James MacDonald Final Report

Alternatives to Methyl Bromide for Control of Soil-borne Fungi, Bacteria and Weeds in Coastal Ornamental Crops

Final Report - July 2001 (Updated May 2003)

Principal Investigator:
James D. MacDonald
UC Davis Dept. of Plant Pathology
One Shields Ave.
University of California
Davis, CA 95616
Ph: (530) 752-6897, Fax: (530) 752-0121
Email: jdmacdonald@ucdavis.edu

Co-investigators:
Clyde Elmore
Department of Veg. Crops/Weed Sci.
University of California
One Shields Ave.
Davis, CA, 95616
530-752-9978 clelmore@ucdavis.edu

Steve Tjosvold
UCCE Santa Cruz County
1432 Freedom Blvd.
Wastonville, CA 95076-2796
408-763-8013 satjosvold@ucdavis.edu

Collaborators:
Howard Ferris
Department of Nematology
University of California
One Shields Ave.
Davis, CA 95616

Inga Zasada
Department of Nematology
University of California
One Shields Ave.
Davis, CA 95616

Location of project:
UC Davis and Santa Cruz County

Commodities:
Ornamentals (bulb/flower)

Funding:
SAREP $71,558
Other Sources: $36,261

Note: USDA funds allowed expansion of the project beyond what was proposed to or supported by SAREP. Only the work done in support of the SAREP project is reported here.

Table of Contents:

Objectives
Summary
Specific Results
Potential Benefits/Impacts on Agriculture
Dissemination of Findings
Future Workplan
Literature
Figures

Objectives

To determine the efficacy of soil solarization (with organic amendments) for the control of selected root pathogens and weed pests of field-grown ornamentals in coastal climates.

Summary

The coastal regions of California represent a highly valued and productive component of California's ornamental industry, but the productivity of these regions is seriously threatened by the pending loss of methyl bromide. Some of the alternatives that have been proposed for strawberries [e.g., chloropicrin plus 1,3-D (Telone)] probably will not be suitable for ornamental production. This is because production of these specialty crops tends to be dispersed on many small parcels of land near homes and business, and cannot easily accommodate the ever-increasing buffer zone requirements. The goal of this project was to research the efficacy of biofumigation, an effect created by the decomposition of Brassicaceae (e.g., broccoli, cauliflower, mustards) in soil to release isothiocyanates (ITCs). In laboratory experiments, ITCs volatized from macerated plant tissues have been shown to kill fungi (Fusarium oxysproum f.sp. dianthi) and nematodes (Tylenchulus semipenetrans and Meloidogyne javanica). Members of the Brassicaceae differ in the amounts and types of ITC precursors produced, so aspects of the research focused on identifying plant species that produce the most biologically-active decomposition products, and whether there are periods in a plant's development when the products peak.

Field experiments have been carried out simultaneously at Davis and Watsonville to determine the efficacy of biofumigation in natural soils. These experiments have generally involved the burying of fungal propagules (Sclerotium rolfsii, Fusarium oxysporum f.sp. dianthi, Rhizoctonia solani, and Verticillium dahliae), nematodes (Tylenchulus semipenetrans [citrus nematode] and Heterodera schactii [cyst nematode]), and weed seeds (Amaranthus retroflexus [rough pigweed], Portulaca oleraceae [common purslane], Malva parviflora [cheeseweed], Convolvulus arvensis [field bindweed] and Poa annua [annual bluegrass]) at different soil depths to expose them to biofumigation or chemical treatments. At intervals of 2-6 weeks following treatment, the buried organisms were recovered to quantify survival. While results have shown a beneficial effect of biofumigation, the effect is inconsistent and efficacy does not approach that of metam sodium, the chemical treatment used as a control standard. In experiments done at Davis, tarping caused a solarization effect that dominated the treatments, although in some experiments a synergistic effect between solarization and Brassicaceae incorporation was detected. In the cooler coastal regions, a solarization effect is difficult to demonstrate, but in combination with Brassicaceae incorporation, a suppressive effect can sometimes be demonstrated. The inconsistency of biofumigation treatments is likely related to a general lack of knowledge of the factors influencing ITC volatization from tissues in soil.

Specific Results

Experiments during the first year of this project tested a range of organic substrates that were purported to release anti-microbial or weed-inhibiting volatile compounds. Many of the materials (bloodmeal, chicken manure, etc.) proved ineffectual. However, we did detect beneficial (although inconsistent) results from broccoli residues. Broccoli residues contain a compound group called glucosinolates that, upon tissue decomposition, release isothiocyanate (ITC). Thus, research over the past year has emphasized broccoli and other members of the Brassicaceae.

Laboratory, greenhouse and field studies were initiated to determine the effects of plant residues (broccoli, mustard, etc.) on the survival and growth of selected fungal pathogens. This work has been aimed at gaining a better understanding of biofumigation so that we can refine this approach to soil treatment and obtain more consistent and reliable results in the field. Following are the experiments and their results.

A. Laboratory and Greenhouse Experiments:

Analysis of the effects of ITCs from different Brassicaceae species on the growth of Fusarium oxysporum f.sp. dianthi.

To analyze the effect of volatile ITCs on fungal growth, a variety of plant materials were used in laboratory assays. The plants included broccoli, cauliflower, radish, wild mustard, Martegena white mustard, cutleaf brown mustard, Florida broadleaf brown mustard, and wheat (as a non-Brassicaceae control). Plant tissues used in experiments were either as powder (following drying and grinding) or as freshly macerated tissue. In both cases, tissues were mixed into sterilized or natural field soil (Yolo Loam), with the amount of plant tissue added normalized to reflect an application rate of 35 tons fresh weight per acre.

Mycelial disks (7 mm diameter) were cut and removed from 7-10 day old cultures of Fusarium oxysporum f.sp. dianthi. One disk each was placed in the center of petri dishes containing 20 ml of potato dextrose agar (PDA) amended with Streptomycin. Each petri dish then had its lid removed under aseptic conditions, so that the bottom of the dish (containing inoculated PDA) could be inverted and placed as a lid over a glass beaker. The beakers were either empty (controls) or contained soil into which Brassicaceae tissues had been incorporated. Some beakers contained only Brassicaceae tissues (i.e., no soil). After placing an inverted petri dish over the top of a beaker, the gap between the two was sealed with a strip of parafilm. There were three replicates per treatment in any given experiment.

After set-up, the beakers were incubated at 25 C under fluorescent lights to favor growth of the fungus. Fungal growth was assessed at 24-hour intervals by measuring colony diameter as the fungus grew away from the 7-mm-dia inoculation plug. In all treatments where no growth was recorded after 7 days, the original inoculation plug was recovered and transferred to a fresh petri dish containing PDA, where it was incubated to determine if it had been killed or merely inhibited by exposure to volatiles emitted by the Brassicaceae tissues.

The data obtained from these experiments is exemplified in Figure 1. This shows the growth of Fusarium oxysporum f.sp. dianthi when exposed to dried tissues. Each set of treatments (dried tissue only, or tissue mixed with sterile or non-sterile soil) shows the colony diameter relative to non-treated controls (i.e., Fusariu oxysporum f.sp. dianthi incubated over an empty beaker, or beakers containing only sterile or non-sterile soil with no added Brassicaceae tissue). Missing bars indicate zero growth. Hence, the Cutleaf, Florida Broadleaf and Martegena mustards all prevented growth in the "no soil" treatment. In sterile soil, Martegena mustard significantly reduced fungal growth, but not when mixed into field soil. On the other hand, the Cutleaf and Florida Broadleaf mustards prevented growth in all treatments. Indeed, recovery of the inoculum plugs and transfer to fresh media showed that the fungal mycelium had been killed, as no growth developed on agar media following the one week exposure to the volatiles emanating from these tissues.

The greater inhibition of Fusarium growth caused by exposure to mustard tissues, relative to broccoli, cauliflower or radish tissues, was consistent across experiments (data not shown).

Evaluation of glucosinolate profiles in selected Brassicaceae over time.

These experiments were initiated to determine the extent to which different species of Brassicaceae differ in the type and amount of glucosinolates that accumulate over time. Such information is critical to plant selection and timing of harvest to obtain (and hence apply) tissues that will yield suppressive effects when added to soil.

This is being determined by growing members of the Brassicaceae (including broccoli, cauliflower, radish, white mustard, brown mustard and wild mustard) in the greenhouse and destructively sampling during (1) active vegetative growth (the 2-, 4-, and 8-leaf stage), (2) immediately prior to flowering, and (3) after seed set. All plants were randomized within the greenhouse and replicated five times. As tissues are harvested, they are weighed and evaluated for glucosinolates by extraction and HPLC analysis. The HPLC analysis was not completed in time for inclusion in this report.

Screening of commercially available isothiocyanates against fungi and nematodes.

Isothiocyanates are formed naturally by the breakdown of glucosinolates-complex compounds that are formed within plant tissues of species of Brassicaceae. Over 100 types of glucosinolates are formed by plants in the Brassicaceae, which result in a wide array of ITCs. Many different types of ITCs are available commercially, so we have initiated experiments to test a number of these for their ability to suppress or kill fungi and nematodes. Knowledge of particularly active ITCs, and concentrations needed for efficacy, is critical to efforts to select plant species, times of harvest, and methods for handling plant tissue to achieve optimum effects.

Laboratory experiments have been initiated to test commercially-available ITCs against the fungus Fusarium oxysporum f.sp. dianthi and the nematodes Tylenchulus semipenetrans and Meloidogyne javanica. The general procedure used in these experiments is to prepare a dilution series of known ITC concentrations and to apply these known amounts into beakers of soil (for fungi) or sand (for nematodes) in which the fungi and nematodes are established. The toxicity of the compounds is determined by subsequent plating of soil suspensions onto agar media (for Fusarium) or suspending soils samples in water and making direct counts of living nematodes. These experiments are still in progress, but should provide more consistent, reproducible results than plant-tissue-based biofumigations, which rely on uncharacterized tissue degradations.

B. Field Experiments:

Microplot experiments at UC Davis.

Microplots were established by burying 20-gal plastic pots in trenches cut into a fallow field (Fig. 2A). Soil was back-filled around the pots so the container walls were not exposed to or heated by sunlight. Resident Yolo clay loam soil (42% sand, 40% silt, 18% clay, and 1.08 OM) was placed in the bottom of each pot, filling them in distinct increments. When sufficient soil had been added to create a layer 30 cm below grade, sachets containing known amounts of nematodes (Tylenchulus semipenetrans [citrus nematode] and Heterodera schactii [cyst nematode]), fungi (Sclerotium rolfsii, Fusarium oxysporum f.sp. dianthi, Rhizoctonia solani, and Verticillium dahliae), and weed seeds (Amaranthus retroflexus [rough pigweed], Portulaca oleraceae [common purslane], Malva parviflora [cheeseweed], Convolvulus arvensis [field bindweed] and Poa annua [annual bluegrass]) were added. More soil (now, also containing chopped broccoli) was added until the soil level was 15 cm below grade, and another series of sachets were added. A final series of sachets were added when the soil/broccoli mix formed a layer 5 cm below grade. This last layer was covered with 5 cm of broccoli-amended soil so that the final level in the pot was equal to surrounding grade (Fig. 2B). Most pots then were covered with a piece of 1.1 mil polyethylene tarp that was sealed in place with a band around the upper rim of the pot-other pots served as untarped controls. The amount of broccoli mixed into the soil that was used to fill the post was adjusted to yield treatment levels of 25, 35 and 50 ton/Acre, fresh weight. Water was added to the soil as it was layered into the pots so that it approximated field capacity. In addition to broccoli-amended soil, there were tarped and untarped controls to which no broccoli was added and a standard treatment of 75 gal/A of metam sodium that was injected into pots using 1/3 doses at each fill layer (30, 15 and 5 cm).

Each treatment was replicated four times in a completely randomized design. Sachets containing the target organisms were removed at 2, 4 or 6 weeks after treatment. Upon removal of the sachets from the field, they were placed in coolers and transported to the laboratory. Populations of living citrus nematode remaining in the sachets were enumerated after Baermann funnel extraction for 72 hours. The samples of cyst nematode were incubated for twenty-one days on a Baermann funnel in water to stimulate egg hatch. Recovered nematodes were counted using a dissecting microscope. Soil samples containing fungal propagules were dried, weighed, suspended in dilute (0.5%) water agar, and aliquots spread onto agar media. Resulting colonies were counted to determine the numbers of surviving propagules per gram of soil. Recovered weed seeds were placed into germination trays to quantify viability (mean weed germination per treatment by species).

Results: Assays of fungal survival are just being completed and are not yet ready for inclusion in this report, so only results of nematode and weed survival are presented.

The juvenile stage of the citrus nematode is free-living and hence, was in direct contact with the soil treatments. In this experiment, we could not detect any surviving nematodes at the 5 cm depth, at any recovery interval (2, 4 or 6 wk) (Fig. 3). At the 15 cm depth, citrus nematode populations were highest at the 2-week sampling period, in the uncovered control and uncovered broccoli treatments. There were lower populations in the covered broccoli treatments, but no significant difference among these treatments. At the 4- and 6-week sampling periods, small numbers of nematodes remained alive in the uncovered control, but all other samples from 15-cm had no surviving nematodes. At the 30-cm depth, metam sodium was the only treatment that provided significant and consistent reductions in citrus nematode populations. Metam sodium provided complete control at all sample depths and times.

Survival of weeds was reduced in all tarp-covered treatments compared to untarped treatments (Fig. 4). Depending on weed species, the loss of viability in plastic tarp-covered treatments ranged from 38 to 86% of the uncovered control. There seemed to be no difference between whether the weed species was annual or perennial, and this result was attributed to solarization alone. In general more weeds germinated after only 2 weeks of treatment compared to 4 or 6 weeks of treatment, with little difference between the latter two. Also there were fewer surviving weed seeds at 5 cm of depth than at deeper depths (data not shown).

As seen in the nematode sampling, the metam sodium treatment was the most effective against weeds. It reduced the germination of annual bluegrass, rough pigweed, and purslane by over 95% compared to the uncovered, non-treated control.

Broccoli incorporated into the soil in containers whether at 5 or 15 cm reduced the viability of all weeds compared to the uncovered control. However, the degree of control was modest. With the exception of purslane (86%) and field bindweed (90%), the incorporation of broccoli residues did not achieve the desired control level of 85% with other weeds. The best control of field bindweed (90%) was with broccoli at 35 T/A incorporated to 15 cm and solarized for 6 weeks. This treatment provided better control than metam at 75 g/A (data not shown).

Field trial at UC Davis.

In this experiment, plots were established on 60-inch beds. After beds were shaped, they were pre-irrigated with sprinklers to wet the soil to field capacity. Unchopped broccoli leaves and stems then were weighed and placed on the bed tops and were incorporated using either a power tiller (to achieve a depth of 15 cm) or a hand spade (to achieve a depth of 5 cm). Sachets containing inoculum of Fusarium oxysporum f.sp. dianthi were then buried at 5, 15 and 30 cm, and weed seeds were broadcast on the soil before a second irrigation. Polyethylene tarps (Chemagro 1.1 mil thickness) were placed over the plots and the edges sealed with soil. Temperatures were recorded at 5, 15 and 30 cm depth in tarped and untarped plots. After six weeks, the tarps were removed and weed control measured by counting weed numbers (by species) in two 0.25m² squares near the center of each plot. Several weeks after tarp removal, the time required to hoe weeds from the plots was recorded as a measure of the weediness of each plot.

To test for any residual phytotoxic effects, transplant seedlings of snapdragon (Antirrhinum sp.) and Godetia (Clarkia amoena) were planted into the beds two weeks after plastic removal (eight weeks after original incorporation of broccoli residues). Plant vigor was evaluated visually two months after planting by counting the number of surviving plants and measuring the height of plants. Evaluations were on a scale of 0 (dead plants) to 10 (maximum growth).

Results: Soil temperatures at 5 cm depth reached 61 C in tarped plots (Fig. 5), and generally ran about 10 C than temperatures in nontarped plots. The incorporation of broccoli tissues did not appear to alter soil temperatures relative to non-amended plots.

Incorporation of broccoli tissues into soil without tarping was ineffective in terms of Fusarium control (Fig. 6). All tarp-covered treatments significantly reduced the numbers of viable Fusarium spores at depths of 5 and 15 cm. However, at the 15 cm depth, spore mortality was greatest in the broccoli plus tarping treatment, indicating a synergistic effect. At a depth of 30 cm, metam sodium was the only treatment that provided effective kill of Fusarium spores.

All treatments that included 6 weeks of solarization gave better weed control than any of the untarped treatments (Fig. 7). Indeed, tarping alone was so effective in these experiments, that a synergistic effect between tarping and broccoli residues could not be detected for most weed species. The only comparisons that revealed a synergistic interaction between tarping and broccoli residues were those involving control of filaree and burclover (Fig. 7). For these weeds, the incorporation of broccoli tissues significantly increased the beneficial effects of solarization (Fig. 7). The greater weed survival in untarped versus tarped plots was reflected in the greater amount of time It took to weed the untarped plots relative to any of the tarp-covered treatments (data not shown).

The snapdragon and godetia seedlings that were planted back into the plot two weeks after removal of the plastic showed that there was a residual affect from the combined broccoli and solarization treatment. The vigor of both plant species was reduced significantly when broccoli and solarization was combined, compared to broccoli alone (Fig. 8), indicating that the plastic tarp had trapped some sort of inhibitory material in the soil. This is contrary to the frequent observation of plant stimulation in solarized soil relative to nonsolarized soil. This result indicates that further research is needed to evaluate plant-back requirements if broccoli and solarization are used together.

Field Trial in Watsonville.

An additional experiment to evaluate soil solarization and soil additives for biofumigation was established in Santa Cruz County. The site was prepared by cultivating to prepare a 40-inch-wide bed top. All plots were sprinkler irrigated prior to treatment application. The following treatments were tested: chicken manure (8 T/A) tarped and untarped; metham (100 gal/A) tarped for 2 weeks and 6 weeks; broccoli tarped at 35, 50 and 75 T/A; broccoli untarped at 35 T/A; and tarped and untarped controls. A Stowaway micrologger with an external thermistor was used to measure soil temperature at 5, 15 and 35 cm depths.

After the application of treatments, sachets containing soil borne pests were buried at 5, 15 and 30 cm. The following organisms were evaluated: weeds [Portulaca oleraceae, Amaranthus retroflexus, Poa annua, Malva parviflora, and Convolvulus arvensis]; fungi [Fusarium oxysporum f.sp. dianthi and Verticillium dahliae]; and nematodes [Tylenchulus semipenetrans]. Plastic tarps were laid over the plots after placement of the test organisms, and the edges secured with soil. Except for the metam sodium 2-week treatment, all treatments were undisturbed for 6 weeks. Sachets were removed at the end of the experiment and were processed similar to the methodology described for the Davis biofumigation experiment. In addition to counts of the aforementioned weeds, calla growth from rhizomes in the soil was measured by counting the number of shoots emerging through the treatments. Survival of old calla rhizomes represents an important contaminant (i.e., weed and pathogen reservoir) in commercial plantings.

Results: The soil temperatures achieved at this coastal location were significantly lower than those achieved at Davis (Fig. 5 and 9). In the case of fungi, the results were highly variable. The only treatment that yielded a significant reduction in the numbers of surviving propagules was metam sodium plus tarping (Fig. 10). Likewise, metam sodium plus tarping controlled all weeds at this location, including the almost complete kill of calla lily rhizomes. Resident prostrate pigweed (A. blitiodes) was reduced with 50 or 75 T/A of broccoli plus solarization compared to solarization alone or the untreated control (data not shown). Very little control was achieved, however, in the untarped broccoli treatment (data not shown). Composted chicken manure at 8 T/A (either tarped or untarped) did not control weeds. The only treatment that gave acceptable weed control at this coastal site was the 6-week-long metam sodium plus tarp treatment (Fig. 11A), although broccoli at 35 T/A plus tarping greatly reduced calla emergence (Fig. 11B). For nematodes, the incorporation and tarping of broccoli at any rate reduced citrus nematode numbers compared to solarization alone at 15 cm. At 30 cm, 75 T/A of tarped broccoli achieved 70% reduction in citrus nematode numbers compared to the untarped control while 35 T/A and 50 T/A tarped broccoli had 42% and 68% reduction, respectively (data not shown). Soil solarization alone at 30 cm did not reduce citrus nematode populations. Both metam sodium treatments suppressed the citrus nematode 100% at all depths.

Evaluation of a range of Brassicaceae in the field.

These experiments were an outgrowth of laboratory experiments (described above) that showed species of Brassicacae differed with respect to antifungal activity. A field trial was established at Davis in which different Brassicacae were grown on beds, and subsequently incorporated into soil as a green manure. The plants selected for use in this experiment included: broccoli, cauliflower, white mustard, brown mustard, radish and wild mustard. Wheat plants served as a non-Brassicacae control, and bare soil served as a plant tissue control. A randomized split plot design was used with the main plots being Brassicacae species and split plots to be tarped with plastic sheeting or left untarped. All treatments were replicated four times. All plots that were to have plant tissues incorporated were seeded during the fall with the appropriate plant species by broadcasting seed over the bed surface. As the plants matured (prior to seed set for the earliest-maturing species) the plant tissues were incorporated with a power tiller to a depth of 15 cm. At this time, sachets containing propagules of Fusarium or the nematode Tylenchulus semipenetrans were buried at depths of 5, 15 and 30 cm. After installation of the sachets, plastic sheeting was laid over tarped treatments and the edges of the sheets secured to the ground with soil. Six weeks after installation, the sachets were recovered and analyzed for survival of fungi and nematodes. Temperatures were recorded during the duration of this experiment at 5, 15 and 30 cm. HPLC and fungal analysis are being conducted at the time of writing.

Results: Data from this experiment was compromised because many of the sachets buried at 5 cm were dug up and damaged by vertebrate pests, so no meaningful data could be obtained from the 5 cm treatment (Fig 12). In comparing the results from the 15 cm sachets, there was a general reduction in propagule survival associated with tarping (Fig. 12 A &B), which is the solarization effect. However, propagule mortality was greatest (i.e., fewest surviving propagules) in treatments that combined solarization with the cutlass and wild mustards (Fig. 12B).

Potential Benefits/Impacts on Agriculture

These experiments are continuing. Over the current (summer, 2001) growing season, we are seeking to learn more about the efficacy of different mustards, and the effects of harvesting tissues at different stages of plant maturity. While biofumigation may have some potential for the control of fungi, nematodes and weeds, our experiments so far have shown that the best alternative to methyl bromide appears to be metam sodium.

Dissemination of Findings

The results of this research will be disseminated through presentations at the annual Methyl Bromide Alternatives conference, the California Ornamental Research Foundation (CORF) tours and newsletters, and the California Cut Flower Commission.

Future Workplan

Work has continued on methyl bromide alternatives. We have continued to study Brassicaceae for their ability to reliably produce ITCs in soil. We also have done experiments, under a USDA grant, to study chemical alternatives. We have found iodomethane plus choloropicrin and metam plus telone C35 to be among the most consistently effective treatments in a variety of filed locations.

A manuscript covering these biofumigation experiments will be submitted soon to Plant Health Progress.

Literature

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Brown, P. D., and Morra, M. J. 1997. Control of soil-borne plant pests using glucosinolate-containing plants. Advances in Agronomy 61:192-204.
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