil-borne plant diseases is most commonly based on synthetic chemicals many of which cause serious negative environmental impact and have been phased out. Biofumigation is an eco-friendly alternative for suppressing soil-borne pests and pathogens. Conventional biofumigation uses macerated green manures from glucosinolate-rich biofumigant plants, such as brown mustard, as soil amendments. This practice suffers from shortcomings that include costs and time associated with cultivating the biofumigant crop. Furthermore, cruciferous cover crops are often hosting common pests of leafy greens, and cover crop rotation is impractical for long-term orchard crops.

In this project, we put forward a novel approach aimed at improving and simplifying the biofumigation practice. We will develop new “off-the-shelf” organic biofumigant products based on papaya seed wastes that are abundantly available in Hawaii to achieve instant and more precise biofumigation without the need for growing biofumigant cover crops. Papaya seeds contain a high level of benzyl glucosinolate that is enzymatically hydrolyzed via myrosinase to form benzyl isothiocyanate, which has potent pesticide activities against a range of soil-borne phytopathogenic nematodes, insect pests, and fungi. 

Our central hypothesis is that ground papaya seeds can be applied as an effective natural soil fumigant, and the efficacy can be enhanced by optimizing seed processing, formulation, and fumigant application regimes. A secondary hypothesis is that the binary nature of the glucosinolate/myrosinase system can be exploited to achieve higher degrees of control over biofumigant delivery and activation, and thus improve dosage and bioavailability of the released isothiocyanate. These hypotheses are supported, in part, by our preliminary study, as well as data in the literature. We will work with our collaborating farmers to conduct a series of field tests with the papaya seed biofumigants on their farms. We will also conduct laboratory studies that integrate with the field tests to help develop a comprehensive understanding of the biofumigation process, and use the knowledge to refine the field tests to achieve high biofumigation performance in managing phytopathogens and promoting plant health.  

The project team will continue to actively involve producers throughout the entire study via research, extension, and outreach activities, with the goal to make the proposed “instant biofumigation” technology readily adoptable by the farmers. Field trials of instant biofumigants will be conducted in collaboration with each of our three core participating producers. Educational workshops and field-day events will be held regularly, and mass and social media technology channels will be employed, to disseminate project findings to farmers and other agricultural professionals, and to solicit their feedback and suggestions.

The key expected outcome of this project is effective management of soil-borne pests, using a natural and renewable fumigant made from underutilized, locally sourced, agricultural waste, based on a simple and environmentally friendly process. Development of papaya-seed biofumigants will reduce reliance on imported pesticides, while endorsing the concept of “reduce, reuse, and recycle” to promote sustainable agriculture and continuing growth of local farm community. 

Project Objectives:

The goal of this proposal is to augment the efficacy of biofumigation, by achieving higher degrees of control over biofumigant delivery and activation to improve dosage and bioavailability of the released isothiocyanate, while keeping the cost of the system down by using raw materials derived from seeds of papaya wastes that are abundantly available in Hawaii. The proposed work on developing the papaya seed biofumigant technology builds on field tests (with papaya, lettuce, and pumpkin as the three test crops) to be conducted on collaborating farmers’ farms with close collaboration with the participating farmers. We also proposed laboratory studies that integrate with and refine the field tests to achieve high biofumigation performance. The proposed research has the following four specific objectives:  

  1. Evaluate the effect of papaya biofumigants in managing plant-parasitic nematodes and Fusarium wilt, and on plant health, in field studies. (Year 1-3)
  2. Optimize the papaya biofumigant system by integrating laboratory studies with the field studies, and assess impact of biofumigant application on soil microbial communities. (Year 1-3)
  3. Determine costs and benefits of the instant biofumigation technology. (Year 2-3)
  4. Disseminate information about the instant biofumigation technology to edible crop producers for soil borne disease management. (Year 1-3)

This is a “long term” project. Our short-term goal, which is to determine field performance of the proposed papaya seed biofumigants (efficacy in managing soil-borne plant-parasitic nematodes and Fusarium wilt, as well as effect on plant health and microbial communities) and to characterize the biofumigation process, will be accomplished within the current proposed 3-year funding period. To bring the technology to full large-scale implementation, we expect additional optimization and field testing would be necessary which will be pursued upon completion of the current proposed 3-year study, for approximately another two years.

Cooperators

Click linked name(s) to expand
  • Michael Kamiya - Producer
  • Owen Kaneshiro - Producer
  • Joshua Silva (Educator)
  • Alec Sou - Producer
  • Jari Sugano (Educator)
  • Jensen Uyeda (Educator)

Research

Hypothesis:

Our central hypothesis is that ground papaya seeds can be applied as an effective natural soil fumigant, and the efficacy can be enhanced by optimizing seed processing, formulation, and fumigant application regimes. A secondary hypothesis is that the binary nature of the glucosinolate/myrosinase system can be exploited to achieve higher degrees of control over biofumigant delivery and activation, and thus improve dosage and bioavailability of the released isothiocyanate. These hypotheses are supported, in part, by our preliminary study, as well as data in the literature. We will work with our collaborating farmers to conduct a series of field tests with the papaya seed biofumigants on their farms. We will also conduct laboratory studies that integrate with the field tests to help develop a comprehensive understanding of the biofumigation process, and use the knowledge to refine the field tests to achieve high biofumigation performance in managing phytopathogens and promoting plant health.

Materials and methods:

Preparation and characterization of papaya ground seeds (PGS) as a soil biofumigant

Before its application in greenhouse trials, preparation of the PGS as a soil fumigant was investigated. The factors examined included (i) freezing seeds prior to drying and grinding; (ii) seed drying temperatures; and (iii) seed drying duration.  Three treatment groups were compared. One group of fresh papaya seeds were frozen at -20°C and then dried at 50°C for 48 h, before being ground in an electric grain mill grinder. A second group of fresh seeds were directly dried at 50°C for 48 h without the pre-freezing treatment. A third group of papaya seeds were dried at 40°C for 24 h, without pre-freezing.  We also tested seeds from two papaya varieties Rainbow and Sunrise. After being homogenized into PGS, equal weight of water was added and allowed to react at room temperature for 6 hours. The oil containing benzyl isothiocyanate (BITC) was extracted using a Soxhlet extractor, with hexane as a solvent. BITC was measured by using a modified 1,2-benzenedithiol (BDT) cyclocondensation assay. Briefly, samples containing BITC were pipetted into glass vials and the volume was made up to 1 mL with a 10 mM potassium phosphate buffer solution at pH 8.5. To these samples, 1 mL of 4 mM BDT in methanol was added. The samples were capped, briefly vortexed, then placed into a water bath at 65°C for 3 h. Then, the reaction mixtures were filtered using a syringe mounted 0.2 mm PES filter, and analyzed using reverse phase HPLC with a Shimadzu Prominence HPLC system. Samples were run using a Shimadzu Premier C18 column with particle diameter 5 mm, pore diameter 120 A, length 25 cm, and internal diameter 4.6 mm, with a mobile phase of 80% HPLC methanol and 20% HPLC water, isocratically, at a flow rate of 1 mL/min. Using a Shimadzu UV/VIS detector, absorbance was measured at a wavelength of 365 nm. Reverse-phase HPLC analysis was used to determine the benzyl glucosinolate (BG) concentration using a Shimadzu Premier C18 column as in BITC measurement. BG standard was purchased from Cerilliant (Round Rock, TX). To monitor BITC after it was amended into soil during the greenhouse trials, triplicate soil samples were collected from the pot at different time points during the trial. The soil samples were then extracted with a mixed solvent consisting of 20% methanol and 80% ethyl acetate.  Frozen soil sample (0.5 g) was transferred into a reinforced Eppendorf tube containing roughly 300 µL of glass beads, 800 µL of ethyl acetate and 200 µL of methanol. The sample was vigorously shaken on a bead homogenizer for 6 min. The tube was then centrifuged at 20,000 g for 5 min. The supernatant was transferred to a new tube, from which 300 µL of supernatant was used for reaction with BDT to determine the amount of BITC in the soil sample. To determine the nutrient profile of the papaya seeds, seeds were first defatted using hexane and methanol, and subject to proximate analysis at the Agricultural Diagnostic Service Center at the University of Hawaii using standard feed quality analysis methods.

Greenhouse trials

Three greenhouse pot trials were conducted to compare the effects of PGS as soil amendment against soil-borne fungi in particular, Fusarium oxysporum f. sp. letucae, and root-knot nematode, Meloidogyne incognita. Brown mustard (BM) was included as a standard biofumigation control. An experiment was conducted using 10-cm diameter pots holding 250 g (dry weight equivalent) of soil per pot. Each pot trial was using soil collected from one of the three sites from Owen Kaneshiro Farm, Waianae, HI with a history of lettuce Fusarium wilt or Rhizoctonia rot.  Mr. Owen Kaneshiro is a participating farmer in this project. Trial I was conducted using soil collected from a lettuce field that was left fallow for some time. The soil was either amended with (1) PGS at 0.5% (dry weight equivalent) (PGS 0.5%); (2) PGS 0.5%+crude aqueous extract 0.5% (PGS+CE); (3) PGS at 1% (PGS 1%); (4) brown mustard at 1% (BM); (5) not amended (NA), or (6) not amended and autoclaved (Auto). In treatment (2), 10 g of PGS was microwaved for 10 minutes to deactivate the myrosinase activity and extracted using 400 mL of boiling water to prepare a crude BG extract from which 50 mL was applied per pot to achieve BG levels equivalent to that of 0.5% PGS powders. Each treatment was replicated 4 times and treatments were arranged in a complete randomized design. In Trial I, after soil amendment, three 3-week-old lettuce (Lactuca sativa) seedlings were transplanted immediately into each pot. Treatment (1) to (5) were inoculated with 100 second-stage juveniles (J2) of M. incognita, whereas the Auto treatment was not. The experiment was terminated 1 month after the nematode inoculation. In Trial II, soil collected from a kai choi (Brassica juncea) field with a history of Rhizoctonia bottom rot was used. Similar treatments in Trial I was imposed. ‘Hirayama’ kai choi seedlings were used as the bioassay crop. Trial III was collected from another lettuce field with a history of Fusarium wilt infestation. Similar treatments as Trial I and II were imposed and 3 Manoa lettuce seedlings were planted into each pot for bioassay over 1 month. In Trails II and III, unlike in Trial I, PGS soil amendment was done 1 week prior to seedling transplanting.

Microbial community analyses by 16S rRNA gene amplicon sequencing

Soil samples were stored at -20oC until DNA extraction. The soil samples (0.2 mg, fully mixed) were subjected to DNA extraction by using the PowerSoil DNA Extraction Kit (Qiagen, USA) following the manufacture’s protocol. The V4 hypervariable region of the 16S rRNA gene was first amplified with the F515/R806 primers (Bates et al., 2010) for 30 cycle. PCR products were then re-amplified by PCR for 15 cycles by using F515/R806 bacterial/archaeal primers linked with the sequencing adapters CS1 (5’-GCTGCGCGCGAACGGCGAAG-3’) and CS2 (5’-TCCCGGCAGAGTTCCCATT-3’). Successful amplification was confirmed by gel electrophoresis of the PCR amplicons and checking for bands with expected DNA size. Library preparation of amplicons, multiplex indexing, and subsequent sequencing on an Illumina Miniseq platform was performed by the DNA service facility at University of Illinois at Chicago. Paired-end sequence reads with the length of 153 bp were generated for each sample. Paired-end sequences reads were quality trimmed and merged using the PEAR software (with parameters: -v 10 -m 300 -n 200 -t 100 -q 20 -u 0.02) (Zhang et al., 2013). Merged reads were denoised and representative sequences of OTU were subsequently picked using the denoise-single command in DADA2 package (Callahan et al., 2016) within QIIME2 (Bolyen et al., 2018) with default parameters. Taxonomic classification of sequences were performed by using the QIIME2 and the Silva 132 99% database (Quast et al., 2012) with default parameters.

References cited:

Bates, S.T., Berg-Lyons, D., Caporaso, J.G., Walters, W.A., Knight, R., Fierer, N. 2010. Examining the global distribution of dominant archaeal populations in soil. The Isme Journal, 5, 908.

Bolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Abnet, C., Al-Ghalith, G.A., Alexander, H., Alm, E.J., Arumugam, M., Asnicar, F. 2018. QIIME 2: Reproducible, interactive, scalable, and extensible microbiome data science. PeerJ Preprints. 2167-9843.

Callahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A., Holmes, S.P. 2016. DADA2: high-resolution sample inference from Illumina amplicon data. Nature methods, 13(7), 581.

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O. 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research, 41(D1), D590-D596.

Zhang, J., Kobert, K., Flouri, T., Stamatakis, A. 2013. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics, 30(5), 614-620.

 

Research results and discussion:

This project started in summer 2020. During this first report period, due to the COVID restrictions and financial challenges facing our farmer collaborators as a result of COVID, we are unable to initiate the proposed field trials on participating farmers’ farms. However, we are able to work with one of our collaborating farmers, Mr. Owen Kaneshiro of the Owen Kaneshiro Farm, Waianae, HI. Using soil collected from various fields on the Kaneshiro farm (picture 1), and through discussions with our collaborating farmer partner, we designed and conducted greenhouse studies to determine the efficacy of the papaya seed biofumigant against Fusarium oxysporum f. sp. letucae and root-knot nematode, Meloidogyne incognita, under different biofumigant formulations and application regimes, using lettuce and kai choi as test crops. Laboratory studies have also been conducted to optimize the preparation of the PGS as biofumigants, and to investigate the impact of PGS application on soil bacterial/archaeal communities.

Picture 1. Soil was collected from lettuce or kai choi field with a history of soil-borne disease problems caused by either Fusarium oxysporum f. sp. letucae or Rhizoctonia solani, and used in our greenhouse pot trials.

Papaya seeds contain a high level of BG that can be enzymatically hydrolyzed via myrosinase in aqueous solution to form BITC which has potent pesticide activities against a range of soil-borne phytopathogenic nematodes, insect pests, and fungi. The binary glucosinolate/myrosinase system requires activation by disrupting the cellular compartments that naturally separate the glucosinote from the myrosinase, and by the inclusion of water to trigger the enzymatic hydrolysis. In our previous study (Han et al., 2018) we noted a significantly elevated level of BITC in the oil extracted from papaya seeds that were previously frozen compared with that found in oil extracted from non-frozen seeds. Since BITC is the active ingredient of the papaya seed biofumigant, we tested whether it was necessary to pre-freeze the seeds to obtain high BITC in the PGS. To that end, prior to the greenhouse pot trials, we investigated processing conditions for preparing the PGS and measured BG and BITC contents in the PGS. The factors examined included (i) freezing seeds prior to drying and grinding; (ii) seed drying temperatures; and (iii) seed drying duration.  One group of fresh seeds were frozen at -20°C and then dried at 50°C for 48 h, before being ground in an electric grain mill grinder (S1). A second group of papaya seeds were dried at 50C for 48 h without a prior frozen treatment (S2), and a third group of seeds were oven dried at 40°C for 24 h without a prior frozen treatment (S3).  The resulting PGS samples were reacted with an equal weight of water at room temperature for 6h to synthesize BITC, and seeds oil containing BITC was finally extracted with hexane and analyzed using the cyclocondensation assay and HPLC quantification as described in the Materials/Methods section. The results are shown in Fig. 1. Samples S1 and S2 gave essentially the same amount of BITC after activated with water, indicating freezing of papaya seeds prior to drying and grinding into PGS is unnecessary. BITC is lower in sample S3 than the other two treatment groups (p< 0.05). Drying the seeds for 24 hours at 40°C did not remove sufficient moisture from the seeds, and the resulting PGS formed small clumps which may have reduced the enzymatic conversion of BG to BITC.  Overall, our results indicate that directly drying the papaya seeds at 50°C for 48 hrs without prior freezing is adequate to generate PGS with reproducible BITC content. Further, activation of PGS with equal volume of water for 6 hours at room temperature is sufficient to convert most of the BG to BITC.

Fig. 1. Effect of PGS-freezing, and PGS drying temperature and duration on BITC content in the PGS. The non-defatted PGS contains approximately 25% (w/w) oil. Refer to the text for description of the three sample groups.

We also measured the BG and BITC contents in seeds of two common papaya varieties Rainbow and Sunrise, and tested whether seed BITC synthesis is affected by dry storage. The BG content was averaged about 20-25 µmole/g dry seeds regardless the papaya varieties. As shown in Figure 2, the BITC generation did not vary considerably between the two varieties.  These results also demonstrate that BITC generation from seeds freshly harvested from fruits is similar to that from seeds dried and stored in an air-tight Ziploc bag for 1 month. 

Fig. 2. Comparison of BITC generation by PGS prepared from two varieties – Rainbow and Sunrise. BITC generation from seeds freshly harvested from fruits (Fresh) is also compared with that from seeds dried and stored for 1 month (Dry).

Based upon these results, PGS for subsequent greenhouse trials was prepared by drying papaya seeds at 50°C for 2 days, milled into powders, and stored in Ziploc bags at room temperature prior to use in the trials. We also analyzed the nutrient profile of the defatted PGS. The proximate analysis result is presented in Table 1.

Table 1. Nutrient profile of defatted PGS.

In greenhouse Trial I, phytotoxicity of lettuce seedlings occurred in all treatments receiving PGS and resulted in plant growth similar to the NA (Picture 2; Fig. 3). However, BM was able to suppress M. incognita and resulted in lettuce growth similar to the autoclaved treatment (without M. incognita inoculation and no fungal infestation) (Fig. 3).

Picture 2. Lettuce seedlings suffering from phytotoxicity when planted immediately after papaya ground seeds were amended into the soil in Trial I. Auto = autoclaved, BM = brown mustard, NA = not amended, PGS 0.5% = amended with papaya ground seeds at 0.5%, PGS 1% = PGS at 1%, and PGS+CE = PGS 0.5% plus drenching with papaya seed crude extract at 0.5%.
Fig. 3. Greenhouse Trial I result. A) Plant height and B) Number of root galls on lettuce planted in soil being autoclaved (Auto), amended with brown mustard (BM), not amended (NA), amended with PGS at 0.5% (PGS 0.5%), 1% (PGS 1%), and PGS 0.5% plus drenching with papaya seed crude extract at 0.5% (PGS+CE). Means (n=12) followed by same letter(s) are not different based on Waller-Duncan k-ratio (k=100) t-test.

In Trial II, PGS was amended to the soil one week prior to the seedling transplanting. While the canopy width of kai choi was increased by BM compared to the NA control, the shoot and root weights of kai choi in BM amended soil were not different from NA control (Fig 4 B, C). All PGS treatments (PGS 0.5%, PGC 1%, PGC+CE) did not pose phytotoxicity on kai choi as the canopy width, shoot and root weight were all not different from the NA control (Fig. 4 A, B, C). Unfortunately, all PGS treatments and brown mustard (BM) amendment did not reduce the plant disease index using a scale of 1-4 (Fig. 4 D). However, all biofumigation (PGS and BM) significantly (P ≤ 0.05) reduced the number of root pieces infected by F. oxysporum (Fig. 4 E) and the number of M. incognita penetrated into kai choi roots (Fig. 4 F). No Rhizoctonia hyphae was observed on the Komada medium. Initial attempts to monitor the fate of BITC in the PGS-amended soil during the trial produced highly varied results and no clear conclusion can yet be drawn. Uniformity in soil sampling is identified as a potential factor that needs further investigation.  It is also noted that PGS has a relatively high oil content (about 25% per dry seed weight), while BITC is hydrophobic, and hence BITC is likely partitioning to the oil phase, and its release may be affected by gradual seed oil biodegradation in the soil over time. These issues will be further investigated to achieve a more effective PGS application regime as a soil biofumigant.

Fig 4. Greenhouse Trial II result. Effect of soil amendment treatment on A) canopy width, B) shoot and C) root weights, and D) disease index (1-4 where 1 is healthy, 4 is least healthy) of kai choi; E) the number of root pieces plated on Komada selective medium showing sign of Fusarium oxysporum, and F) the number of Meloidogyne incognita penetrated the roots on a per g of root basis. The soil was being autoclaved (Auto), amended with brown mustard (BM), not amended (NA), amended with papaya ground seeds at 0.5% (PGS 0.5%), 1% (PGS 1%), and PGS 0.5% plus drenching with papaya seed crude extract at 0.5% (PGS+CE). Means (n=4) followed by the same letter(s) are not different based on the Waller-Duncan k-ratio (k=100) t-test.

Trial III was collected from another lettuce field with a history of Fusarium wilt infestation. Same treatments as Trial II were imposed and 3 Manoa lettuce seedlings were planted into each pot for bioassay over 1 month. Lettuce plants responded with positive growth from the NA control when planted in soil amended with BM or 1% PGS (P ≤ 0.05, Fig. 5 A). Only BM resulted in wider canopy width and total shoot biomass higher than NA (Fig. 5 B, C). It is also promising in this trial that biofumigation treatments resulted in a lower plant disease index compared to the NA control (P ≤ 0.05; Fig. 5D). Deviate from the results in Trial II, PGS 0.5% and PGS 1% were the only treatments that suppressed the recovery of kai choi roots with F. oxysporum on the Komada medium (P ≤ 0.05, Fig 5E) much like the autoclaved soil. Whereas, root gall index (1-5 scale) on lettuce was significantly suppressed by all biofumigation treatment including PGS+CE compared to the NA control (P ≤ 0.05, Fig 5F). From this trial, PGS showed promise as a potential sustainable biofumigant. 

Fig 5. Trial III result. Effect of soil amendment treatment on A) plant height, B) canopy width, C) shoot weight, and D) disease index (1-4 where 1 is healthy, 4 is least healthy; Picture 3) of Manoa lettuce; E) number of root pieces plated on Komada selective medium showing sign of Fusarium oxysporum, and F) root-gall index (1-5 scale) associated with Meloidogyne incognita inoculation. Soil was being autoclaved (Auto), amended with brown mustard (BM), not amended (NA), amended with papaya ground seeds at 0.5% (PGS 0.5%), 1% (PGS 1%), and PGS 0.5% plus drenching with papaya seed crude extract at 0.5% (PGS+CE). Means (n=4) followed by same letter(s) are not different based on Waller-Duncan k-ratio (k=100) t-test.
Picture 3. Kai choi and lettuce disease rating scale of 1-4.

To study the effect of PGS treatment on soil microbial diversity, twelve soil samples, triplicate samples from four different treatments (Table 2), were collected from greenhouse Trial 1 and then subjected to 16S rRNA gene amplicon sequencing. Bioinformatics analysis identified a total of 510 bacterial/archaeal genera across the twelve samples. The 40 most abundant genera, which were those operational taxonomic units (OTUs) with relative abundance no less than 1% in at least one sample, were summarized in Figure 6.

Table 2. Sample ID and treatment conditions for twelve samples subjected to sequencing

Fig. 6. Distribution of the 40 most abundant genera in the twelve soil samples from four treatments.

Analysis of the alpha diversity, including the observed OTUs and Shannon index, were shown in Figure 7A and 7B, respectively. No significant difference among the four treatments were observed with all p-value of Kruskal-Wallis tests higher than 0.1.

Fig. 7. Alpha diversity, observed OTUs (A) and Shannon index (B), in the soil samples from four treatments.

Analysis of beta diversity, including the Jaccard distance and unweighted UniFrac distance, were shown in Figure 8A and 8B, respectively. Different treatments showed significant microbial community dissimilarity, with p-value of 0.021 in PERMANOVA test of unweighted UniFrac distance. Additionally, all the three treatment (0.5 CE, 0.5 PGS and PGS1) showed significant microbial community dissimilarity than the control treatment (CK), with p-value of 0.10, 0.09 and 0.10 in pair-wise PERMANOVA test, respectively.

Fig. 8. Beta diversity, Jaccard distance (A) and unweighted UniFrac distance to CE (B), in the soil samples from four treatments.

Based on the taxonomic classification of 16S rRNA gene, eight previously-reported plant growth promoting bacteria (Genus/Species) were identified in the soil samples, shown in Figure 9. Different treatments resulted in significant difference in the abundance of plant growth promoting bacteria (ANOVA test, p=0.022), shown in Figure 10. All the PGS treatments (0.5 CE, 0.5 PGS and PGS1) showed significant higher total relative abundances of the eight plant growth promoting bacteria than that in the control treatment (CK), with p-value of 0.002, 0.054 and 0.013 in pair-wise ANOVA test, respectively.

Fig. 9. Eight plant growth promoting bacteria (Genus/Species) and their relative abundances in soil samples from four treatments.
Fig. 10. Total relative abundance of eight plant growth promoting bacteria (Genus/Species) in soil samples from four treatments.

References cited:

Han, Z., Park, A., Su, W.W. 2018. Valorization of papaya fruit waste through low-cost fractionation and microbial conversion of both juice and seed lipids. RSC Advances, 8(49), 27963-27972.

Research conclusions:

The PGS biofumigant can be produced easily by simply drying the papaya seeds at 50°C for 2 days, followed by milling into fine powders. Dry seeds stored for over a month under room temperature can produce similar amounts of BITC upon milling into PGS, as those from fresh seeds. Seeds from Sunrise and Rainbow papaya generate similar amounts of BITC. The greenhouse pot experiments revealed that PGS at 0.5 or 1% amendment rate level could pose a phytotoxicity effect on young lettuce seedlings if transplanted immediately after soil amendment, but planting at 1 week after amendment avoided this problem. PGS at 1% rate was more consistent in suppressing F. oxysporum than PGS at 0.5%. Adding papaya seed crude extract (PGS+CE) did not improve either F. oxysporum or root-knot nematode suppression than using PGS alone. Further optimization of crude extract preparation is necessary. Overall, the results indicate that PGS would be safer to use as post-plant treatment or at least 1 week before seedling transplanting. Besides effects on the crop and the target pathogens, it is useful to probe the environmental impacts of PGS by examining its effect on soil microbial diversity. To this end, the 16S rRNA gene amplicon sequencing is shown to be a useful approach. Different PGS treatments (PGS+CE, PGS 0.5%, PGS 1%, and NA) resulted in significantly different microbial composition in soil, as indicated by the statistically different Beta diversity indices, but did not significantly affect the microbial richness, as indicated by the non-statistically different Alpha diversity indices. Eight plant growth promoting bacteria were identified in the soil samples. Different treatments resulted in significant difference in the abundances of these plant growth promoting bacteria. The abundance of plant growth promoting bacteria under the PGS+CE treatment was significantly lower than under other treatments. Interestingly, treatment with PGS at 1% amendment rate showed the highest abundance of plant growth promoting bacteria. 

Participation Summary
1 Farmer participating in research

Education

Educational approach:

The project is still in the research-data collection stage, so there is no outreach to report yet during this reporting period.  However, as we learned more about the characteristics and efficacies of papaya ground seeds as a natural soil fumigant, we will begin to develop outreach activities in year 2 of the project to effectively disseminate the information and knowhow of the biofumigant technology to farmers, producers, and other agricultural professionals, and to solicit their feedback and suggestions. Given the challenges brought by the ongoing COVID pandemic, we need to modify our original outreach plan that emphasizes on in-person educational workshops and field-day events, to incorporate a “hybrid” approach that relies also on webinars, online trainings, social media, web-conferencing, and other digital media. Working in conjunction with the CTAHR Office of Communication Services, the project team will establish a new website dedicated to educating the public about sustainable biofumigation practices, including the PGS biofumigant technology. We plan to host virtual field day events and workshops which feature asynchronous (pre-recorded) and synchronous (live) presentations, interviews, and demonstrations on topics related to this project.  For instance, live demonstrations of PGS biofumigant application will be conducted via Zoom or another virtual platform, and recorded, and technical presentations will be given to educate the public about science/technology, economics, as well as the sustainability aspects of the PGS biofumigant product. Recording of these events will be disseminated via the biofumigation website, social media, and YouTube. Any face-to-face activities to be included in these outreach events will be planed carefully by following Federal, State, County, and University COVID guidelines. The field-day events and educational workshops will target farmers/producers, Cooperative Extension, USDA and state department of agriculture officials, and other agricultural professionals. We will partner with the Western SARE PDP program in Hawaii to recruit field-day participants, and extend technical information transfer to statewide producers, through its multi-agency network. This network brings together extension agents/specialists who work closely with agricultural producers, and other agricultural professionals in agencies such as USDA, Hawaii Farm Bureau Federation, Hawaii Organic Farmers Association, and University of Hawaii at Hilo, Hawaii State Department of Agriculture / Health, county agencies, industry collaborators, Hawaii State Department of Education K-12 programs and other essential public and private organizations. We will make a special effort to recruit and gain the support of new and existing farmers who suffer from production losses linked to plant-parasitic nematodes and other soil borne diseases.

Information and technology dissemination will also be achieved via conventional electronic and paper publications. Research findings will be published in peer-reviewed journals. Technical factsheets and tutorials, as well as other informational handouts and educational videos will be posted online via the internet newsletter of the Sustainable and Organic Agriculture Program (SOAP), Hānai’Ai, which has approximately 1,800 readers. Hard-copies of the Hānai’Ai newsletters will be mailed to selected farmers who do not have regular access to internet. We will also partner with organizations which have existing relationships with culturally diverse communities to extend the new technology into the socially disadvantaged communities.

Educational & Outreach Activities

2 Consultations
1 Curricula, factsheets or educational tools

Participation Summary

1 Farmers
2 Ag professionals participated
Education/outreach description:

Multiple discussions on applying PGS in treating Fusarium wilt with a participating producer, Owen Kaneshiro of Owen Kaneshiro farm. 

Discussions with the county extension service Edible Crops agent Sharon Wages on bringing awareness of the papaya seed biofumigant technology to papaya industry practitioners on the Big Island of Hawaii which is the largest papaya growing area of the state. 

Potential new working collaborations with USDA-ARS (US Pacific Basin Agricultural Research Center, PBARC; contact: Dr. Roxana Myers) on greenhouse/field trials to assess the effectiveness of the PGS biofumigant as a treatment at planting to suppress Coffee Root Knot Nematode, parallel to other treatments being tested.  

Learning Outcomes

1 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation
Key areas taught:
  • Development of a new approach to enable effective management of soil-borne pests by recycling/repurposing agricultural wastes

Project Outcomes

2 New working collaborations
Project outcomes:

The project is still in the research-data collection stage, so there is no major outcome to report yet. However,  the key expected outcome of this project is effective management of soil-borne pests, using a natural and renewable fumigant made from underutilized, locally sourced, agricultural waste, based on a simple and environmentally friendly process. Development of papaya-seed biofumigants will reduce reliance on imported pesticides, while endorsing the concept of “reduce, reuse, and recycle” to promote sustainable agriculture and continuing growth of local farm community.   

Recommendations:

We plan to start the field trials in year 2.  We will ramp up our outreach efforts in year 2 as we learned more about the characteristics and efficacies of papaya ground seeds as a natural soil fumigant.  

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.