The project is entitled “Cover crop 5-in-1 Approach” because we are targeting on selecting cover crops that can possess five mechanisms to suppress plant-parasitic nematodes. Root-knot nematode (Meloidogyne spp.) alone could contribute up to 33% yield loss on cucurbit crops. Our goal is to develop an economically viable cover crop based nematode management tool that can protect cucurbit crops from nematode pests while enhancing soil health through the use of (Brassica spp.) and oil radish (Raphanus sativus) cover crops. Specific objectives of this project are to 1) develop a heat-unit based mustard and oil radish cover crop termination method for efficient trap cropping against root-knot nematodes; 2) determine best termination method for mustard and oil radish for maximum biofumigation effect against plant-parasitic nematodes; and 3) evaluate the effects of mustard and oil radish “Cover Crop 5-in-1 Approach” on soil health. Ph.D. candidate, Waisen, will conduct four field trials at two commercial vegetable organic farms (Bear Claw Farm and 3 Acre in Wailua Farm) to examine best cover crop termination time and method to maximize trap cropping effect against root-knot nematodes, biofumigation effect against root-knot and reniform (Rotylenchulus reniformis) nematodes, while enhancing green manure effect to promote soil health to increase plant tolerance against nematode damage. PI Waisen will fully engage in extension and outreach activities of this project and present research outcomes to farmers during field days to deliver a “Cover crop 5-in-1 Approach” for nematode management. This project hopes to promote good stewardship for farmers to take care of their farm through ecological based low input cover cropping system. The success of this project not only would provide incentive for Hawaii vegetable farmers to practice sustainable and organic farming but would also provide a model system for small-and mid-sized farms in the Pacific to increase their farm profitability.
Objective 1. Develop a heat-unit based mustard and oil radish cover crop termination method for efficient trap cropping effect against root-knot nematodes.
Greenhouse experiment: Two greenhouse experiments will be conducted to determine heat-units required for root-knot nematode (M incognita) to reach an egg-laying female stage on ‘Caliente 199’ mustard and ‘Sodbuster’ oil radish compared to a nematode susceptible crop, ‘Orange Pixie’ tomato (Solanum lycopersicum). All crops will be seeded at 2 seeds per 1-L pot filled with sterile sand-soil mix at 1: 1 ratio (v/v). Experimental design will be a 3 x 11 factorial and treatments arranged in a completely randomized design (CRD) with 4 replications. The 3 treatments are mustard (MS), oil radish (OR) and tomato (TO) each with 11 destructive sampling time points at 4 days intervals starting from O to 40 days. Fourteen-day old seedlings of each test crop will be inoculated with 200 J2 nematodes per plant. Nematode inoculum will be extracted from M incognita cultured on coleus plant (Coleus sp.) maintained in the greenhouse using 0.6% sodium hypochlorite (NaOCI) (Hussey and Barker, 1973), eggs will be hatched, and the second stage infective juveniles ( J2) will be collected at 14 days after extraction to prepare the inoculum. Seven days prior to inoculation, seedlings will be thinned to one plant per pot. A WatchDog Data Logger (Spectrum® Technologies Inc., Aurora, IL) will be buried at I 0-cm deep in the rhizospheres of the potted mustard, oil radish and tomato plants to record soil temperatures at hourly interval for heat unit calculations. We will use the base temperature of M incognita (9.8°C) to calculate heat units (Davila-Negron and Dickson, 2013). Heat units will be calculated means of the maximum and minimum temperature minus a base temperature for a nematode species to develop to egg-laying female (Davila-Negron and Dickson, 2013). Roots will be stained using Acid Fucshin method (Byrd et al., 1983) to check for egg lay-laying female stage. This experiment will be repeated once in the greenhouse condition. Counts of egg-laying female will be subjected to Proc Univariate test to check for normality and analyzed in two-way analysis of variance (ANOVA) in SAS Version 9.4 (SAS Institute Inc., Cary, NC). Means will be separated using Waller-Duncan k-ratio (k=l 00) t-test and only true means will be presented.
Field experiment: Two field experiments will be conducted to determine the best termination time of mustard and oil radish for maximum nematode trapping and biofumigation effects against plant-parasitic nematodes. One field trial will be conducted at Bear Claw Farm in Waimanalo. Cover crops will be direct seeded at 11 kg/ha into 16 plots of I x 7 m2 at four different times, so that the cover crops will be terminated at 14, 28, 42, or 56 days after planting. 6
A bare ground treatment will also be included. Each treatment will be replicated 4 times and arranged in a randomized complete block design (RCBD). Temperature WatchDog Data Loggers will be buried 10-cm deep in soil during the cover crop growing period to monitor heat unit accumulation. Four plants will be randomly selected per plot and stained for egg-laying females (Byrd et al., 1983). Prior to termination of the cover crops, biomass of cover crops will be estimated using a 0.9-m2 quadrant. The cover crops in all treatments will be terminated at once by using a line trimmer to macerate the plant tissues, biofumigation by soil incorporating to 10-cm deep using a hand-held rototiller, followed by tarping with black plastic. Seven days after cover crop termination, 3-week old cucumber seedlings will be transplanted at 6 plants per plot with 1-m spacing between plants. Cucumber growth data including canopy width and chlorophyll content will be measured bi-monthly. Marketable and unmarketable yield of cucumber will be measured weekly. At termination of cucumber crop, severity of root-knot nematode galling on cucumber roots will be rated based on 1-10 scale (Netcher and Sikora, 1990). Soil will be sampled 6 cores per plot prior to cover crop termination and at monthly intervals for 3 subsequent months during the cucumber crop. Plant-parasitic nematodes from 250 cm3 of soil will be extracted by elutriation (Byrd et al., 1976) followed by density-dependent centrifugal sugar flotation (Jenkins, 1964; Barker, 1985). The experiment will be repeated at 3 Acres in Wailua Farm. Counts of egg-laying females, soil populations of root-knot and reniform nematodes, cucumber growth and yield data will be normalized where necessary and subjected to one-way ANOVA in SAS Version 9.4 (SAS Institute Inc., Cary, NC). Means will be separated using Waller-Duncan k-ratio (k=l00) t-test. Only the true means will be presented.
Objective 2. Determine best termination method for mustard and oil radish cover crops for maximum biofumigation effect against plant-parasitic nematodes
Two field experiments will be conducted to compare various termination methods of ‘Caliente 199’ mustard and ‘Sodbuster’ oil radish to maximize biofumigation effects against root-knot and reniform nematodes. The cover crop termination methods to be tested include cover cropping with 1) mustard (MS) or 2) oil radish (OR) followed by tilling; macerating 3) mustard (MSM) or 4) oil radish (ORM) by line trimming followed by tilling; macerating 5) mustard (MSBP) or 6) oil radish (ORBP) followed by tilling and tarp with black plastic; and macerating 7) mustard (MS Sol) or 8) oil radish (ORSol) followed by tilling and tarp with solarization mulch ( clear plastic). A bare fallow tilled treatment will be included as the 9) bare ground (BG) control. The experiment will be arranged in the RCBD and replicated 4 times. The cover crops will be seeded at 11 kg/ha rate on 1 x 7 m2 plots and terminated based on the heat-units determined in Objective 1. WatchDog temperature probes will be buried 10-cm deep in the soil during the cover crop growing period. Seven days after the cover crops termination, 3-weeks old cucumber seedlings will be transplanted, plant growth and yield will be monitored in the similar manner as described in Objective 1. Soil will be sampled prior to cover crop termination, and at monthly intervals during the cucumber growth and subjected to nematode assay similar to the field experiment in Objective 1. To assay for biofumigation effect, myrosinase enzyme activity will be estimated through glucose analysis (Al-Turki and Dick, 2003). Glucose is a by-product of the hydrolysis of glucosinolate, and its concentration had been shown to be proportional to isothiocynate production (AI-Turki and Dick, 2003). Soil will be sampled prior to cover crop termination and at 1, 24, 48, 72, 96, and 168 hours (=l week) after cover crop termination. Soil will be sampled from 6 cores per plot in the similar way as described earlier, composited, homogenized and 1 g 7 of soil subsample will be collected into a vial, brought back to the lab for glucose analysis. Toluene (Sigma-Aldrich, St. Luis, MO) will be added to the soil to kill microbes to avoid glucose metabolism. Glucose (HK) Assay Kit (Sigma-Aldrich, St. Luis, MO) will be used to extract glucose according to the manufacturer’s instructions. Glucose content will be quantified spectrophotometrically using Gen5 software (BioTek Instruments Inc., Winooski, VT) at 340 nm wavelength. The glucose concentration in the soil will be estimated by a regression curve between absorbance readings and standard glucose concentrations. The experiment will be repeated again at Poamoho (3 Acres in Waialua Farm).
Objective 3. Evaluate the effects of mustard and oil radish “Cover Crop 5-in-1 Approach” on soil health using nematodes as bioindicators.
Nematodes extracted from soil samples collected from both field trials described in Objective 2 will be subjected to nematode community analysis as soil health bioindicators. This is based on the fact that nematodes are ubiquitous and play a major role in soil nutrient cycling (Ferris et al., 2001). All nematodes extracted will be identified to genus level under an inverted microscope and will be categorized into one of the five trophic groups: bacterivores, fungivores, herbivores, omnivores, and predators as described by Yeates et al. (1993). Total abundance, percentage of each nematode trophic group, nematode richness in the nematode community will be calculated. Nematode richness will be the total number of different taxa recorded per sample. Simpson’s index of dominance (Simpson, 1949) will be calculated using 11, = l: (pi) 2, where pi is the proportion of each genera present. Simpson’s index of diversity will be calculated as 1/11, (reciprocal of domiance ). The fungivore to fungivore and bacterivore ratio (F IF +B) will be calculated to characterize decomposition and mineralization pathways (Freckman and Ettema, 1993). Total maturity index (MI) will be calculated as l: (Pi Ci), where Ci is the c-p rating of taxon i according to the I to 5 c-p scale (Bongers and Bongers, 1998; Yeates and Brid, 1994) to describe the stability of the nematode fauna. To assess soil nutrient enrichment, nematode fauna will be analyzed using enrichment index (El) which is a weight abundance of opportunistic bacteria feeding nematodes; to measure the complexity of the soil food web, structure index (SI) will be calculated based on weight abundance of the nematodes higher in the soil food web hierarchy including omnivorous and predatory nematodes as described by Ferris et al. (2001). Besides using nematode as soil health indicator, toward the end of the cucumber crop, we will plate cucumber roots from 5 subsamples collected from each treatment plot. Roots will be placed on quarter strength com meal agar for 3 weeks to observe for potential recovery of nematode egg parasites such as Paecilomyces or Trichoderma. The purpose is to examine if amending soil with mustard or oil radish residues also increase the abundance of these nematode antagonists. Statistical analysis: All data collected in Objectives I, 2 and 3 including glucose concentrations, counts of plant-parasitic nematodes, nematode abundance, indices, root-gall index, temperature data, cucumber growth and yield data will be normalized wherever necessary and subjected to one-way analysis (ANOVA) in SAS Version 9.4 (SAS Institute Inc., Cary, NC). Means will be separated using Waller-Duncan k-ratio (k=IOO t-test.
Two greenhouse experiments were conducted to determine heat units required for root-knot nematode (Meloidogyne incognita) to reach an egg-laying female stage on ‘Caliente 199’ brown mustard (Brassica juncea) and ‘Sodbuster’ oil radish (Raphanus sativus), and compared to a nematode susceptible crop, ‘Orange Pixie’ tomato (Solanum lycopersicum). All crops were seeded at 2 seeds per 250-ml pot filled with sterile sand-soil mix at 1:1 ratio (v/v). Experimental design was a completely randomized design (CRD) with 6 replications. An aliquot of100 second stage juveniles (J2) of M. incognita per ml was inoculated in each pot. Destructive samples of 6 plants per crop were collected starting at 9 days post-inoculation and at 3 days interval until egg-laying females were first detected. Roots were stained using Acid Fucshin method to check for egg lay-laying female stage (Byrd et al., 1983). A WatchDog Data Logger (Spectrum® Technologies Inc., Aurora, IL) was buried at 10-cm depth in the rhizospheres of each treatment to record soil temperatures at hourly interval. A base temperature of 9.8°C for M. incognita was used to calculate heat units (Dávila-Negrón and Dickson, 2013) in degree days (DD) according to the equation below. The experiment was repeated once.
DD = ∑(T-Tb)/24 hrs (where T = hourly soil temperature at 10 cm depth; Tb = base temperature of M. incognita.
A field experiment was conducted at Poamoho Experiment Station where ‘Sodbuster’ oil radish (OR) or ‘Caliente 199’ mustard (MS) were grown for 4 weeks and terminated using 3 different methods: 1) cut off cover crop shoots with a sickle followed by covering the residues with a woven weed mat under no-till conditions (ORNT or MSNT); 2) macerating cover crop shoot tissues using a line trimmer followed by tilling into the soil (ORMT or MSMT); or 3) macerated, tilled and covered the soil with black plastic (ORBP, MSBP). A bare ground (BG) treatment was included as a control. The 7 treatments were arranged in a randomized complete block design with 4 replication plots of 1.2 × 3.0 m2 in size. The cover crops were seeded at 11 kg/ha and watered as needed. WatchDog Temperature Data Loggers were buried 10-cm deep in each treatment and temperature was recorded hourly throughout the cover crop season and the subsequent zucchini (Cucumis pepo) crop. Four-week old ‘Felix’ zucchini seedlings were transplanted at 1-m apart. Plants were drip irrigated according to commercial practice. Chlorophyll content of zucchini was measured using a chlorophyll meter (Konica Minolta Sensing Inc., Osaka, Japan) and canopy widths of 4 plants per plot were measured at 3 and 6 weeks after transplanting. Zucchini fruits were harvested at weekly interval.
Soil was sampled right before cover crop termination, one week after, and monthly for 2 months after zucchini planting. Soil from each treatment plot was sampled by taking 4 soil cores of 10-cm deep and composited into one bag. Nematodes were extracted using elutriation followed by centrifugal sugar floatation methods (Jenkins, 1964; Byrd et al., 1976; Barker, 1985).
Glucose analysis to estimate biofumigation activities:
To assay for biofumigation effect, myrosinase enzyme activity was estimated through glucose analysis (Al-Turki and Dick, 2003) from soil sampled at 1, 24, 48, 72, 96, and 168 hours (=1 week) after cover crop termination as described earlier. 1 g of soil subsample was collected into a vial, brought back to the lab and added with toluene (Sigma-Aldrich, St. Luis, MO) to kill off microbes that could metabolize glucose in soil. Glucose (HK) Assay Kit (Sigma-Aldrich, St. Luis, MO) was used to extract glucose and glucose extracted from each soil sample was quantified using 340 nm spectrum of the spectrophotometer (Gen5 software, BioTek Instruments Inc., Winooski, VT). The glucose concentration in the soil was estimated by a regression curve between absorbance readings and standard glucose concentrations.
All nematodes extracted from Objective 2 were identified to genus (family in some cases) using an inverted microscope (Leica Microsystems Company, Wetzlar, Germany). Nematode data were subjected to nematode community analysis (Yeates et al., 1993; Simpson, 1949; Freckman and Ettema, 1993; Yeates and Bird;1994; Bongers and Bongers, 1998; Ferris et al., 2001) as soil health indicators.
Heat units to reach egg-laying female = 283 degree days (equivalent to 3.5 weeks during summer time in Hawaii) for both oil radish and brown mustard. We recommended the farmers to grow the cover crops for 5 weeks to achieve 1.3 tons/acre of biomass, allowing the root-knot nematodes to hatch out from their eggs as they will be most vulnerable to the toxicity from biofumigation (note that egg stage is the survival stage of root-knot nematodes that is not prone to biofumigation).
- Black plastic of mustard (MSBP) resulted in lowest number of root-knot and reniform nematodes among all treatments.
- Black plastic of OR (ORBP) only reduced numbers of reniform nematodes but not root-knot nematodes compared to other termination methods.
- Glucose analysis from OR was complicated by break down of OR.
- Mustard cover with black plastic (MSBP) accumulated higher isothiocynate than the control (BG) and no-till (MSNT).
Thus, brown mustard terminated by macerated, tilled and covered with black plastic (MSBP) was most effective in suppressing both root-knot and reniform nematodes.
- MSBP did not impact bacterivore and fungivores negatively compared to BG.
- ORBP and MST increased bacterivores compared to BG (indicate more bacterial decomposition).
- No significant effects of MS or OR on omnivorous nematodes regardless of termination methods.
- All OR and MS treatments, especially MSBP, increased zucchini canopy width.
- However, zucchini yield was not different among treatments due to heavy pickleworm infestation.
Thus, biofumigation from MSBP did not compromise soil health and in fact it increased zucchini growth.
Educational & Outreach Activities
There were two field days and a workshop conducted during the course of this project period. Field demonstrations were conducted at the experiment site so that farmers were able to see biofumigation effects at first hand. In addition, a presentation on biofumigation for nematode management was shared with farmers during a “WSARE Hot Shot IPM” program at Kahului, Maui to reach out to wider audience away from Oahu.
- Wang, K.-H., Waisen, P. and J. Uyeda. 2018. Sustainable Pest Management Strategies: Biofumigation and nematicide fertigation. Carrot Field Day. Poamoho Experiment Station, Waialua, HI. February 28, 2018 (attendance: 25). https://spark.adobe.com/page/S2pUEUQvhcavo/
- Waisen, P. and Wang, K.-H. 2018. Best termination methods of mustard and oil radish to achieve biofumigation for nematode management. Maui WSARE Hot Shot IPM Workshop. May 23, 2018. Kahului, Maui. https://www.flickr.com/photos/150583970@N07/with/42316804051/
- Waisen, P. and Wang, K.-H. Field demonstration of zucchini crops grown in mustard and oil radish biofumigated plots. Insectary plant for edible crops in Hawaii. Poamoho Experiment Station, Waialua, HI. October 28, 2017 (attendance: 31; https://youtu.be/lNW6BmoQs).
Extension publications/Fact sheets
A pamphlet detailing protocols on effective biofumigation against plant-parasitic nematodes can be accessible online at:
- Waisen, P. and Wang, K.-H. 2018. Trap cropping and biofumigation for plant-parasitic nematode management. HānaiʻAi Newsletter March, April, May 2018. https://gms.ctahr.hawaii.edu/gs/handler/getmedia.ashx?moid=29943&dt=3&g=12
- Waisen, P., K.-H. Wang, Z. Cheng, and B.S. Sipes. 2018. Managing plant-parasitic nematodes and soil health through ecological based biofumigation using brown mustard and oil radish. Society of Nematologists 57th annual meeting, Albuquerque, NM. July 21-25, 2018.
- Waisen, P. and K.-H. Wang. 2018. Best termination methods of mustard (Brassica juncea) and oil radish (Raphanus sativus) cover crops for nematode management. 30th CTAHR Student Research Symposium, Honolulu, HI. April 7, 2018 (Abstract #124).
- Waisen, P., Wang, K.-H., Cheng, Z., and Sipes, B. S. 2017. Effective Termination Methods of Brassica Cover Crops for Suppression of Plant-Parasitic Nematodes While Enhancing Soil Health. Presented at 56th annual conference for Society of Nematologists in Williamsburg, VA. August 12-17, 2017.
Outreach in progress
The PIs are currently synthesizing field trials data into videos and power point presentations to be uploaded into the PI website at https://cms.ctahr.hawaii.edu/wangkh/Research-and-Extension/Cover-Crops.