Advances in agronomy volume 83

VOLUME

83


Advisory Board
John S. Boyer
University of Delaware

Paul M. Bertsch
University of Georgia

Ronald L. Phillips
University of Minnesota

Kate M. Scow
University of California, Davis

Larry P. Wilding
Texas A&M University

Emeritus Advisory Board Members
Kenneth J. Frey
Iowa State University

Eugene J. Kamprath
North Carolina State University

Martin Alexander
Cornell University

Prepared in cooperation with the
American Society of Agronomy Monographs Committee
Diane E. Stott, Chair
Lisa K. Al-Almoodi
David D. Baltensperger
Warren A. Dick
Jerry L. Hatfield
John L. Kovar

David M. Kral
Jennifer W. MacAdam
Matthew J. Morra
Gary A. Pederson
John E. Rechcigl

Diane H. Rickerl
Wayne F. Robarge
Richard Shibles
Jeffrey Volenec
Richard E. Zartman


Edited by

Donald L. Sparks
Department of Plant and Soil Sciences
University of Delaware
Newark, Delaware

Amsterdam · Boston · Heidelberg · London · New York · Oxford · Paris · San Diego
San Francisco · Singapore · Sydney · Tokyo


ELSEVIER B.V.
Sara Burgerhartstraat 25
P.O. Box 211, 1000 AE
Amsterdam, The Netherlands

ELSEVIER Inc.
525 B Street
Suite 1900, San Diego
CA 92101-4495, USA

ELSEVIER Ltd
The Boulevard
Langford Lane, Kidlington,
Oxford OX5 1GB, UK

ELSEVIER Ltd
84 Theobalds Road
London WC1X 8RR
UK

q 2004 Elsevier Inc. All rights reserved.
This work is protected under copyright by Elsevier Inc., and the following terms and conditions apply to its use:
Photocopying
Single photocopies of single chapters may be made for personal use as allowed by national copyright laws.
Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or
systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery.

Special rates are available for educational institutions that wish to make photocopies for non-profit educational
classroom use.
Permissions may be sought directly from Elsevier’s Rights Department in Oxford, UK: phone (+44) 1865 843830,
fax: (+44) 1865 853333, e-mail: Requests may also be completed on-line via the
Elsevier homepage ( />In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222
Rosewood Drive, Danvers, MA 01923, USA; phone: (+1) (978) 7508400, fax: (+1) (978) 7504744, and in the UK
through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road,
London W1P 0LP, UK; phone: (+44) 20 7631 5555; fax: (+44) 20 7631 5500. Other countries may have a local
reprographic rights agency for payments.
Derivative Works
Tables of contents may be reproduced for internal circulation, but permission of the Publisher is required for
external resale or distribution of such material. Permission of the Publisher is required for all other derivative
works, including compilations and translations.
Electronic Storage or Usage
Permission of the Publisher is required to store or use electronically any material contained in this work, including
any chapter or part of a chapter.
Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in
any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written
permission of the Publisher.
Address permissions requests to: Elsevier’s Rights Department, at the fax and e-mail addresses noted above.
Notice
No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or
ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent
verification of diagnoses and drug dosages should be made.
First edition 2004

ISBN: 0-12-000781-9
ISSN: 0065-2113 (Series)
1 The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper).

W
Printed in Great Britain.


Contents
CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix
xi

EFFECTS OF FUMIGANTS ON NON -TARGET
ORGANISMS IN SOILS
A. Mark Ibekwe
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Effects on Biological Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Enzyme Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Substrate-Induced Respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Nitrogen Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Effect on Microbial Activities and Composition . . . . . . . . . . . . . . . . . . . .
A. Changes in Community-Level Carbon Source Utilization by Biolog . .
B. Changes in Microbial Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Changes in Soil Microbial Community Structure and Composition . . .
D. Analysis of Soil Microbial Community Structure
by Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Impact of Recommended Fumigants on Soil Microbial Communities
and Agricultural Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Methyl Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Methyl Isothiocyanate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. 1,3-Dichloropropene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Chloropicrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Effect on Specific Microbial Populations . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2
3
5
5
6
7
8
8
10
12
15
20
22
25
26
27
28
29
29
29

SORGHUM IMPROVEMENT —INTEGRATING
TRADITIONAL AND NEW TECHNOLOGY TO

PRODUCE IMPROVED GENOTYPES
W. L. Rooney
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Variation in Sorghum ssp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Sorghum Improvement—from Landraces to Cultivars . . . . . . . . . . . . . . .
v

38
39
40


vi

CONTENTS

IV. Mechanisms of Controlled Pollination . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Hand Emasculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Genetic Male Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Hot-Water Emasculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Control of Anther Dehiscence Control . . . . . . . . . . . . . . . . . . . . . . . .
E. Cytoplasmic –Genetic Male Sterility . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Improvement Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Population Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Cultivar and Inbred Line Development . . . . . . . . . . . . . . . . . . . . . . .
C. Hybrid Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Use of Exotic Germplasm—Sorghum Conversion . . . . . . . . . . . . . . .
VI. Trait-Based Breeding Efforts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Yield and Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Biotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. Abiotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Grain Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Forage Sorghum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Sweet Sorghum for Syrup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. Broomcorn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Biotechnology in Sorghum Improvement . . . . . . . . . . . . . . . . . . . . . . . . .
VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42
43
44
44
45
46
49
51
52
52
56
59
60
63
75
86
89
92
93
93
95

96

CRITICAL REVIEW OF THE SCIENCE AND OPTIONS
FOR REDUCING CADMIUM IN TOBACCO
(NICOTIANA TABACUM L.) AND OTHER PLANTS
N. Lugon-Moulin, M. Zhang, F. Gadani, L. Rossi,
D. Koller, M. Krauss and G. J. Wagner
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Cadmium in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Cadmium in the Tobacco Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Cadmium Tolerance in Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Root-to-Shoot Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Root and Shoot Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Cadmium in Field-Grown Tobacco Leaves . . . . . . . . . . . . . . . . . . . .
E. Stalk Position Versus Cadmium Accumulation . . . . . . . . . . . . . . . . . .
F. Developmental Stage Versus Cadmium Accumulation . . . . . . . . . . . .
G. Variation Within the Leaf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H. Sub-cellular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Differences Between Varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. External Factors Affecting Cadmium Concentration in Tobacco Leaves . .
A. Soil Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112
113
114
115
117
119
120

121
123
123
124
124
126
127
127


CONTENTS
B. Agronomic Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Additional Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Options to Reduce the Cadmium Content in Tobacco Leaves . . . . . . . . . .
A. Molecular and Biochemical Approaches . . . . . . . . . . . . . . . . . . . . . .
B. Breeding Strategies to Reduce Cadmium . . . . . . . . . . . . . . . . . . . . . .
C. Soil Cadmium Remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii
130
135
137
137
137
153
154

161
162
162

THE IMPACT OF GRAZING ANIMALS ON N2 FIXATION
IN LEGUME -BASED PASTURES AND MANAGEMENT
OPTIONS FOR IMPROVEMENT
John C. Menneer, Stewart Ledgard, Chris McLay
and Warwick Silvester
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Animal Treading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Plant Damage and Burial by Hoof Action . . . . . . . . . . . . . . . . . . . . .
B. Soil Compaction: Mechanical Impedance Effects on Legumes . . . . . .
C. Soil Compaction: Aeration and/or Waterlogging Effects
on Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Significance of Plant and Soil Factors, and Limits of Pasture
Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Animal Grazing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Diet Selection and Defoliation Effects . . . . . . . . . . . . . . . . . . . . . . . .
B. Direct Effects of Defoliation on N2 Fixation . . . . . . . . . . . . . . . . . . .
IV. Animal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Increased Soil N and Grazing Avoidance of Excreta-Affected Areas . .
B. Direct Effects of Excreta N on N2 Fixation . . . . . . . . . . . . . . . . . . . .
V. Strategies to Minimise the Impacts of Grazing Animals . . . . . . . . . . . . . .
A. Pasture Management to Aid Legume Production . . . . . . . . . . . . . . . .
B. Choice of White Clover Cultivar and Companion Grasses . . . . . . . . .
C. Tactical Use of N Fertiliser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Farm-Scale Management Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Soil Management: Preventing Treading and Compaction . . . . . . . . . .
B. Restricted Grazing and Supplementary Feeding

in Winter/spring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Technical Based Decision Making for Improved
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

182
184
186
188
192
198
200
201
204
205
207
209
211
211
216
219
221
222
222
224
227
228
228

viii

CONTENTS

SEED -FILL DURATION AND YIELD OF GRAIN CROPS
Dennis B. Egli
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Seed Filling: Definition and Measurement . . . . . . . . . . . . . . . . . . . . . . . .
III. Variation in Seed-Fill Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Water Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Assimilate and Nutrient Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Flower and Fruit Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Photoperiod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Plant Growth Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. Genetic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Regulation of Seed-Fill Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Regulation by the Seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Regulation by the Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Seed-Fill Duration and Crop Productivity . . . . . . . . . . . . . . . . . . . . . . . . .
A. Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Future Yield Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

244
245

248
248
249
250
251
252
252
253
254
255
256
257
262
262
265
267
268

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

281


Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.

D. EGLI (243), Department of Agronomy, University of Kentucky, Lexington,
KY 40546-0312
F. GADANI (111), Philip Morris USA RD&E Department, PO Box 26583,
Richmond, VA 23261

A. M. IBEKWE (1), USDA-ARS-George E. Brown, Jr. Salinity Lab, 450 W. Big
Springs Road, Riverside, CA 92507
D. KOLLER (111), Philip Morris USA RD&E Department, PO Box 26583,
Richmond, VA 23261
M. KRAUSS (111), Philip Morris USA RD&E Department, PO Box 26583,
Richmond, VA 23261
S. LEDGARD (181), AgResearch Ruakura Research Centre, Private Bag 3123,
Hamilton, New Zealand
N. LUGON-MOULIN (111), Philip Morris International R&D, c/o Philip
Morris Products SA, 200 Neuchaˆtel, Switzerland
C. MC LAY (181), Environment Waikato, PO Box 4010, Hamilton, New Zealand
J. C. MENNEER (181), University of Waikato, Private Bag, Hamilton,
New Zealand
W. L. ROONEY (37), Department of Soil and Crop Science, Texas A&M
University, College Station, TX 77843-2474
L. ROSSI (111), Philip Morris International R&D, c/o Philip Morris Products SA,
200 Neuchaˆtel, Switzerland
W. SILVESTER (181), University of Waikato, Private Bag, Hamilton,
New Zealand
G. J. WAGNER (111), University of Kentucky, Agronomy Department N212
ASCN, Lexington, KY 40546-0091
M. ZHANG (111), Philip Morris USA RD&E Department, PO Box 26583,
Richmond, VA 23261

ix


This Page Intentionally Left Blank

Preface
Volume 83 contains five cutting-edge crop and soil science reviews. The first
review covers aspects of fumigant effects on non-target organisms in soils
including mode of action, biological processes, microbial activities and composition, recommended fumigants on soil microbial communities and agricultural
practices and specific microbial populations. The second chapter is a comprehensive review on sorghum improvement using both traditional plant breeding and
contemporary biotechnology. Chapter 3 is a critical review on the science and
options for reducing cadmium (Cd) in tobacco (Nicotiana tabacum L.) and other
plants. Discussions on Cd in the environment and in the tobacco plant, external
factors affecting Cd concentration in tobacco leaves, options for reducing Cd
content in tobacco leaves and soil Cd remediation are included. Chapter 4 deals
with the impact of grazing animals on N2 fixation in legume-based pastures and
management options for improvement. Chapter 5 is a comprehensive review of
seed-fill duration and yield of grain crops.
I am grateful to the authors for their very fine reviews.
DONALD L. SPARKS
University of Delaware

xi


This Page Intentionally Left Blank


EFFECTS OF FUMIGANTS ON
NON- T ARGET ORGANISMS IN SOILS
A. Mark Ibekwe
USDA-ARS George E. Brown, Jr. Salinity Laboratory,
450 W. Big Springs Road, Riverside, California 92507, USA

I. Introduction

II. Mode of Action
III. Effects on Biological Processes
A. Enzyme Activities
B. Substrate-Induced Respiration
C. Nitrogen Transformation
IV. Effect on Microbial Activities and Composition
A. Changes in Community-Level Carbon Source Utilization by Biolog
B. Changes in Microbial Biomass
C. Changes in Soil Microbial Community Structure and Composition
D. Analysis of Soil Microbial Community Structure
by Molecular Techniques
V. Impact of Recommended Fumigants on Soil Microbial Communities
and Agricultural Practices
A. Methyl Bromide
B. Methyl Isothiocyanate
C. 1,3-Dichloropropene
D. Chloropicrin
VI. Effect on Specific Microbial Populations
VII. Summary and Conclusions
Acknowledgments
References

Soil fumigants are extensively used to control plant-parasitic nematodes,
weeds, fungi, and insects for planting of high value cash crops. The ideal
pesticide should be toxic only to the target organisms; however, fumigants
are a class of pesticide with broad biocidal activity and affect many nontarget soil organisms. Soil microorganisms play one of the most critical roles
in sustaining the health of natural and agricultural soil systems. The ability of
soil microorganisms to recover after treatment with pesticide is critical for
1
Advances in Agronomy, Volume 83

Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved.
DOI 10.1016/S0065-2113(04)83001-3


2

A. M. IBEKWE
the development of healthy soils. In the southwestern United States,
fumigation is used to control pathogens such as Verticillium dahliae,
Pythium, Rhizoctonia, or Cylindrocarpon spp. In addition to pathogen
control, fumigation can also result in enhanced growth response of the
plant by reducing weed pressure. The continued use of fumigants in
agriculture will require more investigations of the different types of
fumigants, soils, environmental conditions, and biological/microbial
communities to establish both the effectiveness on target organisms and
safety to the general public.
q 2004 Elsevier Inc.

I. INTRODUCTION
Agricultural soils are typically treated with pesticides to provide effective
control of nematodes, soil-borne pathogens, and weeds in preparation for
planting high value cash crops. Fumigants are a class of pesticide with broad
biocidal activity and affect many non-target soil organisms (Parr, 1974;
Domsch et al., 1983; Anderson, 1993). Currently, only four registered
fumigants are available in the Unites States: 1,3-dichloropropene (1,3-D),
methyl isothiocyanate (MITC), chloropicrin (CP), and methyl bromide
(MeBr). Methyl iodide (MeI, iodomethane) is another fumigant yet to be
registered that is considered being a promising alternative to MeBr for soilborne pest control in high value cash crops. While most fumigants are known
to have broad biocidal activity, their effects on non-target soil microbial
communities are largely unknown, due to the lack of appropriate methods to

describe microbial soil community composition. Soil microorganisms play
one of the most critical roles in sustaining the health of natural and
agricultural soil systems. They are a significant component of nutrient
cycling, especially of C and N, which are essential for proper plant
nutrition and agricultural productivity. Changes in the microbial
community composition as a result of fumigant applications may lead to
changes in the functional diversity of that community and ultimately, the
overall soil quality.
Because of the strong relationships between microbial diversity and
ecosystem function, soil microorganisms are recognized as sensitive indicators
of soil health. MeBr has the ability to destroy stratospheric ozone (Yung et al.,
1980; Prather et al., 1984) and a ban on its production and importation is to be
completed by 2005 in the United States (USEPA, 1995). 1,3-D, MITC, and CP
have been proposed as the most likely chemical alternatives to MeBr. Since little
ecotoxicological information exists with respect to these (and many other


FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

3

fumigants), it is essential that fumigant effects on non-target microorganisms be
examined.

II. MODE OF ACTION
Fumigants are extensively used to grow strawberries, tomatoes, and other
high valued cash crops in California and Florida. Many studies have
documented the chemistry and air pollution potential of these fumigants in
the environment (Baker et al., 1996; Gan et al., 1998a,b). There are only
four registered chemical fumigants available in the United States: The first is

1,3-D, which is marketed under the trade name Telone and contains an equal
ratio of cis-1,3-D and trans-1,3-D. The second, MITC, is a primary product
of metam sodium (sodium methyldithiocarbamate) metabolism. CP, a third
fumigant also known as trichloronitromethane, is often formulated with
Telone and metam sodium. MeBr is the last fumigant available in the United
States. 1,3-D, MITC, and CP have been proposed as the most likely chemical
alternatives to MeBr. Figure 1 shows the structural formula of metam
sodium, MITC, cis- and trans-1,3-D, CP, and MeBr.
Compared to other pesticides, fumigants were found to have little or no
detectable effects on soil microorganisms at field application rates (Hicks et al.,
1990; Anderson, 1993). Their effects on non-target soil microorganisms at the
field application rate are largely unknown until recently, mainly due to lack of
appropriate methodology to describe the non-target population (Elliot et al.,
1996; Macalady et al., 1998; Ibekwe et al., 2001a; Dungan et al., 2003a).
Fumigants should only be toxic to the target organisms, be biodegradable, and
should not leach into the groundwater, though this is not always the case.
The widespread use of fumigants in the warm climate regions of the United
States is of increasing concern. The mode of action of different classes of
pesticides differs. Some pesticides are designed to affect specific or general
processes in the target organisms and are more suitable for a specific target
population. Fumigants are generally designed to provide effective control of
nematodes and soil-borne pathogens, such as fungi and weeds. Metam
sodium and 1,3-D may be very effective as fungicides, and thus may be
expected to affect non-targeting soil flora. The most used fungicide in
Denmark is fenpropimorph, which specifically inhibits two enzymes
involved in ergosterol biosynthesis (Johnsen et al., 2001). This fungicide
was designed to target leaf-associated fungi and subsequently may have some
effects on the soil fungi. Degradation of fenpropimorph also produces an
intermediate metabolite, fenpropimorphic acid, and it has been shown that
saprotrophic fungi were substantially affected by this intermediate

4

A. M. IBEKWE

Figure 1 The chemical structure of cis- and trans-1,3-dichloropropene (1,3-D), metam sodium,
methyl isothiocyanate (MITC), chloropicrin (CP), and methyl bromide (MeBr).

compound, indicating that the biological activity of the fungicide may be
attributed to both mother compound and the more mobile metabolites
(Bjørnlund et al., 2000).
Changes in the microbial composition as a result of fumigant application may
lead to changes that interfere with the functional diversity and overall soil quality.
The strong relationships between microbial diversity, ecosystem sustainability,
and function are being increasingly recognized as sensitive indicators of soil
health (Turco et al., 1994). Ultimately, the linking of information between
microbial community structure/diversity and crop production will be an
important step in being able to predict soil fertility. There is a significant gap
in information on the effects of fumigants on soil bacterial and their impact on
major soil processes, such as organic matter transformation and pollutant
degradation. This review will elaborate on the most up-to-date data available in
the literature on this topic and describe some of the techniques in microbial
ecology that may be helpful in understanding different processes in soil that are
significantly affected by fumigants.


FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

5

III. EFFECTS ON BIOLOGICAL PROCESSES
A. ENZYME ACTIVITIES
The effect of fumigants on enzyme activities was previously reported by Ladd
et al. (1976) and has been shown to have an inhibitory effect on dehydrogenase
activities (DHAs) (Anderson, 1978; Smith and Pugh, 1979; Tu, 1992).
Fumigation of a field soil with MeBr and CP, either individually or in
combination, decreased soil enzyme activities (Ladd et al., 1976). DHA is an
indicator for potential non-specific intracellular enzyme activity of the total
microbial biomass. Fumigants appear to disturb the membrane-bound processes
of active cells. As noted by Ladd (1978), DHA measurements may be influenced
not only by enzyme concentrations, but also by the nature and concentration of
added C substrates, and of alternative electron acceptors, such as NO2
3 . Zelles
et al. (1997) conducted an extensive study with seven enzymes to determine the
effects of chloroform fumigation on their activities and found that enzymes
bound to the active microorganisms were nearly inhibited completely
(dehydrogenase) or strongly reduced (arginine deaminase). These authors also
reported that fumigation of soil did not change the activities of xylase,
b-glucosidase and saccharase and only induced a low reduction in phosphatase
activity. Recently, Dungan et al. (2003a) showed that the incorporation of
compost manure to agricultural soils significantly increased the DHA of the soil
over a 12-week incubation period when compared to unamended soils. When
these soils were treated with two fumigants, propargyl bromide (PBr) and 1,3-D,
they observed a higher rate of fumigant degradation in the amended soils,
corresponding well with the increased enzyme activity in the amended soil
treatments. When treated with PBr at 10 mg kg21 or 1,3-D at 10 and
100 mg kg21, the DHA in unamended and amended soils was not significantly
reduced. At 100 mg kg21, PBr was more toxic than 1,3-D, as indicated by the
reduced DHA at this concentration. At 500 mg kg21 of PBr and 1,3-D, DHA was

significantly repressed, but by week 8 in the amended soil treatments, DHA had
recovered to levels similar to that of the control. DHA in unamended soils spiked
with 500 mg kg21 of 1,3-D or PBr did not demonstrate significant recovery after
12 weeks. This confirmed the inhibitory effects of fumigants on DHA as
previously reported (Anderson, 1978; Smith and Pugh, 1979; Tu, 1992).
Decreases in DHA, in both unamended and amended soils, were probably a direct
result of the adverse effects of PBr and 1,3-D on soil microbial populations.
Fumigants such as metam sodium may interfere with respiratory enzymes such as
pyruvate dehydrogenase due to their chelating effects on metal cations such as Cu
(Corbett et al., 1984) or to toxic degradation products such as MITC (Staub et al.,
1995). These authors showed that MITC is metabolized to S-methyl metam,
probably by formation of S-(N-methylthiocarbamoyl)cysteine, by cysteine


6

A. M. IBEKWE

conjugated b-lyase. The alternative proposal was that metam sodium might be
very sensitive to oxidation, forming reactive sulfenic and sulfinic acids, which
might contribute to its toxic action. Assessing the toxicity of metam, its oxidative
and methylated metabolites, and their contribution to the toxicity of MITC on
non-target soil bacteria are complicated by their interconnected detoxification
and bioactivation pathways.

B. SUBSTRATE-INDUCED RESPIRATION
Substrate-induced respiration (SIR) is mainly used to characterize microbial
activity and has been used to estimate the size of the microbial biomass
(Anderson and Domsch, 1978). The wide use of SIR to assess the impact of
pesticides on microbial activity and biomass has been reported in the literature

(Wardle and Parkinson, 1990; Harden et al., 1993; Hart and Brookes, 1996; Lin
and Brookes, 1999; Smith et al., 2000; Chen et al., 2001). Results on the
effects of two fumigants from a 24-h SIR experiment, evaluated as CO2 evolved
(mg g21 dry soil 24 h21), were recently reported (Dungan et al., 2003a). SIR was
markedly inhibited by incremental additions of PBr or 1,3-D to either unamended
or manure-amended soil. Other studies show both enhancement and reduction of
CO2 evolution following pesticide application, while others exhibit no effect on
soil respiratory activity (Simon-Sylvestre and Fournier, 1979). In unamended
soil, at the highest concentration of PBr and 1,3-D, SIR was reduced to 61 and
22% of the control, respectively. In amended soil, SIR was reduced to 50 and
25% of the control, respectively; however, SIR was 1.4 (PBr) and 2.2 (1,3-D)
times higher, on average, than in fumigated unamended soil (Dungan et al.,
2003a). This study demonstrated a significant reduction of the impact of fumigant
on non-target soil bacteria with soil amendments. A study by Chen et al. (2001)
has shown that SIR was unaffected over a 56-day experimental period when
treated with benomyl (a fungicide) at a rate of 125 mg kg21. This showed that
soil microbial activity was stimulated by the addition of amendments, even when
treated with PBr (at 10 and 100 mg kg21) or 1,3-D (at 10 –500 mg kg21). The
effect of metam sodium has also been shown to strongly affect SIR (Macalady
et al., 1998). Soil treated with 1.6 g l21 of metam sodium was reduced to
17 – 29% of the controls with no recovery after 28 days. Also, metam sodium
applied at 16 g l21 eliminated respiration of added glucose for all sampling dates.
The authors concluded that metam sodium had inhibitory effects on soil
parameters measured even after 18 weeks. Sensitivity of soil respiration after
repeated exposure to MITC (Taylor et al., 1996) suggested that fumigation with
metam sodium resulted in long-term changes in the composition and activity of
soil microorganisms.


FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

7

C. NITROGEN TRANSFORMATION
The work of Rovira (1976) with CP and MeBr showed an increase in
concentration of NHþ
4 within 28 days after fumigation, followed by a decrease
after 46 days due to increase in activities of nitrifying bacteria. NO3
concentrations in fumigated soil were lower than the untreated soils after 28
days because of a lack of nitrification following fumigation. Malkomes (1995)
also reported that nitrification was inhibited by fumigation for long periods of
time. Metam sodium produced some inhibitory effects on nitrogen transformation
as a result of depression of ammonia-oxidizing activities (Macalady et al., 1998).
These authors showed a decrease in ammonia oxidation potential with increasing
metam sodium dose. In a recent study, the effects of steam sterilization (SS) on
soil microbial properties, including metabolic diversity of the microbial
communities, were examined in a greenhouse study compared to MeBr (Tanaka
et al., 2003). The numbers of nitrifiers, both ammonium-oxidizing bacteria and
nitrite-oxidizing bacteria, were severely affected by the SS and CP treatments,
resulting in their virtual disappearance. The decrease in the levels of microbial
biomass C and N after the treatments suggested that the SS and CP treatments
eradicated the microorganisms more effectively than the MeBr treatment, and
that the influence of the former persisted until the end of the experiment,
4 months after the treatments. Accumulation of NH4-N was observed after the
SS and CP treatments mainly due to the partial decomposition of the dead
microorganisms and the marked decrease in the number of ammonium-oxidizing
bacteria. The numbers of ammonia-oxidizing bacteria were also reduced by more
than four orders of magnitude in soils fumigated with metal sodium and did not
show recovery 105 days later (Toyota et al., 1999). Fumigation decreased the
numbers of nitrite oxidizers by three orders of magnitude, with a slight recovery

after 105 days (still significantly lower than the control). These studies have
shown that several fumigants adversely affect soil N balances through their
temporary inhibition of nitrification.
The process of nitrification involves the conversion of ammonium to nitrite
and to nitrate. The inhibition of this process through the adverse effects of
fumigants on ammonia-oxidizing bacteria may result in some adverse effects on
the overall soil quality. It should also be noted that once the fumigants are
completely dissipated in soil, there is always a regrowth of the bacteria. It has
been shown previously that autotrophic nitrifying bacterium Nitrosomonas
europaea is capable of co-oxidizing numerous halogenated hydrocarbons in the
presence of NHþ
4 through the action of the ammonia-oxidizing enzyme ammonia
monooxygenase (AMO) (Rasche et al., 1990). As alternative substrates for this
enzyme, these compounds inhibit ammonia oxidation through competitive
effects. At lower concentrations these compounds may serve as an alternative
substrate for AMO, or with higher concentrations bacterial population may
decrease, resulting in less nitrification.


8

A. M. IBEKWE

IV.

EFFECT ON MICROBIAL ACTIVITIES
AND COMPOSITION

A. CHANGES IN COMMUNITY-LEVEL CARBON SOURCE
UTILIZATION BY BIOLOG

The Biolog Gram-negative (GN) microtiter plate assay is often used to analyze
the functional diversity through substrate utilization patterns of soil microorganisms. When the functional abilities of the heterotrophic soil microbial
communities were observed over a 12-week period of time following the
application of fumigants, severe alterations were seen during the first week,
especially with MeBr (Ibekwe et al., 2001a). The PCA plot (Fig. 2) from MeBr,
MITC, 1,3-D, and CP-treated soil microbial communities and the control
accounted for 28% of the variance on the first component, with six PCs
accounting for over 80% of the variation. The control and the 1,3-D-treated soils
separated along PC1 with their coefficients positively correlating to the right of
PC1. Analysis of MeBr communities did not show any pattern of groupings
except that communities from the first week of treatments were positively
correlated along PC2 and grouped with MITC after weeks 8 and 12. Pairwise
comparison showed that the MeBr communities differed significantly ðp , 0:05Þ
from the control and 1,3-D communities. The control and 1,3-D treatments were

Figure 2 Principal component analysis performed on Biolog GN fingerprints of soil extracts
treated with methyl bromide (MeBr), methyl isothiocyanate (MITC), 1,3-dichloropropene (1,3-D),
chloropicrin (CP), and non-fumigated soil (C). 1, 8, and 12 after the symbols indicate weeks 1, 8, and
12 and 1, 2, and 3 after the “-” sign indicates 50% below field application rate, field application, and
1000% above field application rates, respectively.


FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

9

similar when compared to the other three fumigants, suggesting a lesser effect of
1,3-D on heterotrophic. In another study, effects of SS on soil microbial
properties, including metabolic diversity of the microbial communities, were
examined in a greenhouse study and were compared to MeBr (Tanaka et al.,

2003). The authors found that the richness and average well color development
(AWCD) values in the microbial communities decreased markedly immediately
after treatment with MeBr but showed a rapid recovery, while those treated
with CP continued to decrease until the transplanting of tomato seedlings.
This was in agreement with Ibekwe et al. (2001a) who showed a sharp decrease in
microbial diversity with MeBr, followed by a quick recovery of the community
after 8 weeks when compared to a more sustained effect for a longer period with
CP. The shifts in microbial communities observed in the Biolog assays were due
to the toxic effects of fumigants on rapid growing microorganisms of high
population in the soils. Analysis of microbial communities from the Biolog GN
assay by DGGE confirmed that carbon source utilization profiles obtained with
Biolog GN plates do not necessarily discriminate the numerically dominant
members of the microbial community used as the inoculum (Engelen et al., 1998;
Smalla et al., 1998).
Under field conditions, natural fluctuations in carbon substrate utilizing
activity and community-level physiological profiles of microorganisms in low
input and conventional rice paddy soils were monitored using Biolog GN plates
for 2 years. The purpose was to establish criteria for assessing side effects of
pesticides on soil microbial ecosystems (Itoh et al., 2002). The activity changed
seasonally showing a regular pattern with more activity observed during late
summer. The level of microbial activities seemed to be directly influenced by soil
temperature and/or redox potential. Soil microbial communities grouped into
three clusters, August – December, January – April, and May – August, based on
the sampling season. Many studies have shown the effects of fumigants on
microbial activities and community structure. The effect of metam sodium
fumigation on community structure after a 5- and 18-week incubation showed
a separation of the two communities along the first principal component (PCA)
based on treatment dose (Macalady et al., 1998). There was a significant
ðp ¼ 0:001Þ dose treatment effect at week 5, whereas at week 18 there was no
dose significant effect ðp ¼ 0:05Þ; but there was a binary variable effect

ðp ¼ 0:001Þ between the treated and untreated samples. Toyota et al. (1999)
compared the AWCD values and richness (number of positive wells) of different
categories of the 95 substrates 105 days after fumigation with metam sodium.
They found that both AWCD and richness in all the substrate groups were
significantly ð p ¼ 0:05Þ lower in the fumigated soils than in the control soils. In
the radish rhizosphere and non-rhizosphere soils fumigated with CP, there was a
significant suppression in AWCD and richness in fumigated soils compared to the
control (Itoh et al., 2002). It was assumed that the bacterial populations with a
high substrate assimilation activity were damaged by CP fumigation and a


10

A. M. IBEKWE

different microbial community was developed in the radish rhizosphere. Cluster
analysis of these communities after 24 h of incubation separated between
rhizosphere and non-rhizosphere samples, and then between fumigated and nonfumigated samples, suggesting the effect of the rhizosphere by CP fumigation.
After 72 h, the Biolog samples showed a clear separation between the fumigated
and the non-fumigated samples in both the rhizosphere and the non-rhizosphere
samples. The authors concluded that fumigants affected mostly the slowsubstrate utilizing rhizosphere microbial communities or that fast growers
seemed to utilize most of the substrates during the early stages of incubation. The
problem with the Biolog system is due first to the respiratory activities of fast
growing heterotrophic bacteria resulting in the stimulation or reduction of the
catabolism of 95 carbon substrates (Engelen et al., 1998; Ibekwe et al., 2001a).
The shifts in microbial communities observed in the Biolog assays were due to
the rapid growth of organisms of their high population in the soils. For example,
Pseudomonas species are found in most soil samples and they respond well in
Biolog assays (Haack et al., 1994; Garland, 1996, 1997).

B. CHANGES IN MICROBIAL BIOMASS
SIR (Anderson and Domsch, 1978) has been the standard method for soil
microbial biomass measurements. It is based on the maximal initial response of the
soil microbial biomass to a substrate amendment. Microbial biomass is assessed
quantitatively by the C, N, ninhydrin-reactive compounds, ATP, quinones, and
phospholipid fatty acid (PLFA) composition of the cells. Total organic carbon in
the microbial biomass (biomass C) is considered as the general indicator of the
amount of microorganisms in the soil, and total nitrogen is considered to be the
indicator of potential available nitrogen in the soil. Ninhydrin-reactive
compounds represent the labile fraction of biomass N and are metabolized to
ammonia by heterotrophic microorganisms in soil. This is one of the major
fractions of available N to plants and microorganisms. The concentration of ATP
in soil is an indicator of the amount of microorganisms that can be readily and
rapidly measured. The total amount of respiratory quinones has been shown to be
an indicator of the microbial biomass since many microorganisms have only one
quinone species. Bacteria contain relatively constant amounts of viable biomass as
phospholipid, so this can also be used as a good biomass indicator.
Many studies have shown the impact of fumigation on microbial biomass
(Zelles et al., 1997; Toyota et al., 1999; Ibekwe et al., 2001a; Suyama et al.,
2001; Itoh et al., 2002). Zelles et al. (1997) reported a decrease in the microbial
biomass-C and -N of about 20% after chloroform fumigation. Griffiths et al.
(2000) examined a technique based on progressive chloroform fumigation of soil
to reduce soil microbial biodiversity, and measured the effects of the reductions


FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

11

upon the stability of key soil processes. The diversity of cultivable and noncultivable bacteria, protozoa, and nematodes was progressively reduced by

increasing fumigation times, with the total microbial biomass less in fumigated
soils than the unfumigated. Specific parameters like nitrification, denitrification
and methane oxidation decreased as biodiversity decreased. Suyama et al. (2001)
looked at the effects of fumigation on paddy rice soil to establish a criteria to
assess the short-term effects of pesticides on soil microorganisms. They
concluded that the degree of fluctuation of microbial biomass and population
in the paddies can be used as references to assess the degree of pesticide effects in
other Japanese paddy soils. In another study, the effects of the pesticides
fenitrothion, chlorothalonil, chloropicrin, linuron, and simazine on microbial
biomass were monitored for 28 days for changes in respiratory quinone profiles
(Katayama et al., 2001). Pesticides were applied to the soil at 10 times the
recommended rates. Application of CP decreased the amount (an indicator of
microbial biomass) and diversity (an indicator of taxonomic diversity of the
microbial community) of the different quinones species during the 28-day
treatment. Continuous change in the structure of the microbial community in the
CP-treated soil was documented by the changes in the dominant quinone species,
and there was no change in the control soil. The authors concluded that quinone
profile analysis is a potential method to detect the effect of pesticide on a soil
microbial community and biomass.
PLFA profiles are often used to study microbial diversity and biomass in
complex communities (Zelles, 1999). PLFAs are components of phospholipids
that are essential parts of membranes found in all living cells. Certain signature
fatty acids in the overall PLFA profile are specific for groups of bacteria, fungi,
and actinomycetes (Tunlid and White, 1992). The biomass of these groups can be
studied once fumigants are applied to any soil because this will represent the
living component of the population. Analysis of PLFA profiles of soils fumigated
with MeBr, MITC, 1,3-D, and CP was carried out over a 12-week period after
application. Biomarker peaks were analyzed and were determined to range from a
minimum of 1.3 nmol g21 dry wt for the four fumigants (week 1) to a maximum
of 55 nmol g21 dry wt for the 1,3-D- and CP-treated samples in week 12 (Ibekwe

et al., 2001a). The biomass contents, as indicated by the total PLFA, were
significantly different at different time points in some treatments ðp , 0:05Þ: At
week 1, the biomass contents in MeBr-amended microcosms were significantly
lower than those in weeks 8 and 12 (Fig. 3), and of the three other fumigants
(Ibekwe et al., 2001a). There was also a decrease in biomass of some of the
Gram-negative (cy17:0, 15:0, and 18:1v7c) and the fungal (18:2v6c) biomarkers, with increases in MeBr concentration during the first week (Fig. 3).
There was a significant increase in biomass for Gram-positive bacteria (a17:0,
i17:0), fungi (18:2v6c), and actinomycetes (10me 16:0) in weeks 8 and 12. 1,3-D
and CP had the strongest effects on actinomycetes, resulting in a significant
decrease in biomass for most treatments. The effects of MITC followed the same


12

A. M. IBEKWE

Figure 3 Biomass contents (nmol of PLFA g21 dry wt of soil) of samples collected after weeks 1,
8, and 12 from MeBr fumigated soils ðn ¼ 3Þ: MeBr1, -8, and -12 indicates samples were taken 1, 8,
and 12 weeks after the start of the experiment. 1, 2, and 3 after the “-” sign indicates 50% below field
application rate, field application, and 1000% above field application rates, respectively. Error bars
represent standard deviation.

trend as MeBr, except that the recovery of Gram-negative bacteria biomass did
not occur during week 8. The effects of fumigants on microbial biomass may be
short term with biomass recovery after a few weeks, as was seen with MeBr,
MITC, and 1,3-D, or it may be long term, as was observed with CP.

C. CHANGES IN SOIL MICROBIAL COMMUNITY STRUCTURE
AND COMPOSITION
Several methods to characterize microbial communities in soils that do not

depend on culturing have been recently developed. These methods were based on
the analysis of biomarkers, such as 16S rRNA genes, PLFA, and respiratory
quinones (Morgan and Winstanley, 1997). Analysis of 16S rRNA genes in soil
was used to detect the long-term effects of phenylurea herbicides on soil
microbial communities (El Fantroussi et al., 1999). The analysis of PLFA
was applied to detect the long-term microbial effects of heavy metals