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V. Impact of Recommended Fumigants on Soil Microbial Communities and Agricultural Practices

V. Impact of Recommended Fumigants on Soil Microbial Communities and Agricultural Practices

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lower than the other treatments even after 12 weeks of incubation. Dungan et al.

(2003a) showed the same effect with PBr and 1,3-D during a 12-week

experiment where the diversity index was very low. Likewise, Schutter and

Ajwa (personal communication) observed a reduction in nitrification potential

in fumigated soils, with recovery in CP-fumigated soils sooner than MeBr, 1,3-D,

or PBr. While these examples have focused on soil microbial populations,

preliminary work with strawberry rhizosphere colonizers in fumigated

compared to native soils suggests that there may be differences in deleterious

and beneficial rhizosphere colonizers following soil fumigation (Martin, 2003).

A basic understanding of the soil and rhizosphere microbiology can simplify

the identification of specific microorganisms that can be used directly for

disease management, enhancement of plant growth or altered crop management

practices to enhance their populations. One excellent example is the

identification of specific bacterial rhizosphere colonizers that are capable of

protecting apple roots from pathogens associated with apple replant disease

and enhancing their soil populations by cropping specific cultivars of wheat

(Mazzola et al., 2002).

Organic amendments may also be useful for management of diseases

commonly controlled by soil fumigation, although questions of cost effectiveness

and field scale practicality may need to be addressed before they are

commercially feasible (Martin, 2003). The benefits of adding organic amendments to soils are well documented. They can improve soil nutrition,

physicochemical conditions, and crop viability (Hungalle et al., 1986;

O’Hallorans et al., 1993). They have been found to be effective in reducing

potentially harmful fumigant emissions (Gan et al., 1998b) and controlling soilborne pathogens by stimulating antagonistic organisms (Akhtar and Malik, 2000)

or by producing toxic volatile compounds (Gamliel and Stapleton, 1997).

Applications of organic amendments have also been shown to increase the soil

microbial biomass and stimulate microbial activity (Perucci, 1990; Bandick and

Dick, 1999; Peacock et al., 2001). In a greenhouse study to evaluate methods for

management of apple replant disorder, Mazzola et al. (2002) observed that soil

amendment with Brassica napus seed meal reduced the incidence of apple root

infection by Rhizoctonia spp. and the lesion nematode Pratylenchus penetrans.

However, in some cases it was found to increase soil populations of Pythium spp.

and the incidence of disease they caused.

Application of fumigants has beneficial effects when the best management

practice is adopted. Soil fumigation generally increases root health, growth,

and fruit yields in strawberries even when major pathogens are not present in

soil (Wilhelm and Paulus, 1980; Yuen et al., 1988; Duniway, 2002).

Temporary inhibition of nitrification and an increase in ammonia-N in the

soil may be partly responsible for the increase in plant growth (Porter et al.,

1999). Soil fumigation has been shown to reduce the incidence of Pythium,

Cylindrocarpon and binucleate Rhizoctonia spp. damaging to strawberry roots



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(Wilhelm and Paulus, 1980; Martin, 1998, 1999). The reduction of pathogens

is the major benefit of fumigation. The process does not result in soil

sterilization but in some cases it results in changes in the microbial community structure. Pseudomonas sp. has been shown to survive in fumigated

soil and recolonize strawberry rhizospheres rapidly and in high numbers after

fumigation (Xiao and Duniway, 1998). The increase in numbers of different

Pseudomonas sp. in the strawberry rhizospheres after fumigation correlated to

a significant increase in the growth of the strawberry plants in field and

greenhouse experiments (Xiao and Duniway, 1998).



A. METHYL BROMIDE

MeBr is a versatile, highly effective and relatively cheap fumigant used for

pre-planting fumigation. It is effective against a wide spectrum of plant pathogens

and pests, including fungi, nematodes, insects, mites, rodents, weeds, and some

bacteria. However, the Br2 residue is left in soil and plants after fumigation.

Bromide residues produced by MeBr fumigations have importance because

excessive uptake of plant materials containing Br2 is considered harmful to

human beings. And in addition, some plants (mainly carnations) are sensitive to

high Br2 levels in the soil (Yates et al., 2003). The concern for Br2 toxicity

from edible plants grown in fumigated fields was the main reason for the

suspension of MeBr in Germany (Anonymous, 1980). The generally accepted

mechanism of MeBr biological activity is through a bimolecular, nucleophilic

displacement (SN2) reaction with functional groups, such as NH2 and SH, in

various amino acids and peptides of the target organisms (Price, 1985). In soil

chemical hydrolysis and methylation through SN2, nucleophilic substitution

with water nucleophilic sites on soil organic matter produces the reactive product

(Gan et al., 1994).

Degradation of MeBr has been demonstrated through cell suspensions of

Methylococcus capsulatus as it mineralized MeBr, by its removal from the

gas phase, the quantitative recovery of Br2 in the spent medium, and the

production of 14CO2 from [14C] MeBr (Oremland et al., 1994). At high

concentrations, biodegradation of MeBr in methanotrophic soils was inhibited

due to the toxicity of MeBr itself, but became significant at concentrations

lower than 1000 ppm. Methyl fluoride (MeF) inhibited the oxidation of

methane as well as that of [14C] MeBr. The rate of MeBr consumption by

cells varied inversely with the supply of methane, which suggested a

competitive relationship between these two substrates. Soil methanotrophic

bacteria, as well as other microbes, can degrade MeBr present in the

environment. Miller et al. (1997) isolated a facultative methylotroph that used

MeBr as a source of carbon and energy. The consumption of MeBr by the

methane-oxidizing bacteria indicates that methane monooxygenases are



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responsible. Shorter et al. (1995) suggested that microbial degradation of

MeBr at low concentrations (ppb) in surface soils may be important in

removing MeBr from the atmosphere, thus reducing its lifetime in the

atmosphere and lowering its ozone-depletion potential. This observation is

limited in applicability since the effectiveness of MeBr will be reduced at

concentrations below the application rate. Studies have indicated that MeBr

oxidation can occur in field-fumigated soil. High rates of 14C-MeBr

oxidation to 14CO2 were observed in the first few days following soil

fumigation where the MeBr concentration was . 9.5 mg g21 soil (Miller et al.,

1997).

Martin (2003) reviewed the ecology of microbial rhizosphere inhabitants

effected by fumigation with MeBr and CP, and how the rhizosphere may

hinder or help the ability to control plant pathogens. In the absence of known

pathogens, many crops have exhibited an increased growth response when

planted into soil that had been fumigated with MeBr (Wilhelm and Paulus,

1980). One of the likely reasons for this observation was that fumigation

altered the microbial composition of the soil, either enhancing beneficial

colonizers or reducing populations of deleterious rhizosphere colonizers. At a

field site where MeBr and CP were used as fumigants, the soils had 10- to

100-fold greater populations of fluorescent pseudomonads and 1000-fold

greater populations of total fungi than the non-fumigated soil (Xiao and

Duniway, 1998). Total bacterial populations were not significantly different in

the fumigated and non-fumigated soils. Although differences in total

fluorescent pseudomonad populations persisted throughout the season, total

fungal populations equilibrated 1 week after fumigation. Given the observed

differences in microbial communities, it is likely that there were also

differences in rhizosphere colonizers as well. Qualitative differences in

rhizosphere colonizers of strawberries grown in MeBr- plus CP-fumigated

and non-fumigated soils were observed. Differences were also observed in

fungal rhizosphere colonizers, in one field in Watsonville, CA, exhibiting

much higher root colonization frequencies by Trichoderma harzianum on

plants from fumigated than non-fumigated soil.

In growth chamber and microcosm studies, the concentrations of human

pathogenic Escherichia coli O157:H7 were significantly higher in the nonfumigated soils during the first 2 weeks after fumigation with MeBr and

MeI (Ibekwe, unpublished). In this study, changes in microbial community

structure in fumigated and non-fumigated soils were examined in microcosms

without plants, in rhizospheres and in non-rhizosphere soils. The effects of

the two fumigants on soil microbial community structure were greater based

on the types of fumigants. Clay soil seemed to protect microorganisms better

than sandy soil, since there were more DGGE bands detected in clay soil

(Fig. 7a and b).



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B. METHYL ISOTHIOCYANATE

MITC is the primary active ingredient of metam sodium in soil and is a broadspectrum fumigant with activity against plant pathogenic nematodes, weeds, and

a range of pathogenic fungi (Kreutzer, 1963). Metam gained notoriety in 1991

when a spill into the Sacramento River in California resulted in human exposure

and an environmental disaster (CA EPA, 1992). The toxic action of this fumigant

involved first the decomposition or metabolism of metam and dazomet to MITC

as the activation product with derivatized critical biological thiols and

amines (Kaufman, 1977; Sinha et al., 1988; Tomlin, 1994; Ware, 1994). In

living cells MITC is metabolized by the mercapturate, N-acetyl-S(N-methylthiocarbamoyl)cysteine via the GSH conjugate. The GSH serves as a

potential carrier for the later release of MITC (Mennicke et al., 1983; Baillie and

Slatter, 1991; Lam et al., 1993). S-methylation is another alternative in which

metam might be methylated and MITC and dazomet metabolized to S-methyl

metam. S-methylation is a bioactivation mechanism for metam and metabolites

of MITC and dazomet in cells (Staub et al., 1995). The conversion of MITC to Smethyl metam and its oxon is believed to involve conjugation with glutathione,

hydrolysis to 50 -(N-methylthiocarbamoyl)cysteine, cleavage by cysteine conjugate, lysis to release metam, and methylation and oxidative desulfuration

(Staub et al., 1995). Riffaldi et al. (2000) evaluated the extent to which metam

sodium (MS) was applied at two different recommended rates and how its

degradation product, MITC, could affect soil respiration. The results suggested

that MS degradation to MITC was complete within 4 h. MITC decomposed

quickly in a few days, except in the soil containing high organic matter where it

was still present after 15 days.

Metam sodium does not move through soil like MeBr, so a thorough mixing of

the soil is needed to ensure even distribution and avoidance of “hot spots” where

high concentrations of MITC do not fully dissipate and can result in phytotoxicity

problems (Martin, 2003). In addition, uniform watering is needed to activate all

products in the soil prior to planting or phytotoxicity problems may be

encountered later when subsequent irrigations activate residual products. Nonuniform distribution may also result in poor degradation of MITC. The

degradation of MITC is a result of both chemical and biological mechanisms,

since degradation of MITC in sterile soil is significantly slower than in non-sterile

soil (Gan et al., 1999; Dungan et al., 2003b,c). Smelt et al. (1989) demonstrated



Figure 7 DGGE analysis of 16S rRNA fragments of duplicate soil samples collected from

microcosm treated with different fumigants in clay and sandy soils. Samples were collected 7 days

after fumigation. Amplified products were separated on a gradient gel of 30– 70% denaturant. (a)

Community structure 7 days after the initiation of the fumigation in clay soil. (b) Community

structures 7 days after the initiation of the fumigation in sandy soil.



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enhanced degradation of MITC in soils that had been previously treated. This

implies that microbial degradation of MITC is occurring. Microorganisms

responsible for the enhanced degradation of MITC may specifically target the

isothiocyanate functional group, which enables the degradation of the

isothiocyanate compounds at an accelerated rate. There is little information

available in the literature about the nature of the degradation products of MITC.

The concern with the products is that they could be more toxic and mobile than

MITC. More research is needed to provide detailed information on the

environmental conditions that may enhance toxicity of the products.



C. 1,3-DICHLOROPROPENE

The fumigant 1,3-D (Telonew, Dow AgroSciences) is an effective nematicide

used either as a stand-alone fumigant (Telone II, 94% 1,3-D) or in mixture with

17 or 35% CP (Telone C-17 and Telone C-35, respectively). The purpose of the

mixture is to improve efficacy against soil-borne fungal pathogens. Registration

of 1,3-D was suspended in California in 1990 because of air quality concerns in

Merced County, but was reinstated in 1994 (Martin, 2003). Concern for safety

and air quality led California’s Department of Pesticide Regulation (2002) to

institute buffer zone requirements and place limits on the amount of this fumigant

that can be applied in a township. In fields that are cropped to strawberry at least

once every 3 years, this can influence the growers’ ability to fumigate some

portions of their fields, especially in agricultural areas faced with urban

encroachment. The township cap varies with area and season, and will restrict the

use of 1,3-D as an alternative fumigant to MeBr. These restrictions take into

consideration potential groundwater contamination, worker exposure, and air

emissions for potential chronic exposure. The toxicity of 1,3-D to soil

microorganisms at the recommended rate may not be a serious concern (Ibekwe

et al., 2001a). What is of concern to farmers is the high degradation rate with

repeated application (Gan et al., 1999).

A decrease in field performance for 1,3-D following repeated application has

been reported (Smelt et al., 1989; Ou et al., 1995, 1997). This loss in efficacy is

associated with an increase in microbial degradation in the adapted soils.

Adaptation of such soils to fumigants may be due to the selection of microbial

populations with high degradative potentials (Van Dijk, 1974; van Hylackama

and Janssen, 1992; Verhagen et al., 1995). Field experiments with continuous

potato cropping found that sustained annual applications of 1,3-D led to

insufficient control of potato-cyst nematodes (Solanum tuberosum L.) (Lebbink

et al., 1989). Pseudomonas sp. may be one of the most abundant bacterial species

in soil. There are many reports of this species’ ability to degrade 1,3-D (Lebbink

et al., 1989; Verhagen et al., 1995). Fifteen bacterial strains with the capacity to

degrade 1,3-D (of which four were Pseudomonas sp.) were isolated from



FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS



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enrichment cultures grown from adapted soils (Verhagen et al., 1995). One strain,

Pseudomonas cichorii 170, was shown to completely degrade 1,3-D to

3-chloroallyl alcohol (Poelarends et al., 1998). The characterization of the

genes involved in the complete metabolism of 1,3-D was identical to the dhaA of

the Gram-positive bacterium Rhodococcus rhodochrous NCIMH13064 and the

dhiA genes from Rhodococcus sp. strain m15-3 (Bosma et al., 1999).



D. CHLOROPICRIN

CP and possibly its dechlorination products are lacrimators, respiratory

irritants, and toxicants and must be used under containment conditions. CP is a

preplant soil fumigant, a warning agent for other fumigants, and a former war gas.

Its mode of action is unknown but presumably related to its facile metabolic

dechlorination on reaction with biological thiols (Sparks et al., 1997). The

reaction of 14CCl3NO2 with GSH yields the di- and monochloro derivatives,

GSSG, and a small amount of nitrite. The toxicity of CP (CCl3NO2) is probably

due to disruption of multiple targets by its cascade of dechlorination products.

The reactivity of CCl3NO2 with biological thiols has been known since the 1940s,

but no products or mechanisms have been identified (Bacq, 1942; Desreux et al.,

1946). CP is known to be metabolized by Pseudomonas (Castro et al., 1983;

Castro, 1993). In aerobic soil CP will be degraded to produce CHCl2NO2,

CH2ClNO2, and CH3NO2 (Wilhelm et al., 1996):

Cl3 CNO2 ! Cl2 CHNO2 ! ClCH2 NO2 ! CH3 NO2

The reactions of CP in cells involve rapid dechlorination to CHCl2NO2 and

conversion of GSH to GSSG, plus possible adduct formation with thiol proteins,

e.g., Hb-SH (Sparks et al., 1997). CP then oxidizes protein thiols with formation of

disulfide bonds that may disrupt multiple targets by its dechlorination products.

CP stability in soil is short term, with microbial degradation primarily

responsible for inactivation of the fumigant (Gan et al., 2000). In these studies

degradation was shown to follow first-order kinetics ðr 2 . 0:87Þ: Total

degradation of CP was higher in compost-amended soil and enriched with CP,

than the unamended soil and it was significantly lower in the sterile soils (Ibekwe

et al., unpublished). The degradation capacity of these samples was predominantly of biological origin. In unamended, compost-amended, and compostamended soils treated with CP, the k values varied from 0.21, 1.12, and 3.50

day21, respectively. This corresponds to half-lives of 3.40, 0.62, and 0.20 days.

Degradation of CP is very rapid compared to other fumigants. For instance, halflives was 2 days for 1,3-D from compost-amended Arlington sandy loam soil

(Ibekwe et al., 2001b). Degradation of CP in sterile soil was significantly

inhibited suggesting an important role of soil microorganisms in CP degradation



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(Gan et al., 2000). Microbial degradation accounted for 70, 57, and 83% overall

CP degradation in compost-amended, unamended, and compost-amended treated

soils, respectively.



VI. EFFECT ON SPECIFIC MICROBIAL POPULATIONS

Fumigants are used for the control of plant pathogens. Some fumigants may be

toxic to some microbes, and this may enhance the selection of others, which may

be beneficial to plants. Since the majority of research has been concentrated on

microorganisms that are easy to manipulate in culture, our understanding of

microbial interactions and their impact on plant health is not yet well understood.

The ecological significance of Mycobacteria, a group of organisms that are not

amenable to standard laboratory enumeration techniques, is currently being

evaluated (Bull, unpublished data). Mycobacteria from fumigated and nonfumigated soil and from strawberry roots were evaluated to determine

interactions with biocontrol agents and pathogens (Shetty et al., 2000; Bull

et al., 2002). Some microorganisms are more amenable to study than others due to

the complexity of the microbial community evaluated. Studies in our laboratory

have shown the effect of MeBr and MeI on heterotrophic bacterial growth to be

insignificant in the rhizosphere and non-rhizosphere of lettuce grown in a growth

chamber and irrigated with E. coli O157:H7 contaminated water. After an initial

decline, there was a fast regrowth of the heterotrophic bacteria (after 3 weeks).

This was also observed with the E. coli O157:H7 colonies. Toyota et al. (1999)

showed insignificant differences in culturable bacteria between the control and

fumigated soils 7 days after fumigation in two Japanese soils. They were able to

quantify a 50% reduction in total number of bacteria 15 days after fumigation, but

at 23 days, the numbers were at the normal level. Both fungal and bacterial

biocontrol agents, including Gliocladium virens, Pseudomonas chlororaphis,

Pseudomonas fluorescens, Pseudomonas aureofaciens, Bacillus cereus, and

Streptomyces isolates, were included in the trials to determine the effects of MeBr

to specific microbes (Martin, 2003). Fumigation promoted the survival of this

species. Ibekwe et al. (2001a) and Dungan et al. (2003a) were able to show that

fumigation with MeBr and PBr resulted in changes in microbial community

leading to the emergence of new communities dominated by Pseudomonas sp.

and Bacillus sp. The approaches that first describe the microbial ecology of

fumigated soils and of the roots in these soils should provide sound information

for targeting screening efforts to identify specific microbes capable of controlling

root diseases. Investigations evaluating individual components of MeBr plus CP

and non-fumigated rhizosphere communities indicated that some strains can have

beneficial or deleterious effects on strawberry plants (Martin, 1997). Some

rhizosphere colonizers were found to enhance strawberry yields in the field.



FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS



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Trials were conducted in test plots managed by a commercial grower in a field

where the predominant pressure was from generalized root pathogens associated

with black root rot, such as Pythium, binucleate Rhizoctonia, and Cylindrocarpon

spp. After 2 years, yields were different from both years due to environmental

conditions. Itoh et al. (2000) showed that Fusarium oxysporum was not detected

in soil 3 weeks after fumigation, but this varies with fumigant, with CP having a

stronger effect than MITC. In another study, Tanaka et al. (2003) showed more

vigorous growth of tomato plants after CP treatments than those treated with

MeBr. The result was attributed to an increase in NH4-N supply at that stage.



VII. SUMMARY AND CONCLUSIONS

The phase-out of MeBr has generated a lot of public awareness of fumigants

and the large use of these compounds in agriculture. Since there is no single,

registered fumigant that is as effective as MeBr, other compounds need to be

developed and tested. The actual registration procedure includes evaluations of

the impact of herbicides on the environment by testing for non-target organism

effects on a single species or on microbial communities. The impact on soil

microbial communities is evaluated in view of their role in sustaining the global

cycling of matter and their varied functions in supporting plant growth.

Internationally, there are various protocols that are required before a new

pesticide is granted registration.



ACKNOWLEDGMENTS

Thanks to Ms Pamela Watt for reviewing and assisting in literature searches, and

Drs Scott Yates, Sharon K. Papiernik, and Frank Martin for providing helpful

materials. This review was supported in part by the 206 Manure and Byproduct

Utilization Project of the USDA-ARS. The mention of trademark or propriety

products in this review does not constitute a guarantee or warranty of the property

by the USDA and does not imply its approval to the exclusion of other products

that may also be suitable.



REFERENCES

Akhtar, M., and Malik, A. (2000). Roles of organic soil amendments and soil organisms in the

biological control of plant-parasitic nematodes: a review. Bioresour. Technol. 74, 35 –47.

Anderson, J. R. (1978). Pesticide effects on nontarget soil microorganisms. In “Pesticide

Microbiology” (I. R. Hill and S. J. L. Wright, Eds.), pp. 313 –533. Academic Press, London.



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