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
Host Institution Award ID: G346-20-W7899
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

Summary:

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.

Timeline:

The project timeline (Figure 2) is organized according to the Tasks and Subtasks outlined in Materials & Methods. The anticipated major milestones are as follows:

  1. Completed formulation and production of large batches of biofumigants for initial field trials; analyzed the biofumigant nutrient profile, evaluated biofumigant release characteristics, and established initial field trial conditions in consultation with the farmer collaborators.
  2. Completed field trial #1 and determined efficacy of instant biofumigation on nematode pest management for papaya.  
  3. Completed field trial #2 and determined efficacy of instant biofumigation on Fusarium wilt pest management for lettuce.
  4. Completed field trial #3 and determined efficacy of instant biofumigation on nematode pest management for pumpkin.
  5. Completed laboratory studies to support field trials for optimizing the biofumigation operation.
  6. Determined cost/benefit of papaya seed instant biofumigation.
  7. Completed all outreach workshop and field-day activities and successfully disseminated information of papaya seed instant biofumigation to farmers and other agricultural professionals.

Cooperators

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  • 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.

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During our second year we collected data pertaining to (1) a field trial of PGS on kai choi in Fusarium-infested soil on a collaborating farmer’s farm (Owen Kaneshiro Farm, Waianae, HI), (2) in-vitro toxicity tests of PGS towards various fungal pathogens, and (3) laboratory studies of biofumigant emission from PGS.

Field trial

In the Fall of 2021, a field trial was conducted in a Fusarium-infested commercial farm (Owen Kaneshiro Farm, LLC). Six preplant soil treatments were installed: 1) Vapam fumigation (grower’s standard practice), 2) sorghum cover crop grown for 1 month then mowed down as surface organic mulch, 3) brown mustard tissue harvested from elsewhere, macerated and buried into soil as biofumigant, 4) 0.5% and 5) 1% papaya ground seed (PGS) based on soil dry weight (dw) as biofumigants, 6) untreated control. Treatments 3, 4 and 5 were based on 3” wide and 3” deep trenches where ‘Hiroyama’ kai choi (mustard green) were to be transplanted. Each field plot was 4×10 ft2 in size with 4 rows of kai choi seedlings. Each treatment was replicated 4 times, and treatments were arranged in randomized complete block design.

Plots were amended with their respective treatments 1 week prior to planting and mustard, PGS, and Vapam amended plots were covered with black tarp to avoid fumigant volatile escape. After 1 week, 3-week-old kai choi seedlings were planted into the plots. Kai choi was harvested 6 weeks after planting.

Soil health analysis: Soil samples were collected from each plot prior to soil amendment, one week after soil amendment, and at the time of experiment termination. Nematodes were extracted from the soil by elutriation followed by centrifugal and sugar flotation method. All nematodes were counted using an inverted microscope. Data was subjected to nematode community analysis (Ferris, 2001).

Halfway through the field experiment, data was collected for level of disease incidence prevalent in each plot. The center two rows for each replicate were observed for the number of diseased plants versus the number of healthy plants. A plant was considered diseased if at least one leaf was exhibiting symptoms of wilt. The representative percent diseased for each plot was then calculated.

At termination, data was collected for total biomass, marketable yield, and percent Fusarium colonization. The center two rows of kai choi from each plot were harvested (roots included) and weighed first for total biomass produced and second for only the marketable yield obtained. Root systems from four plants in each plot were collected, washed for excess soil, and surface sterilized. Three pieces per root system were plated on Fusarium-selective Komada media and incubated at room temperature (citation for protocol). The plated roots were monitored for suspected F. oxysporum colonies (purple) and the percent of roots colonized recorded.

All data was analyzed in SAS (v.9.4) using a Waller-Duncan post-hoc test with α=0.05.

PGS biofumigation plate assay

Two in-vitro bioassay formats were evaluated to assess toxicity of PGS to various fungal species, including mostly known fungal phytophatogens. The assays evaluated are agar plate-based. In the initial assay format, PGS was blended into liquefied warm agar in Petri dishes and allowed to solidify under room temperature. Fungal cultures were inoculated onto the PGS-amended agar plates and incubated for subsequent colony count. In the improved assay, the agar plate was hollowed out in its center and water-amended PGS was used to fill the hole. As a PGS-free control, the hole was either not filled in with the PGS, or without creating the center hole. Based on the improved plate assay format, potato dextrose agar (PDA) in 6-cm diameter petri dishes were prepared with 2-cm diameter wells cut from the center. Wells were filled with 1) 1g PGS + 1g dH2O, 2) 0.5g PGS + 0.5g dH2O, and 3) no PGS control. Two 5 mm mycelial plugs were placed on each plate as pictured in Picture 7 with the following fungal samples used: Fusarium oxysporum, Fusarium solani, and Setophoma sp. (isolated from green onion).  Each fungus was replicated in 3 dishes and were incubated at room temperature for 48 hours. Diameter of mycelial growth from the plugs was measured in cm. Due to positive results, the F. oxysporum mycelial growth was also measured at 96 hours. Data for each fungal set and treatment was analyzed in SAS (v.9.4) using the Waller-Duncan post-hoc test with α=0.05.

Characterization and optimization of biofumigant (BITC) emission from PGS 

To characterize the temporal pattern and extent of BITC emission from water-activated PGS, as influenced by several factors, laboratory tests were designed and carried out in conical centrifuge tubes. In a typical test, 2 grams of PGS was placed in a 50 mL centrifuge tube, and then water at a preset level was blended in. Subsequently, the moist PGS bed was overlaid with either soybean oil or mineral oil to trap the emitted BITC from water-activated PGS. During the incubation, the tube was sealed with an air-tight cap. The factors investigated include: (1) types of oil used as a BITC trap (soybean oil vs. mineral oil), (2) water-to-PGS ratio (0.5, 1, 2, and 3), (3) PGS particle size (fine and coarse, milled using a Cuisinart DBM-8 automatic burr mill), (4) freezing of papaya seeds prior to milling, and (5) inclusion of soil. For the pre-freezing treatment, fresh papaya seeds were frozen at -20°C for one day and then dried at 50°C for 48 h, before being ground into fine PGS. We also took periodic oil samples to determine the temporal pattern of BITC emission from PGS upon water activation. At 2.0 and 3.0 water-to-PGS ratios, a layer of oil (above the PGS bed) was visible after over 40 hours of incubation, and samples were taken directly from the oil layer. At 0.5 and 1.0 water-to-PGS ratios, however, an interstitial air space was present in the PGS packed bed, and hence the oil overlaid initially on top of the PGS bed gradually seeped into the bed during the incubation. Therefore even though 3 mL oil, instead of 2 mL (used in the higher water-to-PGS ratios), was used in these tests, no oil layer was visible at the time of sampling (40 hours). We had to subject the entire PGS tube to centrifugation at 7,000 g to separate the oil and the PGS to allow collection of the oil samples. Moreover, based on the 2-gram PGS test system, we overlaid 4 grams of field soil (from the same field used in the field trial) on top of the PGS packed bed in one treatment, and in another treatment, we blended the soil with the PGS, to evaluate the effect of soil on BITC emission from PGS.  In all treatments, oil samples were taken to determine the extent to which BITC was trapped by the oil phase over a period up to 40 hours. The oil samples collected were analyzed using the modified 1,2-benzenedithiol (BDT) cyclocondensation assay (Zhang 2012) as describe above in the year-1 report.

References cited:

Ferris, H., Bongers, T., & de Goede, R. G. M. (2001). A framework for soil food web diagnostics: Extension of the nematode faunal analysis concept. Applied Soil Ecology, 18(1), 13–29. 

Zhang, Y. (2012). The 1,2-benzenedithiole-based cyclocondensation assay: a valuable tool for the measurement of chemopreventive isothiocyanates. Crit Rev Food Sci Nutr 52(6): 525-532.

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.

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During our second year we collected data pertaining to (1) a field trial of PGS on kai choi in Fusarium-infested soil on a collaborating farmer’s farm (Owen Kaneshiro Farm, Waianae, HI), (2) in-vitro toxicity tests of PGS towards various fungal pathogens, and (3) laboratory studies of biofumigant (BITC) emission from PGS to develop better practices for more efficient use of PGS in the field implementation of PGS-based biofumigation.

Field Trial

A. Biomass and Marketable Yield

Total biomass and marketable yield were measured to determine overall effectiveness of the PGS treatments at preserving the crop for market and maintaining healthy plant growth (Picture 4). All treatments were significantly less in mass for both the total biomass and marketable yield than the conventional Vapam-treated plots within the experiment. However, this included the mustard treatment, which showed no significant difference from the PGS treatments as well (Fig. 11). Considering mustard was an effective biofumigation crop (Waisen et al., 2019) previously tested in the same farm and the positive effects of PGS in our greenhouse pot trials, the current field results suggested that trench application was not an effective approach to deliver PGS or macerated brown mustard.

yield
Picture 4. Marketable yield kai choi (top) collected from Vapam-treated plot compared to unmarketable, diseased kai choi (bottom) collected from untreated field plot.
Fig11
Figure 11. Total biomass measurements and marketable yield mass measurements for each treatment taken at harvest/termination of the field experiment. Treatment groups with the same letter indicate no significant difference (Waller-Duncan, α=0.05).

B. Disease Incidence

Diseased plants were counted to determine the overall effectiveness of PGS and other tested amendments in reducing Fusarium wilt disease incidence in a field infested with F. oxysporum (Picture 5). Of the six treatments, Vapam had the least amount of disease, significantly lower than all other treatments. Both PGS 0.5% and PGS 1% were significantly higher in disease incidence, but comparable to the brown mustard treated plots (Fig. 12). The Sorghum planted plots (not till into the soil) had less diseased than the PGS-treated plots, but not different from the control and mustard. These results suggested that both trench application of PGS and macerated brown mustard and no-till cover cropping of sorghum were not effective biofumigation approaches against the targeted soil-borne pathogens. Future study will examine more effective and feasible biofumigation methods using PGS.

pic5
Picture 5. Representative kai choi plot showing the center two rows that were used for observing disease incidence. Healthy, disease-free plants are marked with a blue check and diseased plants are marked with a red ‘X’.
Fig12
Figure 12. Mean disease incidence percentages observed for each treatment group taken midway through the field experiment. Treatment groups with the same letter indicate no significant difference (Waller-Duncan, α=0.05).

C. Fusarium colonization of kai choi roots

To observe whether or not PGS was able to suppress F. oxysporum and prevent root colonization, kai choi roots collected from the field experiment were sterilized and plated on Fusarium-selective media, then observed for any Fusarium-like mycelial growth protruding from the plated roots (Picture 6).

Control, mustard, Vapam, and 1% PGS treatment groups were all significantly lower in Fusarium growth than the 0.5% PGS and Sorghum treatment groups (Fig. 13). Most surprisingly, there is very little difference between the control (negative) treatment group and the Vapam (positive) treatment group. This is likely due to certain strains of F. oxysporum being known to colonize plant roots as avirulent endophytes in various plants, so we can’t be sure if what emerged from roots on the Komada media are pathogenic or avirulent, symbiotic F. oxysporum strains. In order to verify findings or produce more accurate results, we will develop a qPCR assay specific to the F. oxysporum forma specialis that is pathogenic in kai choi.

pic6

Picture 6. Surface-sterilized kai choi root pieces plated on Fusarium-selective Komada media (left) and same kai choi root pieces on Komada media with suspected Fusarium growth (right).

 

Fig13
Figure 13. Mean percentages of plated kai choi roots observed with suspected Fusarium growth for each treatment group taken after harvest and surface sterilization. Treatment groups with the same letter indicate no significant difference (Waller-Duncan, α=0.05).

D. Nematode community as soil health indicators

A primary reason for assessing alternative biofumigant methods is due to the harm that Vapam inflicts on the natural soil ecosystem when used. To make this comparison and measure the soil health of PGS-treated plots versus Vapam-treated plots, free-living nematodes were extracted and counted. Total free-living nematode counts were significantly higher in the Sorghum-treated plots compared to all other treated plots. The control, mustard, 1% PGS, 0.5% PGS, and Vapam treated plots were all comparable in overall nematode counts (Fig. 14).

Most treatment groups were comparable in species richness (number of different species present) measures, however the Vapam plots were significantly less species rich (Fig. 15). This is likely due to the that Vapam indiscriminately kills everything in the soil that it is used in (nematodes, fungi, beneficial microbes, etc.), thus creating a uniformity in the species present in the soil as the species that are able to bounce back quickly. It’s unlikely in these conditions to observe and record and long generational/large nematodes in the soil samples. So, despite not seeing a large difference among the PGS, sorghum, mustard, and control treated plots, there is already an observable increase in species richness in the soil not treated with Vapam. This is an indicator that in one crop rotation without the synthetic fumigant, the soil is becoming healthier. 

Fig14
Figure 14. Total free-living nematodes counted from elutriation-extracted field soil collected on final day of experiment. C = control, M = mustard, PGSF = 1% PGS, PGSH = 0.5% PGS, S = Sorghum, and V = Vapam. Treatment groups with the same letter indicate no significant difference (Waller-Duncan, α=0.05).
Fig15
Figure 15. Mean nematode species richness calculated from elutriation-extracted field soil collected on final day of experiment. C = control, M = mustard, PGSF = 1% PGS, PGSH = 0.5% PGS, S = Sorghum, and V = Vapam. Treatment groups with the same letter indicate no significant difference (Waller-Duncan, α=0.05).

PGS biofumigation plate assay

In our initial plate assay design, fungal cultures were inoculated onto the PGS-amended agar plates (PGS was blended in and encapsulated in the agar) and incubated for subsequent colony count. We encountered microbial contamination problems with this assay format. Some of the papaya seed indigenous microbes apparently survived the seed drying and milling processes and grew in the PGS-amended agar plates. Subsequently, we developed an alternative plate assay, in which the agar plate was hollowed out in its center and water-amended PGS was used to fill the hole. As a PGS-free control, the hole was either not filled in with the PGS, or without creating the center hole. This assay format worked well, showing little signs of contamination from the indigenous papaya seed microbes, and was adopted for subsequent in-vitro bioassays. Of the three fungi tested, the F. oxysporum plate assay showed greatest overall significant difference in growth, reducing the mycelial growth to almost zero compared to the control group (Picture 7, top left; Fig.16). Thus, it appears that PGS biofumigation is highly effective against F. oxysporum in vitro. However, both F. solani and Setophoma sp. had no significant difference in growth between the control and two treatments groups, with the PGS biofumigation appearing to have no effect on the mycelial growth (Picture 7, top right and bottom; Fig. 17). For Setophoma sp., there was a slight difference between the control group and the group treated with 0.5g PGS + 0.5g water, but this was not easily observed (Picture 7, bottom; Fig. 17). 

pic7
Picture 7. 48 hour old agar plugs with Fusarium oxysporum (left), Fusarium solani (center), and Setophoma sp. (right) mycelium plated on ½ PDA media with wells carved out in the center. From left to right for each fungus, treatments were control (no PGS biofumigation), 0.5g PGS + 0.5g water, and 1g PGS + 1g water.
Fig16
Figure 16. Average growth of Fusarium oxysporum during media plate biofumigation at 48 hours and 96 hours. Control indicates the plates were treated with NO PGS, 1g indicates the plate was treated with 1g PGS + 1g water and 0.5 g indicates the plate was treated with 0.5 g PGS + 0.5 g water. Treatments with the same letter were not significantly different (Waller-Duncan, α=0.05).
fig17
Figure 17. Average growth of Fusarium solani (top) and Setophoma sp. (bottom) during media plate biofumigation. Control indicates the plates were treated with NO PGS, 1g indicates the plate was treated with 1g PGS + 1g water and 0.5 g indicates the plate was treated with 0.5 g PGS + 0.5 g water. Treatments with the same letter were not significantly different (Waller-Duncan, α=0.05).

Emission of BITC from PGS

In intact papaya seeds, cellular membranes separate glucosinolate (the substrate) from myrosinase (the enzyme), and hence efficient breakdown of the membrane barriers is essential to promote the enzymatic hydrolysis upon water addition. One of the simplest approach to break down the membranes is to mill the dried seeds into powders, called “PGS” herein. This method is inexpensive, though it may not completely degrade all membrane barriers in the seeds. In our prior study (Han et al. 2018), we could detect ample amounts of BITC after extracting water-activated PGS using organic solvents. The result indicated that high levels of BITC could indeed be produced by PGS upon water activation. However, since organic solvents were used to extract the BITC, we do not know the extent BITC can be released by itself from the PGS without extraction. In year 2, we designed and evaluated a series of laboratory experimental tests, to gain a better understanding about the temporal pattern and extent of BITC emission from water-activated PGS, and to investigate how this process is affected by PGS water content, PGS particle size, freezing pretreatment, and inclusion of soil. These fundamental characterizations are essential to develop better practices for more efficient implementation of PGS-based biofumigation in the field. Because currently we do not have a gas chromatography system for measure BITC in the vapor phase, we had to develop an alternative assay platform based on measurement using liquid chromatography. In this regard, since BITC is a hydrophobic compound that shows good solubility in plant oil (triacylglycerol) and mineral oil (alkanes), we tested commercial soybean oil and mineral oil as a liquid trap for BITC emitted from PGS, and the oil was then analyzed for BITC using HPLC. The transport of BITC from water-activated PGS to the vapor phase surrounding the PGS involves multiple steps. First, the addition of water to the PGS activates the myrosinase enzyme which catalyzes the hydrolysis of benzyl glucosinolate (BG) in an aqueous phase. Second, the BITC thus generated is hydrophobic, and hence it tends to partition into the seed fat, or emit to the vapor phase (interstitial air space) followed by eventual release into the headspace above the PGS bed. It may also be absorbed onto PGS particle surfaces as it diffuses through the bed of PGS.

In our tests, we typically use 2 g of dried PGS in each test. We first examined the BITC trapping efficiency by soybean oil vs mineral oil. A water:PGS ratio of 3 was used so that a thin layer of water is viable above the PGS bed which was overlaid with 2 mL of oil. The tube was incubated under room temperature for 40 hours before the oil samples were taken and analyzed using HPLC. As shown in Fig. 18, mineral oil appeared to be a more effective BITC trap than the soybean oil. More importantly, this result provided direct proof that not only BITC can be generated by PGS upon water activation, but also it can be released out of the PGS matrix and migrate into the headspace. Next, we investigated the temporal pattern of BITC release from PGS. Using mineral oil as the trap, and with 2 grams of PGS plus 6 grams of water, the BITC continued to partition into the oil trap over the 32-hr testing period, as shown in Figure 19. We also investigated the effect of soil on BITC release. Sorption of released BITC onto soil surfaces could potentially reduce the efficacy of BITC. In one treatment, 2 g of PGS was mixed into 4 g of soil and placed in a 50 mL centrifuge tube, followed by adding sufficient water until a thin layer of water was viable above the soil/PGS bed. In the other treatment, 2 g of PGS was placed in the bottom of a 50-mL centrifuge tube, overlaid with 4 g of soil, followed by adding water until a thin layer of water was viable above the soil/PGS bed.  In both treatments, 2 mL of mineral oil was overlaid on the water layer.  As shown in Figure 20, by dispersing PGS in soil, much higher BITC emission was detected, which could have implications on how to apply the PGS in soil in the field.  Because in these tests, the interstitial space in the PGS bed was filled with water, BITC needs to migrate through this aqueous phase before it can partition into the oil trap on the top. Typically, PGS-amended soil in the field is not waterlogged, the hydrophobic BITC is likely to diffuse more readily in the air space between soil particles and hence greater and faster BITC release is expected in the field. In summary, our results provide direct proofs of BITC release from water-activated PGS, and that the release process likely lasted for several days. The vapor pressure of BITC is two orders lower than that of allyl-isothiocyanate (AITC, active biofumigant in mustard), and hence slower release than AITC is expected.

fig18
Figure 18. Comparison of soybean oil vs. mineral oil for trapping BITC in the emission assay.
fig19
Figure 19. Release of BITC from PGS as a function of time.
fig20
Figure 20. Effect of soil on BITC release from PGS.

Water plays an important role in the conversion of BG to BITC in PGS. On the one hand, water is necessary for the myrosinase to function. However, too much water may rinse out BG (which is water soluble), and may cause BITC degradation. Too much water is also harmful to the crops. We examined BITC release at four different water:PGS ratios (0.5, 1, 2, and 3) using the oil-trap assay. At a 2:1 water:PGS ratio, a higher BITC production was noted (36.05±12.65 mM) compared to our initial treatment (3:1 water:PGS; 22.51±0.43 mM). At further reduced water:PGS ratios of 1 and 0.5, the BITC production was even higher (120.14±1.80 mM and 108.58±36.71 mM, respectively). However, we likely cannot judge the water effect solely on these data. As the water content was decreased, oil on the top seeped into the PGS bed after several hours of incubation. Therefore, some of the oil was in direct contact with the PGS matrix, and BITC likely partitioned into this oil phase once it was produced from BG hydrolysis. This result however suggests addition of some oil (such as vegetable or mineral oil) to the PGS when it is amended to the soil might be beneficial in modulating the release characteristics of BITC. Partition of BITC in an oil phase can help stabilizing the hydrophobic biofumigant and prolong its release from the PGS. This aspect will be investigated further during the next report period.

To investigate two additional potentially important factors, i.e., PGS particle size and seed freezing, we slightly modified the assay. We blended 2 g PGS with 2 g of water and 2 g of mineral oil in a 50 mL centrifuge tube and incubated for 18 hours. The tube was then centrifuged, and the oil sampled for BITC measurement.  As shown in Figure 21, PGS with finer particle size generated more BITC than coarser PGS.  Freezing treatment of papaya seeds prior to drying and grinding into PGS also improved BITC release from PGS (Figure 22). Finer PGS particles likely promoted contact between BG and myrosinase by breaking down tissue/membrane barriers in the papaya seeds and also increased interfacial area for better mass transport. By freezing fresh seeds, further degradation of internal tissues in papaya seeds may occur which can augment breakdown of cellular membrane barriers and promote BITC production.

fig21
Figure 21. Effect of PGS particle size on BITC release.
fig22
Figure 22. Effect of freezing fresh papaya seeds prior to drying and grinding on BITC release from PGS.

References cited:

Waisen, P., B. S. Sipes, and K.-H. Wang. 2019. Potential of biofumigant cover crops as open-end trap crops against root-knot and reniform nematodes. Nematropica 49:254-264.

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. 

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Year 2 concluding remarks:

BITC emission from PGS

Laboratory tests in 50 mL sealed centrifuge tubes using a mineral oil layer to trap PGS-generated BITC allowed rapid comparison of a variety of factors for their effects on BITC release from PGS. Among the factors investigated, PGS particle size, water content, PGS distribution in soil, freezing of papaya seeds prior to drying/milling, and inclusion of vegetable oil in PGS amendment, all showed some degrees of influence on BITC release from PGS. We also showed that BITC release from PGS could last for several days. In year 3, these measurements will be further refined, including comparison of results with direct gas-phase BITC measurements using gas chromatography available in a new collaborator’s lab. Findings from these tests provide insights into developing better practices for more efficient use of PGS in the field implementation of PGS-based biofumigation.

PGS Biofumigation plate assays:

Based on results obtained, PGS biofumigation appears to work well against F. oxysporum in vitro, which is vital as it is the causal agent of Fusarium wilt in various plants and the primary concern for the kai choi fields of participating growers. With further optimization, in the greenhouse and in field trials, there is promise for PGS biofumigation to be recommended as a potential control method of Fusarium wilt. However, right now it’s still unclear whether or not this biofumigation method is effective against other fungal plant pathogens (as seen with F. solani and Setophoma sp.) so to test whether it is the PGS application method that is ineffective to other fungal species or the BITC compound released that is ineffective, another plate fumigation assay will be performed using pure BITC in place of PGS. Pure MITC, the active ingredient in Vapam, will also be tested in vitro to compare its strength in suppressing fungal pathogens to the strength of BITC.

Field biofumigation:

To troubleshoot the PGS amendment and optimize it for future field trials, there are various, small greenhouse experiments planned to determine how best to apply the PGS amendment in the field to get the best results. Most important limiting factor is insufficient amount of PGS can be prepared for entire bed preparation and resulted in uneven distribution of PGS beyond the root zone of kai choi. Other factors that could help to resolve this issue include how deep the PGS needs to be in the soil or how wet the soil needs to be in order to release and dissipate BITC into the surrounding area. Planned experiments to answer this question include basic pot experiments that will measure the amount of BITC captured when PGS is buried at various depths in potting conditions as well as an experiment that will measure volumetric soil moisture and the relative amount of BITC that is released linearly. It is also supposed that not enough PGS was used in the treated plots as well, as the field set up included digging trenches in the plots and only amending those locations. Since the sorghum and Vapam plots were prepared by the grower and employees involved, and the kai choi seedlings were also transplanted by farm employees, the research group was unable to oversee every part of the experimental plot preparation. The trenches were not clearly marked, so it is likely kai choi was not planted directly on PGS and it’s unclear the area that BITC can penetrate. Based on this, it may be beneficial to cover entire plots with the PGS amendment moving forward.

Participation Summary
1 Producers participating in research

Research Outcomes

2 New working collaborations

Education and Outreach

5 Consultations
1 Curricula, factsheets or educational tools
1 On-farm demonstrations
1 Online trainings
4 Webinars / talks / presentations
1 Workshop field days

Participation Summary:

36 Farmers participated
4 Ag professionals participated
Education and outreach methods and analyses:
  • 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. 
  • 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.  

During our second year, the team presented initial findings from the greenhouse study of instant biofumigation with papaya ground seeds (PGS) at a Virtual Soil Health and Sustainable IPM Mini Conference organized by co-PI Uyeda and Wang on Sep 28, 2021. There were 60 participants attended the conference. During a post conference survey, 7 farmers participated in the survey. 100% of the farmers expressed interest to try out PGS biofumigation to manage plant-parasitic nematode problem, however, only 50% of them expressed interest to try PGS biofumigation against soil-born fungal problem. The remaining 50% are interested in using biofumigation from cover crops such as brown mustard or sorghum or continue to use Vapam as a fumigant. Co-PI Wang presented the concept and initial results on PGS-based biofumigation through guest lectures to three cohorts of GoFarm Hawaii (https://gofarmhawaii.org/) which train beginning framers, in the topical area of “sustainable management of nematode and other pests in agroecosystems through cover cropping or biological derived products”. These guest lectures were delivered on: 1) Kauai- Farm Coach: Eric Hanssen (Dec 9, 2021); 2) Waimanalo - Farm Coach: Rachel Ladrig/Coordinator: Laura Ediger (Nov 10, 2021); and 3) Waialua, Oahu - Farm Coach: Dan Caroll (Dec 29, 2021). A total of 36 beginning farmers and 3 farm coaches were reached. Co-PI Wang was also invited as a guest speaker for the University of Hawaii Tropical Plant Pathology Graduate Student Seminar on February 11, 2022. She shared the findings of PGS instant biofumigation studies with 14 participants. A farmer approached our extension co-PI (Silva) and expressed interest to use PGS instant biofumigation for a new fungal disease on onion caused by Setophoma sp. Though our initial petri dish trial did not show significant biofumigant effects against this fungi, we will follow up with seedling treatment in the next progress period.  On March 26 2022, an in-person field day visit was conducted by co-PI Wang and a graduate student with GoFarm Hawaii Waialua Cohort to discuss papaya production. During the discussion, the knowledge on PGS instant biofumigation approach was shared with the participating farmers while demonstrating papaya vegetative propagation to help beginning farmers that are interested to develop their own papaya selection. These farmers were fascinated by making use of papaya seeds to manage diseases on farms.

PI-Su led team meetings to develop an educational/outreach website on biofumigation, with emphasis on the sustainable PGS biofumigation technology that is being developed in this SARE project. A novel “citizen science” idea was proposed to allow the public to participate in the testing and development of the PGS technology. PGS can be made easily using household coffee grinders and papaya seeds are readily available. Moreover, PGS can be tested in participant’s backyards. The project team will develop simple PGS protocols and questions for distribution via website download, while citizen scientists can share their observations and results with the project team via the website for further data analysis. The findings will be disseminated also via the website. We believe this approach may encourage greater interests from the public in this sustainable practice in farming and even home gardening. Additional features of the website will include an online calculator to estimate PGS amounts for given soil amendment rates, and anonymous questionnaires and surveys. In collaboration with the CTAHR IT specialists, we plan to beta test the website in summer 2022.  

Education and Outreach Outcomes

Recommendations for education and outreach:

We completed a field trial in year 2.  During the year-2 field trial, we observed early protection of kai choi seedlings from the fungal disease complex for at least 3 weeks after transplanting. Although the trench application of PGS used in our field trial could not effectively protect kai choi from fungal damages, our laboratory characterization of BITC emission from PGS revealed alternative application schemes of PGS that may improve the PGS efficacy, which will be evaluated further during year 3 in additional greenhouse and field trials. Some of the modifications will include reduction of PGS particle sizes and optimizing the duration of PGS incubation in soil prior to transplanting to maximize soil fumigation while minimizing toxicity to the crop plants. Additional improvements may come from optimizing the way PGS is dispersed in the soil, maintaining soil moisture level during biofumigation, and post plant drenching with refined aqueous PGS extract (to provide additional BG as myrosinase substrate). As we learned more about the characteristics and best application practices of papaya ground seeds as a natural soil fumigant, we will continue to work on outreach to disseminate our findings to the public and to promote adoption of this sustainable plant disease management practice.  

1 Producers reported gaining knowledge, attitude, skills and/or awareness as a result of the project
Key areas taught:
  • Development of a new approach to enable effective management of soil-borne pests by recycling/repurposing agricultural wastes

Success Stories

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Information Products

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.