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

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.

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.

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. 

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).

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.

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. 

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

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.

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.

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.

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.

**********

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.

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.

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.

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).

 

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. 

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). 

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.

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.

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.

***********************

During our third year, we collected data pertaining to (1) in vitro toxicity trials of pure synthetic BITC against various soil-borne fungal pathogens in agar plate assays, (2) in vitro toxicity study of pure BITC, PGS, and PGS extracts against Fusarium fungal pathogen in soil, (3) characterization of gaseous vs dissolved state BITC against Fusarium fungal pathogen, (4) development of a novel assay for live imaging of fungal interactions with PGS in transparent soil, (5) greenhouse trials of PGS biofumigation on kai choi and lettuce in Fusarium-infested field soil collected from a collaborator’s farm, and (6) a greenhouse trial of PGS biofumigation on cowpea in a sterile sand: soil mix artificially inoculated with vermiform reniform nematodes.

BITC in vitro agar plate assays

In order to confirm that BITC released from PGS was the compound involved in suppressing plant-pathogens of interest, synthetic pure BITC was combined with ½ PDA media for two of in vitro experiments with pure fungal isolates. This eliminated any interference of additional microbes or compounds present in the PGS. In both in vitro trials, all four fungal species (F. oxysporum, F. solani, R. solani, and Setophoma sp.) showed significant reduction in growth in both 0.1 mM and 0.5 mM BITC concentration treatments compared to the control (Fig. 23 and 24). Additionally, 0.5 mM BITC plates had significantly lower mycelial growth than the 0.1 mM BITC plates (Fig. 24), with growth completely halted in Trial 1 (Fig. 24A). These results demonstrate that pure BITC is able to suppress a range of fungal pathogens when combined in vitro in agar media, however the level of suppression in this bioassay is known only up to the 48hour time point. Subsequently, we conducted further in vitro testing using an alternative soil-based assay for F. oxysporum, examining an extended range of BITC concentrations and growth periods. The results are presented in the section “In vitro soil-based assays.”

In vitro soil-based assays

Agar-based in vitro plate bioassays were predominantly used in our study to probe the inhibitory effect of BITC on soil-borne fungal pathogens. While it is a convenient assay platform, it does have limitations. Agar is a hydrophilic material, whereas soil surface properties are quite different. It is known that soil biofumigant may be adsorbed onto soil surfaces as it is transmitted through the soil matrix. Therefore, antifungal efficacy revealed in agar-based assays may not completely represent the actual biofumigant performance in soil. We evaluated several alternative in vitro assay formats in which autoclaved field soil (collected from the site the first field trial was conducted) was incorporated. Initially, our approach simply replaced the agar with water-saturated soil, in otherwise the same assay format as the agar/PGS plate assay described above. By placing fungal agar plugs above the soil, we wanted to compare mycelia growth on the plug by varying the PGS amendment rates. In the agar assay, BITC was clearly released from the PGS placed in the center of the agar plate and diffused through the agar to reach the inoculated fungus and inhibit its growth. Unfortunately, we were unable to get consistent results with the soil-based plate assay.  Subsequently, we developed an alternative assay format that offered a simple and reproducible way to semi-quantitatively evaluate the effect of PGS, PGS extracts, and BITC, in inhibiting F. oxysporum mycelial growth in soil. We also invested surfactant treatment of soil on the biofumigant activity. The alternative assay is described in the Research Method section above, under “In-vitro tests of BITC, PGS, and PGS extracts against Fusarium oxysporum in improved soil-based assays.” First, we tested the PGS amendment rates at 0, 1, 2, and 3%, respectively. From Figure 25, significant growth inhibition of F. oxysporum mycelia can be seen at 2% and 3% PGS amendments into the soil. In a typical field soil environment, unlike the selective media used here, nutrients are much scarcer, and hence the extent of fungal inhibition offered by 2% PGS is likely sufficient to suppress the growth of any residual F. oxysporum cells that are not killed by the BITC released by PGS mixed into the soil. The brown patches appeared on the selective agar plates in Figure 25 were from the soil used in the assay. While a very simple assay, it offered consistent and reproducible results. Therefore, we used this assay format to further test the effect of pure BITC and PGS extracts, and soil treatment with a surfactant called Forte (J R Simplot Company, Lathrop, CA). The commercial soil surfactant Forte can alter soil surface properties by alleviating soil water repellency, and possibly reduce soil adsorption of hydrophobic BITC released from PGS. As shown in Figures 26 and 27, respectively, BITC and PGS extracts both performed better with Forte-treated soil than with untreated soil. The effect is especially profound with PGS extracts (Figure 27). Using the improved soil-based assay, pure BITC was shown to be highly effective at 30 mg/L which is equivalent to about 0.2 mM (Figure 26). This is slightly higher than the 0.1 mM BITC revealed from the agar-based assay, although with 0.1 mM BITC, fungal suppression was also not so effective in one of the two trails as shown in Figure 24B.  Surprisingly, PGS extracts performed worse than PGS at 2% and 3%, using untreated soil (cf. Figure 25 vs. Figure 27, day 2), although PGS extract performance was improved considerably when used with Forte-treated soil (Figure 27). In future experiments, we will improve the preparation of the initial mycelia suspension for inoculation to enable quantification of fungal growth upon PGS treatments. We will also apply this assay to examine additional soil-borne fungal pathogens, besides F. oxysporum.

Kai choi greenhouse biofumgation trial

A. Disease Index

Overall disease index was assessed based on a pre-determined kai choi disease scale of 1 to 4, with 1 being healthy and 4 being most diseased (Fig. 28). In the kai choi greenhouse trial, the treatments with the highest disease index ratings were the non-amended (NA) treatment group (both with [+] and without [-] leachate were comparable) and the autoclaved soil (Auto) treatment group, which was comparable to NA (+ and -) (Fig. 29B). As the disease index rating does not discriminate between biotic and abiotic disease symptoms, the high rate of disease in the Auto treatment group was likely due to a lack of nutrients in the autoclaved field soil. To reduce abiotic disease symptoms in autoclaved reps, synthetic fertilizer will be included in the treatment moving forward.

The 0.5% PGS (+) and (-) and the 1% PGS (+) treatments were comparable to each other, however these 3 treatments showed overall reduced disease symptoms when compared to all NA disease index ratings. Additionally, while 1% PGS (-) results were comparable to the NA (+) treatment results, the disease index rating in 1% PGS (-) was significantly less than the disease index rating observed in the NA (-) treatment group (Fig. 29B). Overall, both the 0.5% and 1% PGS amendment groups significantly reduced disease incidence regardless of root leachate drenching used or not. There was also no significant difference in disease incidence between the two PGS concentrations, indicating that the 0.5% PGS amendment rate may be sufficient in trials moving forward. However, additional kai choi greenhouse trials are needed to confirm this hypothesis.

B. Biomass

Overall biomass of combined kai choi shoots and roots was measured in grams (g). Auto treated pots had the lowest biomass, which matches up with the respective disease index ratings. Similar to results seen with the disease ratings, individual PGS treatments were not significantly affected by the use of root leachate or not [i.e. 0.5% PGS (+) did not differ from 0.5% PGS (-) and 1% PGS (+) did not differ from 1% PGS (-)]. However, contrasting with the disease index results, the 1% PGS amendment rate outperformed the 0.5% PGS rate in overall biomass. While the two concentrations were comparable to each other, the 1% PGS treatment had significantly higher mean biomass than the NA treatment group, whereas the 0.5% PGS biomass was comparable to that of the NA treatment group (Fig. 29A).

Interestingly, in terms of biomass, the NA (+) group performed significantly better than the NA (-) group, indicating potential benefits of adding the kai choi root leachate alone (Fig. 29A). As kai choi is a member of the Brassica spp. (Brassica juncea subsp. integrifolia), a genus known for volatile-producing plant species, it is possible that the root leachate contains additional volatiles or compounds toxic to Fusarium spp. and R. solani.

C. Fusarium and Rhizoctonia root colonization

Kai choi root pieces plated on Komada (Fusarium) and KH (Rhizoctonia) selective media were observed periodically over 12 days to check for Fusarium spp. and R. solani mycelial growth. There was a small amount of R. solani growth observed in the Auto treated reps, but due to some Rhizoctonia existing as saprophytic species within plant roots, there is potential for non-pathogenic Rhizoctonia spp. to emerge on the selective media. Otherwise, in this trial, no statistically significant differences between treatments were observed (Fig. 29C-D). This is likely due to a small data set size (4 reps per treatment, with only 3 root pieces plated per rep), so any major variability within treatments may have a larger effect on results. Based on this, no clear conclusions can be derived from root colonization data. For future trials, root pieces will be plated onto selective media per plant growing in each potted rep. Additionally, qPCR assays will be designed and used to quantify levels of Fusarium and Rhizoctonia in the kai choi roots and soil.

Cowpea greenhouse biofumigation trial

A. Disease Index

Disease index was measured based on a pre-determined cowpea disease scale of 1 to 4 (Fig. 30). Based on this scale, the Auto treatment was found to have the greatest overall disease incidence, whereas NA treated pots had a significantly lower mean disease rating compared to the Auto and PGS treatments, which was the opposite of expected results (Fig. 31A).

B. Root and shoot biomass

Cowpea root and shoot biomass was measured separately in grams (g). The NA treated group had significantly greater biomass in both the shoots and roots of the plants, whereas the 1% PGS treatment was significantly the lowest in shoot and root mass, aside from the Auto treatment in shoot mass, in which it was comparable (Fig. 31B-C). These results align with those observed in the cowpea disease index data.

There were confounding factors in this trial. Treatments planted in plastic pots appeared to retain excess moisture, leading to plant roots rotting early in the trial, a lack of uptake in water and nutrients, and premature plant death due to causes unrelated to nematode inoculation and infection. Treatments planted in terracotta pots did not have this issue. For future trials, only terracotta type pots will be used. Additionally, as the sand: soil mixture is autoclaved and without added nutrients in the Auto and NA treatment groups, these pots will be treated with a synthetic NPK fertilizer moving forward to reduce the lack of nutrients as a potential cause of plant death.

C. Number of female reniform nematodes infecting roots

The number of R. reniformis females present per 1 g of roots were counted under a dissecting microscope after acid fusion staining. Infection observed overall was highly variable, with a low number of females quantified in the healthier, NA treatment reps. While it appears that the 1% PGS amended pots had a much higher number of female nematodes present, it was still found to be statistically comparable to the NA treatment (Fig. 31D). This is likely due to large outliers within the 1% PGS data set. Despite outliers, there was generally low infection of roots by reniform females. With questionable health of the plants, it is unclear if infection was low due to bad (rotting) roots or poor transfer of nematodes to the pots during inoculation.

Lettuce greenhouse biofumigation trial

Overall results obtained from this trial displayed no significant interaction between pots that were treated with lettuce root leachate, versus those that were not. All lettuce reps for each treatment group were therefore pooled (n=8) regardless of leachate treatment or not.

A. Disease Index

Disease was measured based on a pre-determined lettuce disease scale of 1 to 4 (Fig. 32). No significant difference was observed between treatments, with the NA treated group having a slightly higher disease rating. However, in general the disease index remained at a relatively constant mid-range across treatments (between 2-2.5 average) (Fig. 33A).

B. Biomass

Lettuce root and shoot mass were measured separately in grams (g). Root biomass was significantly higher in the 0.5% PGS amended reps compared to the NA treatment, however 1% PGS treated reps had significantly lower root mass than NA (Fig. 33C). Similar results were seen in the shoot biomass means, where the 0.5% PGS treatment had had significantly higher biomass, but the 1% PGS treated pots were comparable to the NA treatment (Fig. 33B). Based on these results, rather than being related to disease or lack thereof causing differences in biomass, it is speculated that PGS has a green manure effect at lower concentrations, but phytotoxic effects if PGS concentrations used for amendment are too high. This is supported by the positive growth results seen in the 0.5% PGS data compared to the negative, deleterious effects seen within reps treated with 1% PGS.

In previous, unpublished work, it was determined that PGS contains a high level of nitrogen (N), at 4.14% overall content. While N fertilizer is beneficial to plants, an excess in N is phytotoxic. All-purpose, synthetic store-bought NPK fertilizer commonly contains N at 24% of its overall content, and manufacturers recommend a total application of approximately 15g per 9.3 m². Of this total application, 3.6 g total is N applied over the 9.3 m² area (Miracle-Gro, The Scotts Company LLC., Marysville, OH). At a 1% PGS amendment rate, 25 g PGS is applied to an approximately 0.01 m² area pot, with a total of 1.035 g N applied to a considerably smaller area of soil. Based on this information, it is likely that the N content present in 1% PGS compared to 0.5% PGS amendment is too high and having negative effects on overall plant health.

C. Fusarium root colonization

No significance between treatments was observed from data obtained, likely due to overall low infection rates occurring. The mean number of root pieces demonstrating Fusarium growth on selective media was equal to or less than 0.5 for all treatment groups (Fig. 33D). Field soil collected and used in this trial was obtained from a commercial lettuce and leafy green farm that reported Fusarium wilt disease issues in previous years, however when discussing the problem with the project team, the grower originally described the disease as being an issue during several cropping cycles, but not all.

Characterization of vapor-phase BITC transmission

In biofumigation using mustard greens, the active biofumigant allyl-isothiocyanate (AITC) is transmitted primarily via the vapor phase. However, the active biofumigant compound in PGS, i.e., BITC, is much less volatile. To characterize to what extents BITC is transmitted via vapor vs dissolved state, we set up a series of tests with either pure BIT or PGS placed in air-tight tubes and the gas phase samples were taken and analyzed with gas chromatography. However, we were unable to detect BITC. We can not rule out that some gaseous BITC was still formed but our sampling and analytical method was not sensitive enough to pick up the signal. That said, this result suggests that the vapor-phase transmission of BIT may be low. We then did a simple experiment in which we placed PGS in a small peri dish which was placed inside of a regular sized petri dish at its center and PDA agar was poured afterwards to surround the PGS in the center. F. oxysporum agar plugs were inoculated onto the agar portion of the plate as in typical agar-plat assays.  With this simple design, we were able to create a solid partition between PGS and the surrounding agar, and still allow gaseous BITC to be released from the top surface of PGS. As seen in Figure 34 (note the photos were taken from the back side of the petri dish), PGS without solid partition was capable of inhibiting fungal growth, while a much lower level of inhibition was observed with the solid partition, though still a little better than the control without any PGS. These results strongly suggest that the BITC released from PGS after water activation is transmitted mainly in its dissolved state, instead of its gaseous state. A similar observation has been reported in the literature comparing AITC and BITC as potential antimicrobial agents (Hareyama et al. 2022).

Probing PGS interaction with soil-borne fungi using a novel transparent soil system

In the aforementioned soil-based assay systems, PGS and fungal mycelia were mixed into the soil and incubated for a few days before subsequent analysis based on plating on a selective medium. Therefore, it is not possible to examine what exactly happened during the soil incubation. In recent years, emergence of the transparent soil technology has offered a novel approach for live imaging and analysis of soil microcosms. Here we adopted a transparent soil system that consists of cryolite particles immersed in a Ludox TMA (colloidal silica suspension) solution. By matching the refractive index, the cryolite particles become almost transparent in Ludox TMA, whereas in water, the cryolite suspension looked murky (Figure 35). This transparent soil system was mixed with a dilute F. oxysporum mycelial suspension (stained with a fluorescent dye) and loaded into a PDMS microfluidic chip. The Fusarium fungal mycelia at different depths within the transparent soil matrix could be clearly visualized using a confocal laser scanning microscope (Figure 35). In further studies, this chip will be connected to a fluidic system to introduce BITC, PGS extract, and/or nutrients, and be examined in real time using an inverted or confocal microscope system. Such live imaging analysis will provide highly valuable real-time characterization of fungal inactivation in response to PGS or BITC treatment in a simulated soil environment, and enable development of more efficient PGS application methods.    

References cited:

Hareyama, Yohei, Mitsunori Tarao, Koki Toyota, Tomohiro Furukawa, Yoshiharu Fujii, and Masayo Kushiro. 2022. "Effects of Four Isothiocyanates in Dissolved and Gaseous States on the Growth and Aflatoxin Production of Aspergillus flavus In Vitro" Toxins 14, no. 11: 756. https://doi.org/10.3390/toxins14110756

***********************

Final reporting period results:

Rhizoctonia solani qPCR

In order to accurately measure levels of R. solani in agricultural fields and soil, and diseased kai choi root tissue suspected to be Rhizoctonia-infested, a real-time quantitative PCR assay was designed for use as a quantitative research tool for greenhouse and field experiments.

 

A. R. solani qPCR validation

To validate that the primers/probe used for the detection of R. solani would amplify only the targeted pathogen, R. solani pure culture isolates from anastomosis groups (AGs) 2, 3, and 8 were included as positive controls, and non-target pure culture isolates Fusarium solani, F. pseudograminearum, F. sambucinum, F. culmorum, and Verticillium dahliae were included as negative controls. Nuclease-free water was used as a no template control. To assess the overall sensitivity of the primers/probe, a 10-fold serial dilution of R. solani AG-2 was performed.

It was successfully demonstrated that the assay targeted and amplified various R. solani AGs while avoiding amplification of non-specific targets (Fig. 36, left). Additionally, the assay showed consistent detection sensitivity down to 0.1 pg of DNA (Fig. 36, right).  Both assessments show high sensitivity and specificity of the assay, highlighting its potential as an accurate and sensitive detection tool for R. solani in future experiments. However, DNA samples extracted from soil samples suspected to be infested with R. solani were tested, and very low amplification occurred, indicating that R. solani may not be the primary pathogen causing disease in kai choi (not shown).

Fusarium commune qPCR

A. Fusarium isolate sequencing

In order to determine the causal pathogen of disease in kai choi, after low levels of R. solani was detected in diseased field soil samples, Fusarium single-spore isolates were obtained from diseased kai choi roots and sequenced. Of the sequenced isolates, after BLAST analysis, four Fusarium isolates returned with a 100% match for Fusarium commune, a known species involved in the Fusarium wilt and root rot disease complex. Based on the obtained sequence data, it was suspected that F. commune was a causal pathogen for disease in the kai choi plants rather than R. solani (Fig. 37).

B. F. commune qPCR development

To test for and accurately measure levels of F. commune in agricultural soil and diseased kai choi root tissue alongside R. solani, a preliminary, SYBR green-based RT-qPCR assay was designed for use. To validate that primers were specific to amplifying only F. commune and not other Fusarium species, three sequenced Fusarium isolates with matches for F. commune were included. Non-target, pure culture isolates of Fusarium pseudograminearum, F. culmorum, F. oxysporum f.sp. cubense, F. solani, F. sambucinum, Rhizoctonia solani, and Phytophthora palmivora were included as negative controls, nad nuclease-free water was used as a no template control.

We were able to demonstrate that the designed assay could amplify several F. commune isolates, while also being specific enough to avoid amplifying non-target species, including other pathogenic Fusarium (Fig. 38). Additionally, melting curves for the amplified targets reached their peaks at the same temperature, further confirming all sequenced F. commune isolates used in this assay are of the same species (Fig. 38).

However, initial amplification results showed late amplification of pure F. commune isolates. qPCR assays frequently face low or no amplification signals during reactions due to residual inhibitors present in the DNA template, including phenols, detergents, proteases, or other residuals specific to source material or tissue. Thus, this is one factor that may be contributing to the late amplification of the F. commune template used.

Rhizoctonia solani in vitro PGS biofumigation

To assess the toxicity of PGS biofumigation against pure R. solani, we conducted an additional in vitro PGS assay, performed similarly as the assay on other fungal species in year two of the project. After 48 hours of growth, R. solani mycelial growth was significantly lower in PGS-treated groups versus the untreated, control group. Both 0.5 g PGS and 1 g PGS performed comparably, indicating that lower concentrations of PGS are sufficient enough to slow R. solani growth. However, neither PGS concentration tested was able to completely stop growth (Fig. 39).

Kai choi greenhouse biofumigation (+/- leachate) trial II

Overall results obtained from this trial displayed no significant interaction between pots that were treated with kai choi root leachate, versus those that were not. All kai choi reps for each treatment group were therefore pooled (n=8) regardless of leachate treatment or not.

A. Disease Index

Disease index was measured based on a pre-determined kai choi disease scale of 1 to 4. Based on this scale, there were no differences observed between the treatment groups, including autoclaved soil pots (Fig. 40A). However, due to high heat conditions present in the greenhouse environment at the time of growing, there were complications with preventing water-related wilting in the plants. This is likely the reason that autoclaved treated plants had a visual disease rating that was comparable to the non-amended treatment group, despite being expected to have minimal disease symptoms.

B. Shoot & Root Mass

The autoclave group had the greatest shoot mass despite showing wilting symptoms. All other treatments had significantly less shoot mass, with a decreasing numerical trend shown with increased PGS concentration (Fig. 40B). As we saw this occur with an increase in the amount of PGS used, which subsequently increased the level of BITC produced, it is suspected that phytotoxicity is occurring as it did in early greenhouse trials, due to transplanting kai choi seedlings 48 hours after PGS amendment in this trial rather than 7 days after in previous trials.

However, contrary to the data observed in the shoot mass, the root mass of pots treated with 0.5% PGS were comparable to the autoclaved treatment. The non-amended treatment had the lowest root mass, with 1% PGS having a numerically higher root mass (Fig. 40C). These observed results may indicate that despite initial phytotoxicity, roots in the PGS-treated pots were recovering.

Cowpea greenhouse biofumigation trial II

A. Disease Index

Disease index was measured based on a pre-determined cowpea disease scale of 1 to 4 (Fig. 41). Based on this scale, disease observed was comparable in all treatments, with the non-amended treatment being the numerical lowest (Fig. 42, left). Due to the autoclaved treatment group showing some signs of disease, it is suspected that some abiotic disease symptoms were present.

 

B. Nematodes per gram of root

After acid fuchsin staining roots, the number of vermiform and female reniform nematodes (RN) and egg masses were counted under a dissecting microscope. There was a comparable number of vermiform nematodes and egg masses observed in the PGS-treated versus non-amended pots. However, PGS-treated pots had a lower number of female RN present in roots (Fig. 42, right). This suggests that while there is a larger number of vermiform nematodes present, the BITC produced by PGS appears to disrupt the RN lifecycle and suppress reproduction. Additionally, there was an overall numerical decrease in the number of vermiform nematodes observed in the 1% PGS treated pots compared to the non-amended pots, indicating that the 1% PGS treatment has some suppressive effect on the RN overall (Fig. 42, right).

Kai choi greenhouse biofumigation (+/- surfactant) trial I

In order to assess the effectiveness of using a detergent/surfactant to improve BITC molecule movement through the soil, a kai choi PGS biofumigation trial was performed, in which pots were either treated with 80 mL of a 0.1% Tween80 solution during amendment or 80 mL of water.

A. Biomass

No significant differences were observed between pots treated or not treated with the 0.1% Tween80 solution. All kai choi reps for each treatment group, when assessing biomass, were therefore pooled (n=8) regardless of surfactant treated or not. Overall, biomass observed was not significantly different, however the 1% PGS treatment group was observed to have numerically higher biomass than all other treatments, suggesting the beginnings of a beneficial trend (Fig. 43A).

B. Disease Index

There were some effects of applying versus not applying surfactant in observed disease index ratings. In the 1% PGS treated group, disease index was significantly reduced compared to the non-amended group, and comparable to the autoclaved treatment group (Fig. 43B). Additionally, there was a slight numerical decrease in observed disease index in the 0.5% PGS group, suggesting that the use of surfactant in the watering during amendment may aid in the dispersal of BITC through the soil, especially when using larger concentrations of PGS (Fig. 43B).

Kai choi sorghum and PGS+CE biofumigation field trial

Due to poor results in the field trial performed during year two of this project, and in vitro and greenhouse results that showed the potential for a surfactant to aid in dispersing BITC through the soil, a kai choi field trial was conducted to examine the efficacy of a PGS crude extract (PGS+CE), containing the Tween80 surfactant, on suppressing Fusarium wilt disease in a commercial kai choi field.

A. Disease Index

PGS+CE treated plots showed significantly reduced disease compared to both the untreated, control plots and plots amended with sorghum (Fig. 44A). These results demonstrated that PGS+CE was effective in suppressing disease compared to sorghum amendment and plots that went untreated.

B. Percent healthy plants

As expected, untreated plots had the lowest percentage of healthy plants remaining, where the PGS+CE treated plots showed a greatly increased percentage of healthy kai choi plants, having the highest number of healthy plants compared to both the untreated and sorghum amended groups (Fig. 44B). Additionally, data obtained from the year two kai choi field trial for disease incidence, in which PGS alone was applied, showed the PGS treatment groups as having the highest levels of disease compared to sorghum amended and untreated plots (Fig.12). This further supports the use of PGS+CE as a more effective application method over the use of PGS alone.

Further characterization of fungal growth upon PGS treatments in a soil-based assay

We improved the preparation of the initial mycelia suspension for inoculation to enable quantification of fungal growth upon PGS treatments in the in vitro soil-based assay. We conducted the assay to examine F. solani, in addition to F. oxysporum. The initial mycelia inoculum size in soil was set at about 5-7 x 10⁵ cfu per gram of soil, which is high compared to typical pathogen levels in infested field soil. Even under such a condition, PGS amendment rate at 0.5% was shown to completely suppress the growth of the two Fusarium sp. (Figure 45) This PGS threshold is lower than what was reported in our report last year, and it is mainly because the inoculum size used in last year’s experiment was about an order higher. For F. solani and F. oxysporum, PGS in soil is highly inhibitory.

Direct imaging of Fusarium oxysporum growth inhibition in response to PGS amendment in a transparent soil system

In our last report, we described a transparent soil system that consists of cryolite particles immersed in a Ludox TMA (colloidal silica suspension) solution. By matching the refractive index, the cryolite particles become almost transparent in Ludox TMA, at modest cryolite particle concentrations. Unfortunately, under higher concentrations that mimic natural soil, the system becomes less transparent. Therefore, we evaluated an alternative transparent soil model made of alginate hydrogel particles in water (Ma et al. 2019). This system is simple to set up and remains transparent at high particle packing densities.  By loading the transparent soil inoculated with F. oxysporum into a PDMS microfluidic device which allows the fungus to grow in a soil-like environment, we characterized the effect of 1% PGS amendment by direct imaging of the fungal growth in the microchip using confocal microscopy. The results in Figure 46 provided direct proof that PGS in the soil-like environment can indeed inhibit growth of F. oxysporum. The microfluidic chip coupled with hydrogel transparent soil and confocal microscopy offers a unique system for live cell imaging in a soil-like environment, and it can be used to examine additional aspects of PGS biofumigation, such as effect of PGS extract drenching and diffusion effect of BITC in the soil on fungal growth inhibition.   

Research conclusions:

*************

Year-3 concluding remarks:

in vitro studies

Based on fungal growth on ½ Potato Dextrose Agar supplemented with 0.1 or 0.5 mM pure BITC, we were able to demonstrate that pure BITC was able to suppress various fungal pathogens, including F. oxysporum (the causal pathogen of Fusarium wilt) and R. solani (the causal pathogen of Rhizoctonia rot), especially at the higher concentration of 0.5 mM (74.6 mg/l). Further tests with F. oxysporum using PGS in soil-based in vitro tests clearly demonstrated inhibitory effects on mycelial growth at PGS amendment rates above 1%, and showing strong inhibition at or above 3%. Moreover, dissolved state rather than gaseous state BITC released from PGS is most responsible for the anti-fungal activity in soil. These findings are significant, because they provide direct evidence that BITC can be released from PGS and transmitted efficiently through soil (and not just in agar media) to reach the phytopathogen for inactivation. Because dissolved state BITC is likely most responsible for PGS’s anti-fungal activity, soil moisture content plays a role in PGS’s efficacy as a soil biofumigant. It is also better to disperse PGS in the soil rather than applying it as an agglomerate. Similar soil-based tests will be conducted to examine PGS effect on additional soil phytopathogens such as F. solani and R. solani. These simple in-vitro assays can inform approximate PGS dosage for field applications.   

Kai choi greenhouse biofumigation trial

It was speculated PGS biofumigation was less effective in the field due to the presence of fungal survival structures (sclerotia of R. solani, chlamydospores of Fusarium) which are less prone to allelopathy of BITC. The hypothesis was that root exudates could signal fungal pathogens to exit the survival state, making the PGS more effective in suppressing the vulnerable stage of the fungi. Initial results showed no noteworthy improvement of PGS by root leachate against F. oxysporum but did show total suppression of R. solani colonization on kai choi roots when soil was amended at 1% PGS after receiving root leachate 2 days prior to soil amendment. The experiment needs to be repeated to verify this finding. On the other hand, nitrogen content in PGS contributed to some green manure effects on kai choi growth.

Lettuce greenhouse biofumigation trial

Overall Fusarium infection during this trial was low, rendering the test of PGS on lettuce inconclusive.  However, shoot and root biomass were different among treatments despite the lack of disease symptoms and infection rate among treatments. Therefore, it is believed that outside of disease suppression, PGS can provide some green manure benefits at an optimal concentration but can also prove to be phytotoxic and stunt growth if the concentration is too high. This is suspected to be related to nitrogen toxicity from the high nitrogen content of PGS, but further experimentation is necessary to confirm this.  

Additional trials are needed to obtain data from lettuce using soil with higher population densities of F. oxysporum, and to verify the green manure benefits vs phytotoxicity of PGS to lettuce.

Cowpea greenhouse biofumigation trial

Results from this trial was inconclusive due to premature death of plants unrelated to reniform nematodes. In order to elucidate the effectiveness of PGS amendment against reniform nematodes, additional cowpea greenhouse trials are needed. To assess the effectiveness of BITC and PGS biofumigation against nematodes in a controlled lab experiment, simple in vitro assays like those performed for F. oxysporum and R. solani can also be completed.

*********

Final report concluding remarks:

R. solani qPCR

The project team successfully developed and validated a real-time qPCR assay for the detection of R. solani in clay-like soils in Hawaii, but due to overall low detection levels of R. solani from soil samples collected from an infested field with high disease pressure, it was concluded that a different pathogen was the primary cause of infection and disease in the affected kai choi.

F. commune qPCR

Fusarium isolates obtained from diseased kai choi roots were successfully sequenced and identified as Fusarium commune. Thus, we suspected F. commune as the pathogen causing primary disease symptoms in kai choi, and designed a preliminary qPCR assay for the detection of F. commune.

However, in initial qPCR reaction runs for F. commune, amplification curves appeared late, which can be an issue for detection of lower pathogen concentrations in root or soil samples. Prior to using the designed qPCR assay as a quantification tool in other experiments, further optimization and validation of the thermal protocol, the reaction buffer concentrations, and the DNA extraction method is required.

R. solani in vitro assay

Based on our results, PGS biofumigation in an in vitro setting demonstrated effective suppression of R. solani growth, but did not completely stop mycelial growth. Therefore, while PGS has the potential as an alternative treatment against R. solani, more in vitro tests are needed to elucidate the concentrations of BITC, and subsequently PGS, required to completely halt R. solani growth.

Kai choi greenhouse biofumigation +/- leachate – Trial II

During this trial, the time between PGS amendment and seedling transplant was reduced from 7 days to 48 hours. However, due to a general downward trend observed in shoot mass, with both concentrations of PGS used having decreased mass compared to the untreated reps, it was determined that mild phytotoxicity was occurring in the plants. Based on this, we concluded that 48 hours post-amendment was still too short.

However, with an increase in root mass observed in PGS treated reps (especially with 0.5% PGS) compared to untreated reps, it was found that despite initial phytotoxicity, roots in the PGS treated reps were recovering, whereas the untreated reps continued to show low root mass due to disease.

Cowpea greenhouse biofumigation – trial II

Observed aboveground disease symptoms in this trial were not consistent with data collected on nematode counts in the roots, likely due to abiotic stress factors. When analyzing results obtained from nematode count data, 1% PGS treated reps showed low level suppression of vermiform nematode infection as well as a greater suppression of female RN infection on cowpea roots. Due to an overall low level of suppression, it is suspected that a higher concentration of PGS has potential for greater effect in suppressing RN. As the most significant decrease observed in nematodes infesting plant roots was in female RN counts, it is also believed that BITC was disrupting the RN lifecycle in some way, preventing vermiform RN from reaching female reproductive maturity. Therefore, PGS biofumigation has potential as an alternative control method against RN, but more understanding is needed on the mechanism of BITC suppression on RN in order to better design future greenhouse and field experiments for its application and use.

Kai choi greenhouse biofumigation +/- surfactant – Trial I

When assessing overall disease symptoms observed in kai choi plants, the use of a 0.1% Tween80 surfactant solution during PGS amendment showed to be effective in decreasing disease symptoms in reps using 1% PGS.

Due to BITC being a large, hydrophobic molecule, it was initially hypothesized that the use of a detergent or surfactant would aid in moving BITC through the soil. Based on preliminary data collected from this trial, it was determined that the combined use of the surfactant with a 1% PGS amendment has potential as an optimal method of PGS application, however additional trials demonstrating similar results need to be completed to further validate this assumption.

Sorghum & PGS+CE field trial

In this field trial, it was demonstrated that using PGS in a crude extract form increased disease suppression, subsequently increasing overall healthy, marketable kai choi yield.  When compared to the previous kai choi field trial in year two of this project, where PGS only-treated plots had more incidence of disease than sorghum-treated plots, and were comparable to the untreated plots. There was significant improvement in kai choi health when using the PGS+CE application method.

It is believed that the use of 0.1% Tween80 as a surfactant in the CE preparation process, combined with the drenching method used when applying the extract, assists in uniform movement of the BITC molecules through field soil, as similarly concluded from the kai choi greenhouse trial conducted using 0.1% Tween80 when amending using dry PGS. We conclude that PGS performs better as a treatment against disease caused by Fusarium when used with a surfactant, especially in the production of crude extract.