Nematodes As Bioindicators of Soil Food Web

NEMATODES AS BIOINDICATORS OF SOIL FOOD WEB

HEALTH IN AGROECOSYSTEMS: A CRITICAL ANALYSIS

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By SHABEG SINGH BRIAR * * * * *

The Ohio State University 2007

Dissertation Committee: Professor Parwinder S. Grewal, Adviser Professor Sally A. Miller, Adviser Professor Casey W. Hoy Professor Landon H. Rhodes Approved by Advisers

______

Plant Pathology Graduate Program

Abstract

Nematodes occupy a central position in the soil food web occurring at multiple trophic levels and, therefore, have the potential to provide insights into condition of the soil food webs. I hypothesized that differences in management strategies may have differential effects on nematode community structure and soil properties. This

hypothesis was tested in three different replicated experiments. In the first study a

conventional farming system receiving synthetic inputs was compared with an

organically managed system and in the second study four different farming strategies

with and without compost application transitioning to organic management were

compared for nematode communities and soil characteristics including soil bulk

density, organic matter, microbial biomass and mineral-N. The third study was aimed

at assessing the indicative value of various nematode measures in five habitats.

Nematode food webs were analyzed for trophic group abundance and by calculating

MI, and enrichment (EI), structure (SI) and channel indices (CI) based on weighted

abundance of c-p (colonizer-persister) guilds.

Bacterivore nematodes were more abundant in the organic than the conventional

whereas the conventional system had higher population of the root lesion nematode,

Pratylenchus crenatus compared with organic system. Organic hay plots had the lowest

populations of P. crenatus compared to corn, soybeans and oats. Enrichment

opportunists c-p 1 and general opportunists c-p 2 bacterivores were higher in composted than the non-composted strategies. Population of Pratylenchus sp. was significantly lower but that of c-p 2 plant parasites Tylenchus and Filenchus were higher in the composted compared with non-composted amended soils. Principal components analysis indicated that MI, SI and EI contributed the most to differences between the habitats. Forests and shrublands had highly structured food webs as expected but their high enrichment may be unique due to the muck soils. Croplands showed a clear distinction from other habitats with less structured food webs. Turfgrass lawns occupied an intermediary position between disturbed and unmanaged habitats. Lack of increase in SI in the organic farming system could be due to the high tillage requirement, which might have suppressed the population of high c-p value tillage- sensitive nematodes.

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DEDICATED TO MY PARENTS

ACKNOWLEDGMENTS

During the course of my thesis work, there were many people who were

instrumental in helping me. Without their guidance, help and patience, I would have never been able to accomplish the work of this thesis. First, I wish to thank my advisers, Dr. Parwinder Grewal and Dr. Sally Miller for intellectual support, encouragement, and enthusiasm, which made this thesis possible, and for their patience in correcting both my stylistic and scientific errors.

I thank my advisory committee members, Dr. Casey Hoy and Dr. Landon

Rhodes for their guidance, help, academic support and constructive suggestions that

opened my eyes to much deeper insight in my research project. Also I want to thank all

members of Dr. Grewal’s lab for their compassion, friendship and help, especially Dr.

Ganpati Jagdale. Also I want to thank all the faculty members and staff of the

Department of Plant Pathology for their help, time and the highest level of education. I

would like also, to express my deep respect and gratitude for all members of the

Department of Entomology, where I conducted my entire research project. I do not

want to forget to thank my wife (Ruby), and two sons (Robin and Monty) for their

patience during stressful times.

VITA

April 3, 1973...... Born-Faridkot, India.

1995 -1997………………………………Master of Science, Nematology and Plant

Pathology, Punjab Agricultural University,

Ludhiana, Punjab, India.

2003-Present…………………………….Graduate Research Associate, The Ohio State

University.

EMPLOYMENT

September 1998-March 2003…………...Assistant Professor of Nematology, Punjab

Agricultural University, Punjab, India.

September 1997-August 1998………...Research Associate, Department of Plant

Pathology, Punjab Agricultural University,

Ludhiana, Punjab, India.

FIELD OF STUDY

Major: Plant Pathology

TABLE OF CONTENTS

Page

Abstract ...... ……..ii

Dedication ...... ……..iv

Acknowledgments...... ….....v

Vita...... …...... vi

List of Tables...... …....ix

List of Figures...... ……....xii

Chapters:

1. Introduction...... ………………………....1

2. Soil nematode community, organic matter, microbial biomass and nitro gen dynamics in field plots transitioning from conventional to organic management………...... …….…...... 11

2.1 Introduction……………………………………………………………….……...... 11

2.2 Materials and methods….…………………………………….…………………....14

2.3 Results…………………………………………………………………..……….....18

2.4 Discussion……………………………………………………………………….....20

2.5 Summary…………………………………………………...…………………...... 24

2.6 References……………………………………………………………………...... 26

3. Effect of compost or non-compost based organic transitioning strategies on soil nematode community, organic matter, microbial biomass and nitrogen dynamics…...44

3.1 Introduction……………………………………………………………………...... 44

vii

3.2 Materials and methods……………………………………….………………….....47

3.3 Results………………………………………………………………………..……51

3.4 Discussion………………………………………………….……………...………55

3.5 Summary……………………………………………...…………………………...59

3.6 References………………………………...…………………………………….…61

4. An assessment of the indicative value of nematode trophic group abundance

and food web indices in differentiating habitats with a gradient of anthropogenic

impact…...... ….79

4.1 Introduction………..……………………………………………………………...79

4.2 Materials and methods…..………………………………………………………..82

4.3 Results……………………………………………………………………………85

4.4 Discussion..………….……………………………….…………………………...88

4.5 Summary………………………………………………………………………….93

4.6 References………………………………………………………………………...94

5. Synthesis and future directions……………………………………………….……..109

Bibliography………………………………………………………………………..122

viii

LIST OF TABLES

Table Page

2.1 List of nematode genera identified at the experimental site in conventional and

organic farming system plots …………………………………………………….35

2.2 Effect of farming systems on nematode trophic groups, root lesion nematode

Pratylenchus crenatus and nematode community indices including

enrichment (EI), structure (SI), and channel index (CI) from spring 2000

to spring 2004…………….....…………………………………………..………..36

2.3 Effect of farming systems on soil bulk density (BD), total organic

matter % (TOM), particulate organic matter (0.05-2 mm) (POM), % mineral

+ associated organic matter (MAOM) (<0.05 mm), mineral-N (NH4 -N

- -1 and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N).……….……....37

2.4 Mean soil bulk density (BD), total organic matter % (TOM), particulate organic

matter (0.05-2 mm) (POM), % mineral associated organic matter

+ - -1 (MAOM) (<0.05 mm), mineral-N (NH4 -N and NO3 -N (µg g dry soil), and

microbial biomass-N (MB-N) in conventional and organic arming

systems…………………………...... 38

3.1 Description cropping sequences of four main plot management strategies:

fallow, mixed species hay, low intensity vegetables as open field

vegetables and intensive vegetable production under high tunnels...…….………69

3.2 Effect of main plot strategies and compost on nematode c-p groups

and food web indices [enrichment (ei), structure (si), and channel

index (ci)]..…….….…...... …...... 70

3.3 Comparison between main plot strategies with and without compost on

nematode c-p guilds (Bf: bacterivore; Ff: fungivores; Om: omnivores;

Pr: predatory and Pp: plant-parasites) from 2003 and 2006 soil

sampling..…………………...... …………………....71

3.4 Effect of main plot strategies and compost on soil properties [bulk density

(BD), organic matter % (TOM), % mineral associated organic matter (MAOM)

(<0.05 mm), particulate organic matter (0.05-2 mm) (POM), mineral-N

+ - -1 (NH4 -N and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N)

(µg g-1)]……………...... ……..73

3.5 Mean (SE) of soil properties [bulk density (BD), organic matter % (TOM), %

mineral associated organic matter (MAOM) (<0.05 mm), particulate

+ - -1 organic matter (0.05-2 mm) (POM), mineral-N (NH4 -N and NO3 -N (µg g dry

soil), and microbial biomass-N (MB-N) (µg g-1)] in various

strategies….….……...... ……….....74

4.1 Description of different habitats at Huron and Wayne Counties in Ohio……...... 101

4.2 Mean (± SE) values of different nematode indices including maturity (MI),

plant parasitic (PPI), enrichment (EI), structure (SI), channel (CI)

and Shannon diversity indices (H’) and trophic groups used in the multivariate

analysis.…………...... …...102

4.3 Multivariate test (Pillai’s trace) based on different nematode indices

including MI, PPI, EI, SI, CI and H’ comparing the habitats within muck

soils……………...... …...103

4.4 Multivariate test (Pillai’s trace) based on different nematode indices including

MI, PPI, EI, SI, CI and H’ comparing turfgrass lawns vs croplands habitats

within mineral soils and with muck soils turfgrass lawns and

croplands………...... ……..104

4.5 Eigenanalysis of the correlation matrix for nematode indices including

MI, PPI, EI, SI, CI and H’ for all

habitats………………………...... ……………….……………..105

4.6 Eigenanalysis of the correlation matrix for nematode trophic groups for all

habitats…………………………………………………………………………..106

5.1 List of genera of plant-parasitic nematodes………..……………………………118

LIST OF FIGURES

Figure Page

1.1 Framework of nematode faunal analysis as an indicator of the likely conditions

of the soil food web…………………………………………….……...... 4

2.1 Bars represent mean precipitation in inches per month from year 2000-2004. Line

graph shows a mean of 5 years air temperature each month from January to

December…………………………………………………………………………..39

2.2 Mean (± SE) nematode trophic groups: bacterivore (A), fungivore (B), omnivore

(C), predatory (D), total plant-parasitic nematodes (E) and root lesion nematode

Pratylenchus crenatus (F) per 10g soil in organic and conventional farming

systems from spring 2000 to spring 2004.…………………...... …...... 40

2.3 Mean (± SE) soil population of Pratylenchus crenatus from year 2000 to 2003

comparing corn, soybean, oats and hay. Treatments within a group with the same

letter are not significantly different (p ≤ 0.05) by Fishers’s LSD test between the

crops…………………………………………………………………………….....41

2.4 Mean (± SE) values of nematode food web indices: enrichment (A),

structure (B), and channel (C) indices per 10g soil in organic and conventional

farming system from spring 2000 to spring 2004...... 42

xii

2.5 Comparison and temporal progression of the food web indicated by nematode

faunal analysis in organic and conventional farming systems from spring 2000 to

spring 2004. Numbers 1-9 represent the progression of changes in the fauna

in each farming system...……….…………………………………………………43

3.1 Effect of various strategies on abundance of root lesion nematode

Pratylenchus and root hair feeders (Tylenchus + Filenchus).……………………...75

3.2 Food webs indicated by nematode faunal analysis in 8 sub plot management

strategies in 2003-2006.……………………..………………...... …….76

3.3 Canonical correlation analysis of soil factors vs nematode c-p guilds………...... 77

3.4 Bars represent month mean soil temperature (January-December)…………...... 78

4.1 Principal components analysis of nematode indices.………………………….....107

4.2 Principal components analysis of nematode trophic group abundance.….…...... 108

5.1 Percent ectoparasites, free-living and endoparasites in total nematode

community in different habitats………...... ……………….…...109

5.2 Comparison of the abundance of ectoparasitic nematodes in corn, soybeans, oats

and hay………………………………………………………………………...... 120

5.3 Effect of composted and non-composted farming strategies on Pratylenchus sp.

and plant-parasitic nematode c-p guild 2 consisting mainly of Tylenchus and

Filenchus……………………………………………………………………….....121

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CHAPTER 1

INTRODUCTION

Nematodes or roundworms inhabit virtually all ecosystems including marine,

freshwater, and terrestrial environments. They encompass the phylum Nemata that

includes plant, insect, animal and human parasites and predatory forms that consume

microorganisms including bacteria, fungi, algae, and other nematodes (Platt, 1994). In

general nematodes are small, usually less than a millimeter in length, with the exception

of some animal parasitic nematodes, which are rather large and can be seen by the naked eye (Wallace et al., 1996). Several nematode genera are important as pests of crops and animals while others contribute to nutrient mineralization (Ferris and Matute, 2003) and

control of plant pests and pathogens (Grewal et al., 2005).

The soil food web a plethora of soil organisms including bacteria, fungi,

nematodes, annelids and arthropods is dependent primarily on autotrophic input from

plants or other external sources including organic matter (Ferris and Bongers, 2006).

Assessment of soil health through complete analysis of such a diverse group of organisms

may require several extraction techniques, which is technically daunting (Ritz and

Trudgill, 1999; Ferris and Bongers, 2006). Alternate to this is the use of biotic indicators

of soil ecological health but any indicator used should reflect the structure and function of

ecological processes and must respond to soil conditions (Neher, 2001).

Nematodes have been evaluated for their use as indicators due to several attributes they possess. They are frequently the most numerous component of the microfauna and are considered as the most abundant and diverse invertebrates present in the soil (Yeates,

1979). Although, nematodes represent a relatively small amount of biomass in the soil, their occurrence across multiple trophic levels is vitally important in the soil environment

(Barker and Koenning, 1998). Since their feeding habits are clearly related to oral structure, their trophic roles are readily inferred. Each soil sample contains an abundance

and diversity of nematodes and, consequently, has high intrinsic information value

(Bongers and Ferris, 1999; Yeates et al., 1993). In addition to this, nematodes are easy to

extract from the soil using simple extraction procedures (Ritz and Trudgill, 1999).

Yeates et al. (1993) categorized nematodes into five generally recognized trophic

groups: bacterivores, fungivores, predators, omnivores and plant parasites. Bongers

(1990) classified nematodes along a colonizer-persister (c-p) continuum of 1-5.

Nematodes with c-p value equal to one are short lived, have high fecundity, feed on

enriched media whereas those of c-p value five are have large body size, longer life span,

low fecundity, susceptible to disturbance and are predominantly omnivores and predators

(Bongers, 1990). C-p classifications of nematodes lead to the formation of the maturity

index (MI), which is a weighted mean frequency of c-p scaling across the entire

nematode community and provides the information of the likely condition of the soil

environment (Bongers, 1990). The development of MI represented a significant advancement in interpreting the relationships between the ecology of nematode communities and functions of the soil (Neher et. al., 2005). However, Ferris (1993) argued that use of trophic groups could often lead to ambiguous results since the trophic groups encompass an enormous diversity of life history and physiological characteristics.

Calculation of the MI index also assumes a progression of soil conditions from stressed or polluted to pristine exactly congruent with the continuum of nematode life history characteristics in the c-p classification as suggested by Bongers (1990). Ferris et al.

(2001) observed that the most abundant nematode taxa under stressed conditions are those in c-p 2, while the enrichment opportunists (c-p 1) respond positively to disturbances that result in enrichment of the food web. Therefore, in an attempt to improve the indicator capabilities of nematodes, Ferris et al. (2001) assigned weights to indicator nematode guilds representing basal, enriched and structured conditions of the food web. This concept leads to the development of food web indices including enrichment (EI) and structure index (SI). EI is based on the expected responsiveness of the opportunistic guilds (bacterivore nematodes with c-p value equals one) to organic resources enrichment. Therefore, EI describes whether the soil environment is nutrient enriched (high EI) or depleted (low EI). SI represents an aggregation of functional guilds with c-p values ranging from 3-5 and describes whether the soil ecosystem is structured with greater trophic links (high SI) or degraded (low SI) with fewer trophic links (Ferris et al., 2001). Plotting of EI and SI provide a model framework of nematode faunal analysis as an indicator of the likely conditions of the soil food web (see figure 1.1).

Further, Ferris et al. (2001) also proposed the channel index (CI), which is a percentage

of fungivores among the total fungivores and c-p one opportunists bacterivores. CI

indicates predominant decomposition channels in the soil food web, a high CI (> 50 %)

indicates fungal decomposition channels whereas low CI (< 50 %) suggests bacterial

decomposition channels (Ferris et al., 2001). In later studies use of these indices provided critical information about below ground processes in distinct agroecosystems

(Bulluck et al., 2002b; Ferris and Matute, 2003; Neher et al., 2005).

From Ferris et al., 2001.

Figure 1.1: Functional guilds of soil nematodes characterized by feeding habit (trophic group) and by life history characteristics expressed along a colonizer-persister (cp) scale

(cp scale proposed by Bongers and Bongers, 1998). Bax (bacterivores), Fux (fungivores),

Cax (carnivores), Omx (omnivores) (where value of x = 1-5 on the cp scale) represents various functional guilds. Indicator guilds of soil food web condition (basal, structured, enriched) are designated and weightings of the guilds along the structure and enrichment trajectories are provided, for determination of the enrichment index (EI) and structure index (SI) of the food web.

Synthetic fertilizers, pesticides, and herbicides are important inputs in

conventional agricultural systems and have been shown to impact diversity and abundance of nematode trophic groups (Yeates and Bongers, 1999). However, organic

farming depends on the addition of organic matter to the soil in the form of green manure

cover crops and decomposed or partially decomposed animal wastes such as poultry or

beef manure. These alternate crop raising and management strategies aim at reduction or

elimination of synthetic inputs (McSorley and Frederick, 1999), environmental pollution

due to chemical fertilizers and pesticides, and losses emanating from diseases and pests.

Organic amendments have been shown to reduce soil bulk density and increase soil

nitrogen and carbon supply (Drinkwater et al., 1995, Bulluck et al., 2002a). Although various organic amendments can have differential effects on soil properties and nematode communities (Nahar et al., 2006), all tend to increase availability of nutrients, such as nitrogen, microbial biomass and abundance of bacterivore and fungivore nematodes

(Bulluck et al., 2002; Ferris et al, 1996). An increase in organic matter in the soil increases microbial biomass by providing an enlarged food base for free-living nematodes (Papatheodorou et al., 2004). We hypothesized that differences in inputs and management strategies employed in organic and conventional farming would have

differential effects on soil properties and nematode community structure that may have

consequences for the sustainability of the agroecosystems.

In the present study we intended to thoroughly investigate the model proposed by

Ferris et al. (2001) for the assessment of the soil food webs in different farming strategies

practiced in Ohio. Specific objectives were to 1) evaluate the effects of crops and different management strategies on the dynamics of nematode communities (free-living and plant parasitic) in replicated field plot trials during transition from conventional to organic management, 2) determine the relationship between nematode c-p (colonizer- persister) guilds and soil properties and 3) quantify the relative importance of nematode community measures to discriminate habitats using multivariate analysis.

THESIS

Nematodes are a diverse, highly speciated group of organisms, occupy key positions at most trophic levels in soil food webs and therefore, have the potential to provide insights into soil health. Differences in management strategies may have differential effects on nematode community structure and soil properties. These changes are effectively reflected by the nematodes. A development of practical understanding from the research generated would help in choosing farming strategies for increasing beneficial free-living nematodes and suppressing harmful plant parasitic nematodes.

REFRENCES

Bongers, T., 1990. The maturity index: an ecological measure of environmental

disturbance based on nematode species composition. Oecologica 83, 14-19.

Bulluck III, L.R., Brosius, M., Evanylo, G.K., Ristaino, J.B., 2002a. Organic and

synthetic fertility amendments influence soil microbial, physical and chemical

properties on organic and conventional farms. App. Soil Ecol. 19, 147-160.

Bulluck III, L.R., Barker, K.R., Ristaino, J.B., 2002b. Influences of organic and synthetic

soil fertility amendments on nematode trophic groups and community dynamics

under tomatoes. Appl. Soil Ecol. 21, 233-250.

Ferris, H., Bongers, T., 2006. Nematode indicators of organic enrichment. J. Nematol.

38, 3-12.

Ferris, H., 1993. New frontiers in nematode ecology. J. Nematol. 25, 374-382.

Ferris, H., Venette, R.C., Lau, S.S., 1996. Dynamics of nematode communities in

tomatoes grown in conventional and organic farming systems and their impact on

soil fertility. Appl. Soil Ecol. 3, 161-175.

Yeates, G.W., Bongers, T., De Goede, R.G.M., Freckman, D.W., Georgieva, S.S., 1993.

Feeding habits in soil nematode families and genera–an outline for soil ecologists.

J. Nematol. 25, 315-331.

Barker, K.R., Koenning, S.R., 1998. Developing sustainable systems for nematode

management. Ann. Rev. Phytopathol. 36, 165-205.

Drinkwater, L.E., Letourneau, D.K., Workneh, F., van Bruggen, A.H.C., Shennan, C.,

1995. Fundamental differences between conventional and organic tomato

agroecosystems in California. Ecol. Appl. 1098-1112.

Ferris, H., Bongers, T., de Geode, R.G.M., 2001. A framework for soil food web

diagnostics: extension of the nematode faunal analysis concept. Appl. Soil Ecol.

18, 13-29.

Ferris, H., Matute, M., 2003. Structural and functional succession in the nematode fauna

of a soil food web. Appl. Soil Ecol. 23, 93-110.

Grewal, P.S., Ehlers, R.U., Shapiro-Ilan, D.I. (Eds.). 2005. Nematodes As Biocontrol

Agents. CABI Publishing, CAB International, Oxon, U.K., pp. 1-505

McSorley, R., Frederick, J.J., 1999. Nematode population fluctuations during

decomposition of specific organic amendments. J. Nematol. 31, 37-44.

Nahar, M.S., Grewal, P.S., Miller, S.A., Stinner, D., Stinner, B.R., Kleinhenz, M.D.,

Wszelaki, A., Doohan, D., 2006. Differential effects of raw and composted

manure on nematode community, and its indicative value for soil microbial,

physical and chemical properties. App. Soil Ecol. 34, 140-151.

Neher, D.A., 2001. Role of nematodes in soil health and their use as indicators. J.

Nematol. 33, 161-168.

Papatheodorou, E.M., Argyropoulou, M.D., Stamou, G.P., 2004. The effects of large-

and small-scale differences in soil temperature and moisture on bacterial

functional diversity and the community of bacterivorous nematodes. App. Soil

Ecol. 25, 37-49.

Platt, H.M., 1994. In The Phylogenetic Systematics of Free-living Nematodes, S.

Lorenzen, (Ed.) The Ray Society, London, pp 383.

Porazinska, D.L., Duncan, L.W., McSorley, R., Graham, J.H., 1999. Nematode

communities as indicators of status and processes of a soil ecosystem influenced

by agricultural management practices. Appl. Soil Ecol. 13, 69-86.

Ritz, K., Trudgill, D.L., 1999. Utility of nematode community analysis as an integrated

measure of the functional state of soils: perspectives and challenges. Plant Soil

212, 1-11.

Wallace, R.L., Ricci, C., Melone, G., 1996. A cladistic analysis of pseudocoelomate

(aschelminth) morphology. Invertebrate Biology 115, 104-112.

Yeates, G.W., 1979. Soil nematodes in terrestrial ecosystems. J. Nematol. 11, 213-229.

Yeates, G.W., Bongers, T., 1999. Nematode diversity in agroecosystems. Agric. Ecosys.

Environ. 74, 113-135.

Neher, D.A., Wub, J., Barbercheck, M.E., Anas, O., 2005. Ecosystem type affects

interpretation of soil nematode community measures. App Soil Ecol. 30, 47-64.

CHAPTER 2

SOIL NEMATODE COMMUNITY, ORGANIC MATTER, MICROBIAL

BIOMASS AND NITROGEN DYNAMICS IN FIELD PLOTS TRANSITIONING

FROM CONVENTIONAL TO ORGANIC MANAGEMENT

2.1 INTRODUCTION

Nematodes are abundant and diverse invertebrates present in the soil (Yeates,

1979). They are considered an important component of the soil biotic community and

assessment of nematode fauna provides a unique insight into soil biological processes

(Ritz and Trudgill, 1999). Nematodes play a critical role in decomposition and nutrient cycling (Ferris et al., 2004; Ferris and Matatue, 2003; Ingham et al., 1985; Wright, 1998).

Free-living nematodes that feed on bacteria and fungi (as opposed to plants) contribute as much as 27% of the readily available nitrogen in the soil (Ekschmitt et al., 1999) and also promote rhizosphere colonization of beneficial rhizobacteria (Kimpinski and Sturz, 1996;

Knox et al., 2003). Nahar et al. (2006) observed a negative correlation between free-

living and plant parasitic nematodes in organically grown tomatoes. Therefore, one of

the major goals of sustainable agriculture should be to enhance populations of free-living

nematodes and reduce that of plant-parasitic nematodes.

Synthetic fertilizers, pesticides, and herbicides are important inputs in

conventional agricultural systems. Insecticide and mineral fertilizer applications have

been shown to impact diversity and abundance of nematode trophic groups (Yardim and

Edwards, 1998; Yeates and Bongers, 1999; Yeates et al., 1999; Sohlenius and

Wasilewska, 1984; Sarathchandra et al., 2001; Sohlenius, 1990; Sohlenius and Boström,

1986; Neher and Olson, 1999). By contrast, organic farming depends on the addition of

organic matter to the soil in the form of green manure cover crops and decomposed or

partially decomposed animal wastes such as poultry or beef manure. These alternate crop

raising and management strategies aim at reduction or elimination of synthetic inputs

(Mian and Rodríguez-Kàbana, 1982; Abawi and Widmer, 2000; McSorley and Frederick,

1999), environmental pollution due to chemical fertilizers and pesticides, and losses

emanating from diseases and pests. Organic amendments have been shown to reduce soil

bulk density and increase soil nitrogen and carbon supply (Doran, 1995; Drinkwater et

al., 1995, Bulluck et al., 2002a; Werner, 1997). Although various organic amendments

can have differential effects on soil properties and nematode communities (Nahar et al.,

2006), all tend to increase availability of nutrients such as nitrogen, microbial biomass

and abundance of bacterivore and fungivore nematodes (Gunapala and Scow, 1998;

Freckman, 1988; Griffiths et al., 1994; Bulluck et al., 2002b; Ferris et al, 1996, 1999).

Increase in organic matter in the soil increases microbial biomass by providing an enlarged food base for free-living nematodes (Papatheodorou et al., 2004; Alon and

Steinberger, 1999; Bongers and Ferris, 1999; Ferris et al., 1999; McSorley and Frederick,

1999). The U.S. National Organic Standards Act requires an organic farming certification process to manage soil fertility through crop rotation and cover crops,

supplemented with animal and crop wastes, while strictly restricting synthetic chemical

inputs. Although addition of organic amendments improves soil health, frequent tillage is

required for mixing organic inputs and to manage weeds in certified organic farms.

Frequent tillage may affect the soil ecosystem by changing the microbial and nematode

trophic structure (Parmelee and Alston, 1986; Fu et al., 2000; Ferris et al., 2001, 2004;

Wang et al., 2004). Berkelmans et al. (2003) observed that organic and low synthetic

input farming practices favored multiplication of bacterivore, omnivore and predatory nematodes and suppressed root lesion nematode (Pratylenchus thornei) compared to conventional management. They also observed that severe disruption caused by frequent tillage operations in the organic farming system decreased the structure index (SI) (an indicator of greater trophic links and healthy ecosystem; Ferris et al., 2001) to the level of a conventional system. Other factors such as soil compaction, irrigation and crop rotation may also impact the soil nematode community (Bouwman and Arts, 2000; López-Fando and Bello, 1995; McSorley and Gallaher, 1994).

This paper compares the long-term effect of two farming systems, which differ

primarily in external inputs and crop rotation cycles under a field crops agroecosystem.

We hypothesized that differences in management and cropping strategies employed in organic and conventional farming would have differential effects on soil properties and nematode community structure thus impacting the sustainability of the systems. This research is part of the “Organic Food and Farming Education and Research” (OFFER) program at the Ohio Agricultural Research and Development Center (OARDC), Wooster,

Ohio. The conventional farming system had a corn-soybean rotation and the organic farming system had a 4-year corn-soybean-oats-hay rotation. Crop rotations were

selected keeping in mind the dominant farming practices on agricultural landscapes of

Ohio. To compare between the farming systems we chose the following indicators:

nematode feeding groups (trophic abundance) and nematode food web indices

(enrichment, basal, structure and channel indices), soil organic matter, microbial biomass

and nitrogen dynamics. Assessment of different nematode trophic groups provide

comprehensive insight into soil biological processes (Ritz and Trudgill, 1999) and food

web indices provides critical information about below ground processes in

agroecosystems (Bulluck et al., 2002b; Porazinska et al., 1999; Freckman and Ettema,

1993; Bongers, 1990; Ferris, et al., 1996; Ferris et al., 2001). Nematode faunal analysis developed by Ferris et al. (2001) provides a graphical representation of the ‘basal’,

‘structural’ and ‘enrichment’ conditions of the soil food web. Our specific objectives in this study were to evaluate the effects of crops and management strategies on the dynamics of organic matter, microbial biomass, nitrogen, and nematode communities

(free-living and plant parasitic) in replicated field plots during transition from conventional to organic management.

2.2 MATERIALS AND METHODS

2.2.1. Site description, treatments and field plot design

A long-term field crop transition experiment was established in spring 2000 at the

field crops research area at the OARDC, Wooster, Ohio (40° 47' N, 81° 55' W; 310 m

elevation). Monthly average precipitation and air temperature for the period of study are shown in Figure 2.1. The soil at the experimental site was classified as Wooster silt loam. The experiment had a split block design with two farming systems as main plots,

replicated 6 times and all phases of the rotation were present every year. Each block consisted of four subplots representing complete rotation in organic and two subplots representing complete rotation in conventional farming system. The organic farming system comprised of a corn-soybeans-oats-hay (mixture of red clover and timothy) rotation and the conventional system had corn-soybeans rotation. Individual plots measured 18 x 18 m. Organic corn received raw straw pack beef manure at the rate of 27

Mg/ha and poultry compost manufactured by DayLay at the rate of 28 Mg/ha. Oats received raw straw pack manure at the rate of 18 Mg/ha and poultry compost 1.8 Mg/ha.

The conventional corn received starter fertilizers (19:19:19) at the rate of 192 kg and side dressed with liquid fertilizer (28% N) 385 L per ha. Crops in both systems were rainfed and there was no external source of Irrigation. Primary tillage in organic plots was given

by chisel plow, disked or harrowed 6” deep twice and tined. Weed control was

mechanical and typical operations included at least two diskings and or field cultivations before planting, two rotary hoeings or tine weedings soon after planting and two row cultivations. Conventional soybeans were sown without cultivation and primary tillage

was applied only in corn plots. In the conventional farming system weeds were managed

using herbicides Accent (nicosulfuron) at the rate of 162g + 70g Exceed (prosulfuron)

and 625ml Round-up [N- (phosphonomethyl) glycine] per ha in corn and soybeans, respectively.

2.2.2. Soil sampling

Soil samples were collected twice each year during spring before addition of

organic matter at the beginning of cropping season and in autumn after crop senescence or at the end of the cropping season. Three soil cores (5 cm diameter and 15 cm deep)

were removed from each plot using a soil auger from three positions: i) in row, ii) in-

between rows and iii) mid way between the two. Samples were mixed thoroughly to

form a composite sample to reduce the variance associated with aggregated spatial

patterns of nematodes in the soil (Barker and Campbell, 1981). Large plant parts or

stones were removed from the samples by passing them through a soil sieve (6 mm

mesh). All soil samples were stored in the dark overnight at 5oC and existing field

moisture was maintained to minimize changes in nematode population (Barker et al.,

1969). Soil samples were collected nine times from spring 2000 to spring 2004.

2.2.3 Nematode extraction, identification and counting

Nematodes were extracted from a 10 g subsample taken from each composite soil

sample using the Baermann funnel technique (Flegg and Hooper, 1970). Nematodes

were collected after 72 h, heat killed, and fixed with triethanolamine formaldehyde (TAF)

solution (Shepherd, 1970). Specimens were identified to the genus level using an

inverted microscope at 40X magnification. Diagnostic keys by Goodey (1963), Siddiqui

(1986), and Jairajpuri and Ahmed (1992) and the University of Nebraska Lincoln nematode identification website (http://nematode.unl.edu/konzlistbutt.htm) were used.

All identified nematode genera were assigned to a trophic group (plant-parasitic, fungal

feeder, bacterial feeder, omnivore, or predatory) according to Yeates et al. (1993).

Nematode genera were also assigned a colonizer-persister value (c-p value) according to

Bongers (1990). Numbers of nematodes were not corrected for extraction efficiency.

2.2.4. Soil properties

Soil bulk density, organic matter, mineral associated organic matter (<0.05 mm),

+ - particulate organic matter (0.05-2 mm), mineral-N (NH4 -N and NO3 N), and dissolved

organic-N, were analyzed at the Field Crop Ecology Laboratory, OARDC, Wooster,

using standard methods (Sims et al., 1995). Microbial biomass-N was determined using modified chloroform fumigation method (Brookes et al., 1985).

2.2.5. Food web evaluation

The enrichment index (EI), structure index (SI) and basal index (BI) were

calculated according to Ferris et al. (2001), with basal components (b) of the food web

(fungal and bacterial feeders in the c-p 2 guild) calculated as b = ∑kbnb where kb is the

weighted constant for the guild, and n is the number of nematodes in that guild.

Enrichment (e) and structure (s) components were similarly calculated, using nematode

guilds indicative of enrichment (bacterivores of c-p 1, and fungivores of c-p 2), and

guilds supporting structure (bacterivores of c-p 3-5, fungivores of c-p 3-5, omnivores of

c-p 3-5, and predatory nematodes of c-p = 2-5). Finally, the EI was calculated as 100x

e/(e + b), and the SI as 100x s/(s + b). Channel Index (CI), which provides an index of

nature of decomposition, was calculated as 100 x (0.8 fungivores cp 2/ (3.2 of

bacterivores of c-p 1 + 0.8 fungivores of c-p 2) where the coefficients are the ke

enrichment weightings for the respective guilds (Ferris et al., 2001).

2.2.6. Statistical analysis

Farming systems were compared for nematode trophic groups, various food web

indices, and soil properties, using repeated measures analysis of variance (PROC GLM,

SAS Ver. 9.00, SAS Institute, Cary, NC) and F-values for the split block experimental

design were obtained using an appropriate error term in the model. Results from both

multivariate and univariate repeated measures are presented. No significant interaction

was observed between crops and farming systems except for population abundance of

root lesion nematode Pratylenchus crenatus. Therefore the comparison between the crops was made only for this nematode using Fisher’s LSD mean separation test. Nematode

population data were transformed as ln (x+1) prior to statistical analysis to normalize the

variance in the data.

2.3. RESULTS

2.3.1. Effect of farming systems and crop rotation on nematode communities

Nematode genera identified are shown in Table 2.1 in both farming systems.

Among the most abundant genera were Rhabditis, Cephalobus, Acrobeloides,

Monhystera, Mononchus, Aphelenchoides, Aphelenchus, Pratylenchus, Paratylenchus,

Tylenchus and Filenchus. A small number of juveniles of Heterodera and Longidorus

were found, and only until 2001. Mesodorylaimus, Wilsonema, Placodera, Psilenchus,

Hoplotylus, Helicotylenchus, Trichodorus and Tylenchorynchus were found in low

numbers in both farming systems. Although farming systems differed in abundance of

trophic groups, all genera identified occurred common in both farming systems.

Populations of bacterivore nematodes were significantly higher in organic than

the conventional farming system for most of the study periods (Fig. 2.2A). Overall test

for comparison also showed that farming systems differed significantly for the abundance

of bacterivore nematodes (F = 13.94, P = 0.01) (Table 2.2). No significant differences

were observed between the farming systems for fungivore nematodes (F = 3.57, P =

0.10), omnivore (F = 0.33, P = 0.59) and predatory nematodes (F = 0.04, P = 0.84)

(Table 2.2, Fig. 2.2 B, C and D). Both multivariate and univariate analyses yielded a

significant time x farming system interactions for fungivore and bacterivore nematodes

while the interaction was non-significant for other trophic groups (omnivores, predatory

and plant-parasites) (Table 2.2). This suggests that farming systems exhibited different

patterns of population change for bacterivore and fungivore nematodes and not for other

trophic groups. Although no significant differences were observed between the farming

systems for total abundance of plant-parasitic nematodes (PP), root lesion nematode

Pratylenchus crenatus a dominant plant-parasitic nematode species, was significantly

higher in the conventional than the organic farming systems (F = 8.33, P = 0.03).

Population of P. crenatus however, increased over time in both farming systems (Fig.

2.2F and Table 2.2). In general, hay plots had lower population of dominant plant parasitic nematode P. crenatus than corn, soybeans and oats (Fig. 2.3).

2.3.2. Effect of farming systems on soil properties and microbial biomass-N

Soil bulk density measured as an indicator of soil compaction did not differ

between the farming systems (F = 0.67, P = 0.49) (Table 2.3). Total soil organic matter,

mineral associated organic matter and particulate organic matter showed a higher trend in

organic farming than the conventional farming system most the sampling times but

overall comparison between the farming systems revealed no statistical significant

- differences (F = 0.52, P = 0.50) (Table 2.3). Soil mineral-N (NO3 ) was higher in

conventional than the organic farming system (F = 0.11.39, P = 0.01) whereas soil

+ mineral-N (NH4 ) (F = 2.19, P = 0.21) and dissolved organic nitrogen (F = 0.96, P =

0.37) did not differ between the farming systems (Table 2.3). Significant interaction

between time and farming systems was only observed for total organic matter (F = 3.57,

- P = 0.003) and soil mineral-N (NO3 ) (F = 3.94, P = 0.001) while the interaction was

non-significant for other soil parameters (Table 2.3). Microbial biomass-N was

significantly higher in the organic as compared to conventional farming system (F =

25.10, P = 0.004) (Table 2.3). In spring 2003, both the farming systems showed a drop in

microbial biomass compared to all other sampling times which was later bounced back in

the next season autumn 2003 (Table 2.3).

2.3.3. Food web evaluation

The structure (F = 2.19, P = 0.14), enrichment (F = 0.47, P = 0.52) and channel

indices (F = 0.69, P = 0.44) were not significantly different between farming systems

(Table 2.2; and Fig. 2.4A, B and C). The enrichment index showed abrupt changes over

time in both farming systems, with lowest values in autumn 2002 and spring 2003 in

conventional and organic, respectively, and highest in spring 2004 in both systems

compared. The structure index increased from spring 2000 to spring 2001 and thereafter declined until spring 2003 and slightly increased in autumn 2003 in both systems (Fig.

2.4A, B and C). Nematode faunal analysis based on plotting enrichment and structure

indices revealed values in quadrat A and B (Fig. 2.5) in both conventional and organic

farming systems. Significant interaction between the farming system and time was only

observed for channel index (F = 3.10, P = 0.008) and not for enrichment (F = 2.05, P =

0.06) and structure indices (F = 1.02, P = 0.43) (Table 2.2).

2.4. DISCUSSION

Soil bulk density monitored as an indicator of soil compaction did not differ

between the two farming systems in this study. Frequent use of agricultural machinery

usually results in increase in soil compaction (Bouwman and Arts, 2000). Therefore, higher frequency of tillage operations in organic compared to the conventional

management should have resulted in higher bulk density in the organic system. The lack

of differences in soil bulk density in the two systems may be attributed to the buffering effect of the large amounts of organic matter added in the organic system. Organic inputs

including the addition of compost, animal manures and cover crops have been shown to

reduce soil bulk density (Werner, 1997; Drinkwater et al., 1995; Bulluck et al., 2002a).

We found that total organic matter, mineral associated organic matter and particulate

organic matter contents were trended higher in the organic than in the conventional

system. These findings are consistent with other studies where applications of organic

amendments have been shown to increase soil organic matter (Clark et al., 1999; Borken

et al., 2002; Nahar et al., 2006; Tu et al., 2006). Therefore, it is possible that the potential

effects of extra tillage on soil bulk density in organic farming are mitigated by the

addition of organic matter.

This study clearly showed that microbial biomass-N was significantly higher in

the organic compared to the conventional farming system. In contrast, N in the mineral

pools was higher in the conventional than the organic system as indicated by significantly

- higher NO3 N pools at least at four sampling times. These findings demonstrate clear

shifts in nitrogen pools between the two systems. Other studies have also observed shifts

in nitrogen pools in organic and conventional systems (Tu et al., 2006; Clark et al., 1999;

Poudel et al., 2001). Although N levels generally start out lower in organic systems,

they increase gradually exceeding those in conventional farming systems by the end of

transition period (Clark et al., 1999). We found that after the transition period was completed, the organic system had slightly higher N than the conventional farming system (although not statistically significant). Therefore, to prevent yield losses

resulting from low availability of N, Tu et al. (2006) suggested gradual or partial

reduction in synthetic inputs in farming systems transitioning to organic instead of

immediate withdrawal of all conventional inputs during the transition period. The fact

that the organic system recovers from N drop after the transition period indicates the

long-term sustainability of the system as the N pools are predominantly controlled by

- microbial biomass compared to highly labile NO3 N in case of the conventional system.

This microbial control of N availability results in slow release of N and reduces N- leaching substantially (Dalal, 1998; Friedel et al., 2001).

The abundance of free-living nematodes, mainly represented by bacterivores, was

higher in the organic than the conventional system for most of the study period. Other

trophic groups including fungivore, omnivore and predatory nematodes were different

between the systems only from autumn 2000 to autumn 2001. Generally, external

organic matter inputs in the form of compost, animal manures and cover crops increase

energy availability for the soil microbes thereby enhancing microbial activity and

biomass (Lundquist et al., 1999; Gunapala and Scow, 1998; Powlson et al., 1987; Alon and Steinberger, 1999). Therefore, the observed increase in the abundance of bacterivore nematodes could be attributed to the increase in the microbial biomass (Ferris et al.,

1996). Further, evidence of the positive relationship between abundance of free-living nematodes and microbial biomass-N comes from the coupled dynamics of these two parameters observed in our study. Both the free-living nematodes and microbial biomass-N showed a sharp decline in spring 2003 in both the farming systems in this study, which was most likely due to extreme dry conditions during the summer of 2002.

Interestingly, microbial biomass levels bounced back to 2002 levels in autumn 2003

followed by bacterivore nematode increase in spring 2004. The delayed response of free-

living nematodes to increase in soil microbial biomass-N has been previously reported by

Papatheodorou et al. (2004) who observed that bacterivore nematodes increased only months after the increase in populations of bacteria in the soil.

Although total populations of plant-parasitic nematodes were not reduced in the

organic system, root lesion nematode P. crenatus was significantly suppressed in the

organic farming compared to the conventional system. Suppression in populations of

plant parasitic nematodes has been shown to be either due to the addition of organic

amendments (Nahar et al., 2006; Abawi and Widmer, 2000; McSorley and Frederick,

1999; Rodríguez-Kàbana et al., 1981; Hominick, 1999) or to the addition of cover crops

into the soil (Sohlenius and Sandor, 1989; Wang et al., 2001), or both. In our study, only

hay reduced P. crenatus population as compared to other crops in the organic rotation.

Therefore, suppression in population of P. crenatus seems to be primarily due to the

inclusion of hay in the organic system. The lack of effect of organic amendments on root

lesion nematode Pratylenchus sp. and root-knot nematode Meloidogyne incognita was

also reported by Bulluck et al. (2002b). However, Nahar et al. (2006) reported that

organic amendments in the form of compost or raw manure can have differential effects

on plant-parasitic nematodes. Nevertheless, our study shows that transitioning to organic

management can reduce populations of the predominant plant-parasitic nematode, P.

crenatus provided appropriate rotational crops are included.

None of the food web indices differed significantly between the two farming

systems. The construction of faunal profiles revealed that food webs either lay in quadrat

A or B in both farming systems suggesting that the food webs were highly enriched and

moderately to highly structured and the decomposition channels were bacterial in both

the systems. Although increase in bacterivore nematodes were observed in the organic

system, no corresponding increases in predatory or omnivorous nematodes were found.

This lack of increase in the higher trophic links in the food web resulted in no differences

in structure index between the two systems. This could be due to frequent tillage

requirement during the preparation of seedbed for mixing of organic manures and cover

crops into the soil and hoeing operations for weed management. It has been reported

previously that several omnivore and predatory nematode genera are more tillage

sensitive than bacterivore nematodes (Fiscus and Neher, 2002; Freckman and Ettema

1993; López-Fando and Bello, 1995). Therefore, it is clear that high tillage in the organic farming system is counterproductive to the beneficial effects of organic amendments and cover crops, as it breaks the natural progression of the soil food web towards maturity.

Therefore, we conclude that the organic farming can result in more mature and highly functional food webs if tillage intensity can be reduced.

2.5 SUMMARY

Dynamics of soil bulk density, organic matter, microbial biomass, nitrogen, and

nematode communities were assessed for a period of four years in field plots

transitioning from conventional to organic farming practices. A rotation of soybeans,

corn, oats and hay was used as an organic transitioning strategy and the conventional

farming system had a corn and soybean rotation for comparison. Organic corn received

raw straw pack beef manure and poultry compost at the rate of 27 and 28 Mg/h

respectively, and organic oats received raw straw pack beef manure and poultry compost

at the rate of 18 and 1.8 Mg/h, respectively while conventional plots received synthetic

fertilizers. All crops in the organic system received primary tillage (chisel plow, disked

and tined) whereas only corn received primary tillage in the conventional system but

soybeans were no-till. Weed control was mechanical (twice diskings, rotary hoeings and

row cultivation) in the organic system whereas herbicides were used in the conventional system. Soil bulk density did not differ in the two systems over a four-year period but organic farming had slightly higher organic matter, mineral associated organic matter and particulate organic matter. The conventional system had more N in the mineral pools as

- indicated by higher NO3 -N whereas organic system had higher N in the microbial

biomass indicating shifts in nitrogen pools between the two systems. Bacterivore

nematodes were more abundant in the organic than the conventional system for most of

the study period. In contrast, the conventional system had significantly higher

populations of the root lesion nematode, Pratylenchus crenatus, than the organic system

after completion of the rotation cycle (transition period) in spring 2004. The organic hay

plots had the lowest populations of P. crenatus compared to corn, soybeans and oats.

Nematode faunal profile estimates showed that the food webs were highly enriched and

moderately to highly structured and the decomposition channels were bacterial in both

systems. The lack of differences in structure index between the organic and conventional

systems is probably due to the excessive tillage in the organic farming system, which

may have prevented the build up of tillage-sensitive omnivorous and predatory

nematodes that contribute to the structure index. We conclude that transition from

conventional to organic farming can increase soil microbial biomass-N and populations

of beneficial bacterivore nematodes while simultaneously reducing the populations of

predominant plant-parasitic nematode, P. crenatus. Our findings also underscore the potential benefits of reducing tillage for the development of a more mature soil food web.

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Bacterivores Plant parasites Omnivores Fungivores Predatory Rhabditis Pratylenchus a Dorylaimus Aphelenchoides Mononchus Mesorhabditis Paratylenchus Eudorylaimus Aphelenchus Rhabdolaimus Tylenchus Pungentus Pelodera Filenchus Mesodorylaimus b Placodera b Xiphinema Labronema Cephalobus Longidorus c Aporcelaimellus b Acrobeloides Hoplolaimus Acrobeles Helicotylenchus Wilsonema b Hoplotylus b 35 Monhystera Tylenchorhynchus b Panagrolaimus Psilenchus b Plectus Trichodorus b Turbatrix Heterodera c Alaimus Diplogasteroides Diplogaster a Identified to species level (Pratylenchus crenatus), bobserved in low numbers, cobserved only until year 2001 in low numbers.

Table 2.1: Nematode genera identified at the experimental site in conventional and organic farming system plots.

Univariate repeated measures Multivariate repeated measures Farming systems Time x Farming system Time x Farming system (df 1, 5) (df 8, 40) (df 8, 17) Trophic groups F P F P* F P Bacterivore 13.93 0.01 5.32 0.0001 7.98 0.0002 Fungivore 3.57 0.10 2.92 0.01 3.21 0.02 Omnivore 0.33 0.59 1.56 0.16 5.39 0.001 Predatory 0.04 0.84 0.87 0.54 0.96 0.50 36 Plant-parasitic 0.46 0.52 1.36 0.24 2.03 0.14 P. crenatus 8.33 0.03 1.87 0.06 2.12 0.09 Indices EI 0.47 0.52 2.05 0.06 1.23 0.34 SI 2.19 0.14 1.02 0.43 2.44 0.057 CI 0.69 0.44 3.10 0.008 0.60 0.76 Farming systems compared using repeated measures ANOVA. *Adjusted Greenhouse-Geisser P-value

Table 2.2: Effect of farming systems on nematode trophic groups, root lesion nematode Pratylenchus crenatus and nematode community indices including enrichment (EI), structure (SI), and channel index (CI) from spring 2000 to spring 2004.

Univariate repeated measures Multivariate repeated measures Soil parameter Farming systems Time x Farming system Time x Farming system (df 1, 5) (df 8, 40) (df 8, 17) F P F P* F P BD 0.67 0.45 1.17 0.34 2.93 0.02 TOM 0.52 0.50 3.57 0.003 3.43 0.01

37 MAOM 0.33 0.58 0.85 0.56 2.09 0.09 POM 1.94 0.22 1.93 0.08 3.08 0.02 + NH4 -N 2.19 0.21 1.53 0.18 1.42 0.27 - NO3 -N 11.39 0.01 3.94 0.001 4.39 0.005 DON 0.96 0.37 1.77 0.11 0.81 0.60 MB-N 25.10 0.004 2.10 0.056 3.13 0.02 Farming systems compared using repeated measures ANOVA. *Adjusted Greenhouse-Geisser P-value

Table 2.3: Effect of farming systems on soil bulk density (BD), total organic matter % (TOM), particulate organic matter + - -1 (0.05-2 mm) (POM), % mineral associated organic matter (MAOM) (<0.05 mm), mineral-N (NH4 -N and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N).

+ - Year Farming system BD TOM MAOM POM NH4 -N NO3 -N DON MB-N Sp. 2000 Conventional 1.34 2.49 1.89 0.61 1.03 5.67 3.20 55.00 Organic 1.35 2.41 1.82 0.61 0.93 5.81 2.65 53.70 Aut. 2000 Conventional 1.25 2.61 2.03 0.58 1.73 13.03 8.84 64.00 Organic 1.18 2.60 1.95 0.63 1.22 6.75 6.42 78.87 Sp. 2001 Conventional 1.42 2.65 1.27 0.58 1.43 4.38 10.90 37.60 Organic 1.41 2.73 1.41 0.63 0.82 4.91 9.80 54.30 Aut. 2001 Conventional 1.38 2.78 2.20 0.50 1.50 7.77 7.22 31.40 38 Organic 1.37 2.96 2.24 0.64 1.45 8.78 7.34 48.00 Sp. 2002 Conventional 1.38 2.65 1.97 0.62 2.16 9.90 4.14 33.46 Organic 1.38 3.00 2.20 0.74 1.86 8.73 4.73 46.60 Aut. 2002 Conventional 1.44 2.88 2.07 0.72 1.40 9.99 6.55 54.00 Organic 1.44 2.88 2.09 0.70 0.90 4.06 5.68 60.00 Sp. 2003 Conventional 1.51 2.78 1.99 0.69 1.50 10.48 8.51 20.10 Organic 1.50 2.93 2.11 0.76 0.40 6.47 8.31 30.10 Aut. 2003 Conventional 1.44 2.58 1.90 0.62 0.89 3.27 5.70 40.60 Organic 1.38 2.75 2.04 0.67 0.94 2.56 6.10 48.50 Sp. 2004 Conventional 1.56 2.32 1.64 0.60 1.17 4.7 7.60 32.87 Organic 1.54 2.54 1.83 0.72 1.17 5.3 6.61 47.01

Table 2.4: Mean soil bulk density (BD), total organic matter % (TOM), particulate organic matter (0.05-2 mm) (POM), % + - -1 mineral associated organic matter (MAOM) (<0.05 mm), mineral-N (NH4 -N and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N) in conventional and organic arming systems.

0.3 2000 80 2001 2002 70 0.25 2003 2004 60 Air temperature F) 0 0.2 50

0.15 40

30 0.1

20 Mean air temperature ( Mean precipitation (inches) 0.05 10

0 0 Jan Feb Mar Apr May June July Aug Sep Oct Nov Dec

Fig. 2.1: Bars represent mean precipitation in inches per month from year 2000-2004. Line graph shows a mean of 5 years air temperature each month from January to December.

Fig. 2.2: Mean (± SE) nematode trophic groups: bacterivore (A), fungivore (B), omnivore (C), predatory (D), total plant-parasitic nematodes (E) and root lesion nematode Pratylenchus crenatus (F) per 10g soil in organic and conventional farming systems from spring 2000 to spring 2004. F-values from analysis of variance comparing the farming systems are shown in table 2.2.

40 Corn a Soybeans a a Oats a Hay ab 30

a ab ab a 20 ab b a a a a a ab a a a 10 a a b ab ab a a b b ab

Pratylenchus crenatus population / 10 g soil ab b

0 2000 2001 2002 2003

Fig. 2.3: Mean (± SE) soil population of Pratylenchus crenatus from year 2000 to 2003 comparing corn, soybean, oats and hay. Treatments within a group with the same letter are not significantly different (p ≤ 0.05) by Fishers’s LSD test between the crops.

farming systems areshownintable2.2. from spring2000to2004. (B), andchannel(C)indices Fig. 2.4:

Basal Index Enrichment index Mean(±SE)valuesofnematode food web indices:enrichment (A),structure 10 15 20 25 30 35 S 50 55 60 65 70 75 80 85 90 p 5 r Au ing (C)

-20 (A) tumn-2 00 S pri 0 A ng 00 u -20 Conventional Organic tumn-2 01 Sprin 00 A g 1 u -200 tum n 2 Sprin -2 002

per10gsoilinorganicandconventionalfarmingsystem A u g tu -2003 m n Spring-2004-2 003 F- values from analysis ofvariance comparingthe

42 Channel Index Structure Index 10 20 30 40 50 60 70 80 Sp 10 20 30 ri 0 A ng-2

u (B) tumn-200 (D) 00 Spring-2001

A 0 utumn-20010

S pring-2002 Autumn-2002

S pri n Autum g-20

0 Conventional Organic S n 3 pri -200 n g-20 3 04

90 9 Quadrat A 9 Quadrat B 80 3 8 5 3 5 1 70 7 8 2 4 6 4 2 60 6 7 1

Enrichment IndexEnrichment 40 Quadrat D Quadrat C

30 Organic Conventional 20

10 10 20 30 40 50 60 70 80 90 Structure Index

Fig. 2.5: Comparison and temporal progression of the food web indicated by nematode faunal analysis in organic and conventional farming systems from spring 2000 to spring 2004. Numbers 1-9 represent the progression of changes in the fauna in each farming system.

CHAPTER 3

EFFECT OF COMPOST OR NON-COMPOST BASED ORGANIC

TRANSITIONING STRATEGIES ON SOIL NEMATODE COMMUNITY,

ORGANIC MATTER, MICROBIAL BIOMASS AND NITROGEN DYNAMICS

3.1. INTRODUCTION

Greater non-farming land use (often leading to higher land prices) shrinking

agricultural infrastructure for small-scale conventional farms, low commodity prices and

less tolerance for conventional farming practices, constrain agricultural systems near

urban areas. Therefore, farmers in periurban areas have started to adopt organic farming

methods to fetch premium prices of their produce. The U.S. National Organic Standards

Act allows certification only after 3 years have passed since the last use of restricted substances. During this period crop yields are unstable, premium prices unavailable, and

pests (insects, parasitic nematodes, pathogens, and weeds) may be severe since the use of

conventional pesticides is withdrawn (Clark et al., 1999; Tu et al., 2006). In addition to

economical and agricultural aspects, scientific support should be considered while

choosing the field management and crop system to be used during the transition period

(Tu et al., 2006).

Nematodes are abundant and diverse invertebrates present in the soil (Yeates,

1979). They are considered an important component of the soil biotic community and assessment of nematode fauna provides a unique insight into soil biological processes

(Ritz and Trudgill, 1999). Nematodes can be grouped into five major trophic groups; bacterivores, fungivores, plant parasites, predatory and omnivores (Yeates et al., 1993).

Plant-parasitic nematodes are considered as primary consumers and they affect food web resources through direct herbivory (Ferris and Bongers, 2006). Bacterivore and fungivore nematodes graze on decomposer microbes such as bacteria and fungi and significantly contribute to nutrient mineralization (Ferris and Matatue, 2003; Ferris et al.,

1996; Ingham et al., 1985; Ferris et al., 2004). These nematodes also promote rhizosphere colonization of beneficial bacteria (Kimpinski and Sturz, 1996; Knox et al.,

2003). Predatory nematodes regulate the food web by preying on nematodes and other invertebrates in the soil (Grewal et al., 2005). Nematode communities are sensitive to changes in soil environment caused by different agroecosystem management practices

(Yardim and Edwards, 1998; Yeates and Bongers, 1999; Yeates et al., 1999; Sohlenius and Wasilewska, 1984; Neher and Olson, 1999). Nematode faunal analysis based on the relative weighted abundance colonizer-persister (c-p) guilds provides a graphic representation of the probable conditions of the soil food web (Ferris et al., 2001). This nematode faunal analysis includes food web indices including enrichment (EI), structure

(SI), basal (BI) and channel (CI), which provide critical information about below ground processes in distinct systems (Bulluck et al., 2002b; Porazinska et al., 1999; Freckman and Ettema, 1993; Bongers, 1990; Ferris, et al., 1996; Ferris et al., 2001).

Organic amendments have been shown to reduce soil bulk density and increase

soil nitrogen and carbon supply (Doran, 1995; Drinkwater et al., 1995, Bulluck et al.,

2002; Werner, 1997). All organic amendments tend to increase availability of nutrients

such as nitrogen, microbial biomass and abundance of bacterivore and fungivore

nematodes (Gunapala and Scow, 1998; Freckman, 1988; Griffiths et al., 1994; Bulluck et

al., 2002a; Ferris et al, 1996, 1999). Increase in organic matter in the soil increases

microbial biomass by providing an enlarged food base for free-living nematodes

(Papatheodorou et al., 2004; Alon and Steinberger, 1999; Bongers and Ferris, 1999;

Ferris et al., 1999; McSorley and Frederick, 1999).

This study compares the long-term effect of four organic transitioning strategies,

with and without compost amendment, on the soil nematode community and how this

relates to soil characteristics including bulk density, mineral nitrogen, organic matter,

particulate organic matter, and microbial biomass-N. Four main plot management

strategies were fallow (FA), a single planting of mixed perennial hay (HA), low intensity vegetables as field vegetables (FV) and high intensity vegetables under high tunnels

(HT). Half of each main plot received an annual addition of compost and the other half received no amendment. High tunnels were constructed for Ohio vegetable growers for extended duration vegetable production with partial control over environmental variables.

At the end of the transition period tomatoes were grown in all the plots to allow for comparison. The purpose of this project was to describe the benefits of eight strategies that differ in input intensity. Various strategies leading to organic vegetable production may have differential impacts on the soil characteristics and nematode communities.

Therefore, the primary objective of this study was to monitor the effects of strategies on

nematode c-p guilds, food web indices (enrichment, structure and channel) and soil

characteristics (organic matter, microbial biomass and nitrogen dynamics). A sub

objective was to determine the relationship between nematode c-p guilds and soil

characteristics.

3.2. MATERIALS AND METHODS

3.2.1. Field site description and management strategies

An organic transition field experiment was established at the Ohio Agricultural

Research and Development Center (OARDC) in Wooster, Ohio (40° 47' N, 81° 55' W;

310 m elevation). Soil at the site was a moderately well drained Wooster silt loam initially with approximately 1.2% organic matter. The field was previously in a

conventional corn and soybean rotation. Transition to organic farming started in 2003

and four management strategies were studied. Four management strategies (main plots)

were tilled fallow (FA), single planting of mixed species perennial hay (HA), low

intensity open field vegetables (FV) and intensive vegetable production under high

tunnels (HT) (Table 3.1). Half of each main plot received an annual addition of 18.1 t ha-

1 (dry weight) composted dairy manure. The compost mix was incorporated in three

summer additions in tilled fallowing, spread over the surface after hay harvests in mixed

species hay, and incorporated prior to the main summer crop in low-intensity vegetables

and intensive vegetable production under high tunnels. The same quantity of compost

was incorporated prior to planting tomatoes in the fourth year. The main plots were

replicated four times in a randomized split plot design. Main field plots measured 17.1 x

18.3 m long. After 3 years of transition period the field was certified as organic by the

Ohio Ecological Food and Farm Association (OEFFA) as per national organic standards

in 2006. High tunnels were constructed according to Lamont and Orzolek, 2000 as

plastic enclosed structures (6.4 m x 14.6 m) (capable of accommodating field sized

equipment) with partial control over environmental variables for extending vegetable

crops growing period. In FA strategy weed seedlings were removed throughout the year

and a cover crop (rye or wheat) was planted in the fall every year to help prevent erosion

and was incorporated into the soil every spring to add organic matter back into the soil.

In the HA plots, 2 or 3 cuttings were taken each year by mowing off the foliage, allowing

it to dry on the ground and then the foliage was removed from the plots. Foliage was

incorporated into the soil of the HA plots only in spring of 2006 in preparation for the

tomato transplanting. The irrigation system for the project was drip irrigation. The drip

tape used all four years was 510-12-450 TSX (hole spacing 0.30 m apart in the tape) with

water being released at a rate of 1.70 L /minute/30.48 m of tape. Drip irrigation was in

place in the HT and FV plots each year from April 1 to November 1, and from November

1 to April 1 irrigation was done in the high tunnels by hand with Dramm watering cans.

Irrigation was done as needed for the first 3 years based on visual appearance of the soil

and the weather. In 2006, plots were on a regular irrigation schedule of 3 day/week for 4-

6 hours per day, unless it had rained.

3.2.2. Soil sampling

Six soil cores (5 cm diameter and 15 cm deep) were removed randomly from the

central portion of each sub-plot using a soil auger and total of four composite soil samples (two per subplot) were collected per main plot. Therefore, a total of sixty-four

soil samples were analyzed for nematode and soil properties at each sampling time.

Nematodes and soil properties were analyzed the first three times in March of 2003-2005

and the fourth time in October of 2006 at the time of tomato harvest. Soil samples were

mixed thoroughly to form a composite sample to reduce the variance associated with

aggregated spatial patterns of nematodes in the soil (Barker and Campbell, 1981). Large

plant parts or stones were removed from the samples by passing them through a soil sieve

(6 mm mesh). All soil samples were stored in the dark overnight at 5oC and existing field

moisture was maintained to minimize changes in nematode populations (Barker et al.,

1969). The soil samples were divided into sub samples for nematode and soil properties

analyses.

3.2.3 Nematode extraction, identification and counting

Nematodes were extracted from a 10 g subsample taken from each composite soil

sample using the Baermann funnel technique (Flegg and Hooper, 1970). Nematodes

were collected after 72 h, heat killed, and fixed with triethanolamine formaldehyde (TAF) solution (Shepherd, 1970). Specimens were identified to the genus level using an inverted microscope at 40X magnification. Diagnostic keys by Goodey (1963), Siddiqui

(1986), and Jairajpuri and Ahmed (1992) and the University of Nebraska Lincoln nematode identification website (http://nematode.unl.edu/konzlistbutt.htm) were used.

All identified nematode genera were assigned to a trophic group (plant-parasitic, fungal

feeder, bacterial feeder, omnivore, or predatory) according to Yeates et al. (1993).

Nematode genera were also assigned a colonizer-persister value (c-p value) according to

Bongers (1990). Numbers of nematodes were not corrected for extraction efficiency.

3.2.4. Soil analysis

Soil bulk density (BD), total organic matter (TOM), mineral associated organic matter (MAOM) (<0.05 mm), particulate organic matter (POM) (0.05-2 mm), mineral-N

+ - (NH4 -N and NO3 -N), and dissolved organic-N (DON), were analyzed at the Field Crop

Ecology Laboratory, OARDC, Wooster, using standard methods (Sims et al., 1995).

Microbial biomass-N (MB-N) was determined using modified chloroform fumigation

method (Brookes et al., 1985).

3.2.5 Soil temperature measurements

Soil temperature (6 cm deep) was recorded using a data logger and soil

thermometer installed inside high tunnel. Outside field soil temperature data was

obtained from the Ohio Agricultural Research and Development Center weather station.

3.2.6. Data analysis

The enrichment (EI), structure (SI) and channel (CI) indices were calculated

according to Ferris et al. (2001), with basal components (b) of the food web (fungal and

bacterial feeders in the c-p 2 guild) calculated as b = ∑kbnb where kb is the weighted

constant for the guild, and n is the number of nematodes in that guild. Enrichment (e)

and structure (s) components were similarly calculated, using nematode guilds indicative

of enrichment (bacterivores of c-p 1, and fungivores of c-p 2), and guilds supporting

structure (bacterivores of c-p 3-5, fungivores of c-p 3-5, omnivores of c-p 3-5, and

predatory nematodes of c-p = 2-5). Finally, the EI was calculated as 100x e/(e + b), and

the SI as 100x s/(s + b). CI provides an index of the nature of decomposition and was

calculated as 100 x (0.8 fungivores cp 2/ (3.2 of bacterivores of c-p 1 + 0.8 fungivores of

c-p 2) where the coefficients are the ke enrichment weightings for the respective guilds

(Ferris et al., 2001).

Management strategies were compared for nematode c-p (colonizer-persister)

guilds, food web indices, and soil properties using PROC mixed model. Relationship

between soil parameters and nematode c-p guilds were analyzed by canonical correlation

analysis. All data were analyzed using SAS Ver. 9.00, SAS Institute, Cary, NC.

Nematode population data were transformed as ln (x+1) and food web indices as square

root prior to statistical analysis to normalize the variance in the data. The probability

level of P ≤ 0.05 was regarded as significant.

3.3. RESULTS

During the course of this study twenty-seven nematode genera were identified

from the experimental plots. Nematode genera identified in the bacterivore guilds were

seven in c-p 1 guild (Bf1) (Rhabditis, Mesorhabditis, Rhabdolaimus, Panagrolaimus

Diplogasteroides, Teratocephalus and Monhystera), four in c-p 2 (Bf2) (Cephalobus,

Acrobeloides, Acrobeles, Plectus,) and one in c-p 4 (Bf4) (Alaimus). Two fungivore

genera (Aphelenchoides and Aphelenchus) were observed in c-p 2 guild (Ff2). Only one

predatory genus in c-p 4 guild (Pr4) (Mononchus) and three omnivores in c-p 4 (Om4)

(Dorylaimus, Eudorylaimus and Pungentus) were observed. Plant-parasitic genera observed were three in c-p 2 guild (Pp2) (Tylenchus, Filenchus and Psilenchus), five in c-

p 3 (Pp3) (Pratylenchus, Paratylenchus, Tylenchorhynchus, Helicotylenchus and

Hoplolaimus) and only one in c-p 5 (Pp5) (Xiphinema). Tylenchus and Filenchus

dominated in the Pp2 guild while root lesion nematode was dominant among the

nematodes of Pp3 guild. Rhabditis, Panagrolaimus, Cephalobus, Acrobeloides,

Aphelenchoides, Aphelenchus, Tylenchus, and Filenchus were the most abundant genera

while Helicotylenchus, Hoplolaimus, and Psilenchus were observed in low numbers.

3.3.1. Effect of management strategies on nematode communities

Analysis of soil samples prior to the sowing of crops and application of compost in spring 2003 showed no significant differences between the plots for nematode c-p

(colonizer-persister) guilds (Table 3.2). Bf1 were significantly higher in the composted

compared to the non-composted management strategies in 2006 (P = 0.0008, F = 12.7).

In 2006 composted plots had a higher abundance of Bf2 nematode guild than the non-

composted plots (P = 0.001, F = 22.6) (Table 3.2). HA composted and HA non-

composted plots however, had similar level of Bf2 nematodes (Table 3.3).

Abundance of fungivore nematodes differed significantly between the main plot

at the end of the transitioning period (P = 0.04, F = 3.7 for 2006) while compost showed no significant effect (Table 3.2). HT tended to have higher populations of fungivore nematodes compared to all other strategies, however in 2006 both composted and non- composted HA strategy had similar levels of fungivore nematodes (Table 3.3).

Population of omnivore (Om) and predatory (Pr) nematodes declined over time

compared to year 2003 baseline soil sampling and no significant effect of main plot

strategies and subplot treatments (compost) was observed on the abundance of omnivore

nematodes throughout the study period (Table 3.2). Predatory nematodes differed

significantly between composted and non-composted plots in 2006 (P = 0.009, F = 7.4)

(Table 3.2).

Main plot strategies showed non-significant differences for Pp2 guild throughout

the study period but consistent differences were observed for Pp3 and Pp5 guilds and total abundance of plant-parasites among the strategies from 2004 to 2006 (Table 3.2).

Compost favored the abundance of Pp2 guild dominated by Tylenchus and Filenchus

while suppressed root-lesion nematode Pratylenchus at the end of the experiment in 2006

(Figure 3.1).

3.3.2. Effect of management strategies on nematode faunal indices

EI was the only index, which differed significantly among the main plot strategies

(P = 0.03, F = 3.9) at the end of the transition period in 2006 while effect of compost was

observed to be significant only for CI (P = 0.0006, F = 13.9) and not fro EI and SI (Table

3.2). Comparison for CI among all the 8 sub-plot strategies from 2003-2006 showed

discernible differences only in 2006 when composted strategies had higher CI compared

to the non-composted strategies. However, HA non-composted showed a similar level of

CI as the other composted strategies.

Figure 3.2 shows the faunal profiles in all the 8 sub-plot strategies. At the time of

establishment of experiment in 2003 food webs were enriched and moderately structured

and no discernible differences were observed between the plots. SI declined in all the

strategies except for year 2005 when HA (composted and non-composted) had the

highest SI compared to other strategies. Although, in year 2006, food webs in all

strategies had low SI and EI in general, HT clearly distinguished from other strategies

(FV, HA and FA) by having the poorly structured and enriched food webs.

3.3.3. Effect of strategies on soil properties and microbial biomass-N

Soil BD was consistently lower in the composted than the non-composted plots

from 2004-2006 (Table 3.4 and 3.5). POM and MAOM were consistently higher from

2004-2006 in the composted plots, than the non-composted plots (Table 3.5). Although

+ soil mineral-N NH4 showed no significant differences between the composted and non-

- composted, significant differences were observed for NO3 -N (P = 0.0001, F = 21.6) and

DON (P = 0.01, F = 6.1) (Table 3.4). Although composted management strategies favored MB-N over the non-composted strategies throughout the study period, composted HT and HA favored MB-N the most (Table 3.4 and 3.5).

3.3.4. Relationship of organic matter, mineral-N and microbial biomass with nematode c-

p guilds

Figure 3.3A and B show the results of canonical correlation analysis performed

for determining the relationship between nematode c-p guilds and soil parameters (BD,

+ - MAOM, POM, mineral-N: NH4 -N and NO3 -N, and MB-N) for the first soil sampling in

2003 and the last soil sampling in year 2006, respectively. In year 2003 MB-N, mineral-

- N (NO3 -N) and POM were the major factors explaining the first canonical variate

whereas POM and DON had important loadings on the second canonical variate. Plant

parasitic nematodes in c-p 3 guild mainly represented by Pratylenchus sp. showed the

highest correlation with the first canonical variate followed by bacterivores Bf1 and Bf2 guilds while Bf1 and Bf2 were also correlated with the second canonical variate. In year

2006, first canonical variate was represented by MAOM, MB-N and various forms of

- + nitrogen (NO3- N, NH4 -N and DON). BD also had an important loading on both first

and second canonical variates in this sampling time. Canonical structure further revealed

that in year 2006 bacterivores (Bf1 and Bf2) followed by Pp2 were correlated with the first canonical variate while Ff2 showed correlation with the second canonical variate. Pp3 showed the maximum change in relationship with the soil parameters from 2003 to 2006.

3.3.5 Soil temperature inside high tunnels and outside field plots

The average soil temperature under high tunnels increased than the outside field soil in vegetable plots from November to May (Figure 3.4).

3.4. DISCUSSION

This study compared soil characteristics and nematode communities in four oranic transition strategies with and without the addition of compost. In the beginning of the fourth year in 2006, tomato was transplanted in all the plots. Annual addition of compost increased organic matter, mineral-N and MB-N in the composted compared to the non- composted plots throughout the study period. These findings are consistent with other studies where applications of organic amendments have been shown to increase soil organic matter, mineral-N and MB-N (Clark et al., 1999; Borken et al., 2002; Tu et al.,

2006; Werner, 1997; Drinkwater et al., 1995). Nematodes communities were also sensitive enough to respond to changes in soil properties due to addition of compost as observed in other studies (Nahar et al., 2006; Bulluck et al., 2002b). However, the effect of compost on soil properties and nematode communities varied due to main plot strategies in our study.

Pp2 was the dominant plant-parasitic nematode guild consisting mainly of

Tylenchus and Filenchus, and root lesion nematode Pratylenchus was dominant in Pp3 guild. Addition of organic amendments suppressed plant parasitic nematodes. A

probable mechanism of suppression is the accumulation of nitrogenous compounds in the

soil after compost addition (Nahar et al., 2006; Abawi and Widmer, 2000; McSorley and

Frederick, 1999; Rodríguez-Kàbana et al., 1981; Hominick, 1999). However, our study clearly indicated that compost may not have the same effect on all the plant parasitic nematode guilds, especially when the biology and host range of nematode guilds are contrasting (Zunke, 1990; Okada et al 2002; Vestegard, 2004). At the end of the experiment in 2006 when tomato was planted in all the plots, the population of

Pratylenchus sp. was significantly lower in the composted than the non-composted strategies while that of the Pp2 guild was higher. Migratory endoparasites like

Pratylenchus spp are considered harmful to the host plant, as they cause cell death during feeding and migration through root tissue, and also predispose the cortex to attack by other plant pathogens (Zunke, 1990). On the other hand ectoparasites like Tylenchus,

Filenchus and Psilenchus are either considered weak root hair feeders (Yeates et al.,

1993) or fungal feeders (Okada et al., 2002). Therefore, we hypothesize that

accumulation of nitrogenous compounds due to the addition of organic amendments may

have harmful impact on endoparasitic nematodes like Pratylenchus spp but not on

ectoparasites such as Tylenchus, Filenchus and Psilenchus.

Quick responses of fungal feeders and bacterial feeders, to organic amendments

have been attributed to increases in their food availability (Ferris et al., 1999 and 2001;

Griffiths et al., 1994; Nahar et al., 2006; Bulluck et al., 2002a). We also observed that abundance of bacterivore nematodes dominated by the two c-p guilds Bf1 (mainly

rhabditids) and Bf2 (mainly cephalobids) was significantly higher in the composted than

the non-composted strategies but the compost did not affect abundance of fungivore

nematodes. It appears possible that well decomposed compost applied to soil in our

study supplied more labile low C/N organic material and favored only bacterial

communities that provided the base for bacterivore nematodes and not the recalcitrant

high C/N organic material that favors fungal decomposition thereby showing increase in

fungivores (Ferris and Matute, 2003).

The main aim of the HT management strategy was to protect the vegetable crops

from frost damage that occurs during the beginning of spring and end of autumn season in Ohio. HT in general had the highest total abundance of bacterivore and fungivore

nematodes compared to all other vegetable strategies. Soil analysis also revealed higher

MB-N and mineral-N in both composted and non-composted HT compared to other

management strategies. Marginal increase in average soil temperature might have

enhanced the decomposition processes thereby making the soil food web biologically more active in HT compared to the other strategies (Bakonyi and Nagy, 2000).

At the time of establishment of experiment food webs were highly enriched and

moderately structured in all the plots. However, by the end of the experiment food webs

in all management strategies had low EI and SI. Organic transition strategies where

compost was applied did not show higher enrichment or structure as was expected.

Increase in the abundance of general opportunists along with enrichment opportunist

bacterivores in the composted strategies reduced the overall level of EI and SI in our

study. However, frequent tillage may have also lowered the SI by preventing the build

up of higher c-p value nematodes in the composted strategies, which are sensitive to

physical disturbance (Fiscus and Neher, 2002; Freckman and Ettema 1993; López-Fando

and Bello, 1995). Evidence of the sensitivity of high c-p value nematodes to physical

disturbance also comes from the fact that HT management strategy showed the lowest SI

compared to other strategies where the vegetable crop period was extended compared to

the other strategies. This might explain why addition of organic amendments alone is not

sufficient to increase the trophic links in soil food webs.

External organic matter inputs in the form of compost, animal manures and cover

crops increase energy availability for the soil microbes thereby enhancing microbial

activity and biomass (Lundquist et al., 1999; Gunapala and Scow, 1998; Powlson et al.,

1987; Alon and Steinberger, 1999). In an organic farming system, N availability depends primarily on the decomposition processes carried by the soil microbes (Laakso et al.,

2000). However, microbivorous organisms such as nematodes feed on the microbes and

enhance mineralization of nutrients, which may otherwise be immobilized in the body of

microbes (Griffiths, 1994; Chen and Ferris, 1999; Bardgett, et al., 1999). In our study

bacterivore nematode c-p guilds including Bf2, Bf2, showed correlation with POM and

MB-N in year 2003 sampling time before the addition of organic matter and the

relationship of these c-p guilds remains the same in year 2006 after the transition period was over. A positive correlation was also reported between bacterivore nematode, amount of organic matter and microbial biomass in the soil (Ferris et al., 2004; Villenave et al., 2003). In our study we also observed changes in relationship of plant parasitic nematode guilds with soil parameters due to application of organic amendments. Pp3

(mainly Pratylenchus) that showed the highest correlation with MB-N in 2003, was observed to be more correlated with mineral-N and DON in 2006. However, in 2006 correlation of Pp2 nematodes with MB-N might suggest that this group of nematodes

become functionally more active after the addition of organic matter to the soil in our

study. Root hair feeders such as Tylenchus and Filenchus have been shown to contribute to the increase of microbial activity by eliciting leakage of root metabolites into the soil and are thus actively involved in nutrient mineralization (Verschoor, 2002; Bardgett et al., 1999; Yeates et al., 1999).

Higher CI (>50) indicates fungal decomposition channels whereas low CI (< 50)

suggest bacterial decomposition channels (Ferris et al., 2001). Therefore, higher CI in

the non-composted strategies may suggest some level of fungal decomposition channels

in this study. Calculations of food web indices require numerator and denominator and

therefore, increase in the value of index does not necessarily reflect that the numerator

would be higher (Ferris and Matute, 2003). In this case abundance of Bf2 was lower in

non-composted strategies and as a consequence the CI value is inflated (inferred as

fungal decomposition). However, abundance of fungivores did not differ between the

composted and non-composted strategies but the differences were observed between the main plot strategies. HT and hay strategies showed a higher level of fungivore nematodes compared to other strategies. Nevertheless, neither abundance of fungivore nematodes nor CI values suggested absolute fungal decomposition channels in our study.

3.5. SUMMARY

Four main plot crop management strategies including tilled fallow (FA), a single

planting of mixed hay species (HA), low intensity open field vegetables (FV) and

intensive vegetable production under high tunnels (HT) with and without annual addition

of compost were established as a 3-year organic transition experiment. Half of each

main plot received composted dairy manure at the rate of 18.1 t ha-1 dry weight and the

other half received no amendment. At the end of the transition period tomatoes were grown in all the plots to allow for comparison between treatments. Soil bulk density

(BD) was lower while various forms of organic matter [total organic matter (TOM), particulate organic matter (POM) and mineral associated organic matter (MAOM),

+ - mineral-N (NH4 -N and NO3 -N), dissolved organic-N (DON) and microbial biomass-N

(MB-N)] tended to be higher in composted than the non-composted strategies throughout the study period. However, composted HT and HA favored MB-N more than all other management strategies. Enrichment opportunists c-p 1 bacterivores and general opportunists c-p 2 bacterivores were higher in composted than the non-composted strategies in general. Main plot HT and HA strategies tended to have higher populations of fungivore nematodes compared to other strategies. HT had the highest total abundance of bacterivore and fungivore nematodes compared to the other strategies. At the end of the experiment, root lesion nematode Pratylenchus sp. was significantly lower in the composted strategies while the compost favored c-p 2 plant-parasites dominated by

Tylenchus and Filenchus over the non-composted strategies. Nematode food webs in all strategies had low SI and EI and no consistent differences were observed between composted and non-composted strategies. Lack of increase in EI in the composted compared to the non-composted strategies was mainly due to the higher abundance of general opportunist c-p 2 bacterivores while both low abundance of higher c-p value

(omnivores and predatory) nematodes and higher abundance of c-p 2 bacterivores contributed for the lower SI in composted strategies. CI values suggest bacterial driven decomposition channels in all the strategies. Canonical correlation analysis revealed that bacterivore nematodes were correlated with the MB-N and organic matter. The

correlation of c-p 2 plant-parasites with MB-N suggests that this guild was also functionally active in these organic transitioning plots.

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Year Management Crop strategy

2003 Fallow Rye Hay Mixed species hay FV Potatoes-mixed vegetables HT Potatoes-mixed vegetables 2004 Fallow Rye Hay Mixed species hay FV Butternut Squash-wheat

69 HT Mixed vegetables-zucchini squash- mixed vegetables 2005 Fallow Wheat Hay Mixed species hay FV Green beans-lettuce-wheat HT Mixed vegetables-green beans-mixed vegetables 2006 Fallow Tomatoes Hay Tomatoes FV Tomatoes HT Tomatoes Mixed vegetable consisted of lettuce, spinach, swiss chard, radishes and beets.

Table: 3.1: Description cropping sequences of four main plot management strategies: fallow (FA), mixed species hay (HA), low intensity vegetables as open field vegetables (FV) and intensive vegetable production under high tunnels (HT) from 2003-2006.

. Parameter 2003a 2006* Strategy (df 3, 53) Strategy (df 3, 53) C vs. NC (df 1, 53) Nematode c-p ¶ Bf1 0.89 (0.2) 0.0003 (7.32) 0.0008 (12.68) Bf2 0.12 (2.4) 0.0001 (29.7) 0.001 (22.6) Bf4 0.66 (0.5) 0.01 (4.02) 0.55 (0.3)

Fungivores (Ff2) 0.14 (1.8) 0.04 (3.77) 0.51 (0.43) Omnivores (Om4) 0.30 (1.2) 0.64 (0.6) 0.10 (0.75) Predatory (Pr4) 0.38 (1.1) 0.06 (3.42) 0.009 (7.40)

70 Pp2 0.74 (0.4) 0.11 (2.06) 0.05 (3.42) Pp3 0.53 (0.7) 0.01 (6.8) 0.08 (2.5) Pp5 0.40 (1.0) 0.0001 (11.28) 0.94 (0.0) Indices EI 0.25 (1.6) 0.03 (3.90) 0.31 (0.60) SI 0.16 (0.9) 0.10 (2.20) 0.45 (0.5) CI 0.06 (2.6) 0.50 (0.80) 0.0006 (13.6) Numbers are P and F-values (Numbers in parenthesis are F-values) Bf: bacterivores; Ff: Fungivores; Om: Omnivores; Ca: Carnivores and Pp: Plant parasitic nematodes, C: Composted, NC: Non-composted, aMain plot management strategy, aSoil sampling before the establishments of organic strategies, *Soil sampling after a uniform crop tomato was grown in all strategies in 2006.

Table 3.2: Effect of main plot strategies and compost on nematode c-p groups and food web indices [enrichment (EI), structure (SI), and channel index (CI)].

Year Strategy a Bf Ff Om Pr Pp

¶ Bf1 Bf2 Bf4 Total Bf Ff2 Om4 Pr4 Pp2 Pp3 Pp5 Total Pp

2003b 79.4 3.7 43.9 24.6 50.8 4.0 19.0 5.0 42.3 1.4 0.2

2004 FA-C 14.8 11.4 0.25 26.5 2.6 1.1 0.1 3.7 3.3 0.3 7.5 FA-NC 12.1 10.7 0.12 23.0 5.0 2.1 0.1 5.3 2.4 0.0 7.6 FV-C

71 34.0 19.7 0.12 53.8 5.8 2.4 0.2 3.6 3.0 0.1 6.7 FV-NC 22.1 22.3 0.0 44.3 4.5 4.7 0.0 16.5 7.7 0.2 24.5 HA-C 17.2 22.6 0.0 39.8 7.2 10.1 0.0 16 17.0 0.1 33.1 HA-NC 7.8 14.1 0.0 22.0 4.3 1.0 0.1 18.8 9.6 1.1 29.6 HT-C 43.5 43.7 0.1 87.4 23.6 1.5 0.0 14.0 14.3 0.1 28.5 HT-NC 27.7 59.0 1.0 87.7 32.7 2.2 0.1 9.5 9.1 0.2 18.8 bSoil sampling before the establishments of organic strategies, ¶ Numbers in the subscript represents c-p values, a Sub plot strategies, *Soil sampling after a uniform crop tomato was grown in all strategies in 2006.

Table 3.3: Comparison between main plot strategies with and without compost on nematode c-p guilds (Bf: bacterivore; Ff: fungivores; Om: omnivores; Pr: predatory and Pp: plant-parasites) from 2003 and 2006 soil sampling.

34.0 29.2 44.1 22.1 28.0 27.8 Total Pp 7.1 27.2 20.1 19.5 22.2 32.7 7.2 16.0 10.5 28.6 5 1.2 3.5 0.0 0.8 4.0 0.0 0.1 0.0 0.5 0.8 Pp 0.1 0.2 0.0 0.0 0 0.5 3 9.5 12.6 25.5 8.1 9.0 0.6 5.0 1.1 3.6 0.1 1.7 10.7 1.0 10.1 Pp 4.5 5.6 2 23.15 13.0 18.6 13.2 15.2 14.0 7.0 6.5 22.1 19.3 15.4 11.5 9.5 28.2 17.0 Pp 22.9 Pp

4 1.1 1.3 1.1 1.3 1.3 0.3 0.5 0.1 1.0 0.0 0.0 2.7 0.7 0.1 Pr 0.4

0.9 Pr

4 4.0 2.6 2.0 5.1 2.6 1.6 1.0 2.7 2.5 2.3 3.6 0.7 1.5 2.5 Om 1.5 2.1 Om

2 0.7 11.6 14.1 10.8 2.6 6.6 3.7 6.1 1.6 2.2 5.2 5.1 5.0 5.5 Ff 9.4

Ff 9.7

52.2 103.4 156.4 15.0 127.0 170.5 16.8 43.8 57.5 43.4 81.0 130.6 74.6 39.1 188.0 Total Bf 69.0 4 3.2 1.0 1.0 0.3 0.3 1.2 0.6 0.6 1.0 0.6 0.7 0.6 0.3 0.4 2.2 Bf 1.2 2 21.7 79.7 102.7 12.8 9.6 23.1 31.1 93.0 14.7 22.6 39.5 88.2 43.0 27.8 154.7 Bf 58.8 1 27.2 22.6 52.6 20.2 25.4 3.6 5.1 155.1 33.2 20.2 40.7 41.7 31.2 10.8 31.12 Bf Bf 9.0 Strategy FA-C FA-NC FV-C FV-NC HA-C HA-NC HT-C HT-NC FA-C FA-NC FV-C FV-NC HA-C HA-NC HT-NC HT-C

Year 2005 2006 Table 3.3: continued 72 Year Soil parameters

+ - BD TOM POM MAOM NH4 -N NO3 -N DON MB-N

2003 Strategy a (df 3, 53) 0.06 (3.1) 0.38 (1.1) 0.62 (0.6) 0.37 (1.2) 0.69 (0.5) .09 2.7) 0.55 (0.7) 0.09 (2.8)

73 2006* Strategy (df 3, 53) 0.41 (1.1) 0.62(2.1) 0.01 (5.1) 0.03 (4.6) 0.89 (0.2) 0.47 (0.9) 0.59(0.6) 0.74 (0.4) C vs NC (df 3, 53) 0.001 (20) 0.1 (2.2) 0.0001 (519) 0.0001 (37) 0.27 (1.2) 0.0001 (21.6) 0.01(6.1) 0.10 (2.8) Numbers are P-values and F-values are in parenthesis, C: Composted, NC: Non-composted, aMain plot strategy, bSoil sampling before the establishments of organic strategies, * Soil sampling after a uniform crop tomato was grown in all strategies in 2006.

Table 3.4: Effect of main plot strategies and compost on soil properties [bulk density (BD), organic matter % (TOM), % + mineral associated organic matter (MAOM) (<0.05 mm), particulate organic matter (0.05-2 mm) (POM), mineral-N (NH4 -N - -1 -1 and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N) (µg g )].

+ - BD OM POM MAOM NH4 -N NO3 -N DON MB-N

2003 Fallow 1.54 (0.01) 2.01 (0.07) 0.44 (0.02) 1.53 (0.06) 1.68 (0.11) 1.98 (0.51) 5.85 (0.61) 40.43 (1.12) FV 1.53 (0.01) 1.99 (0.02) 0.48 (0.01) 1.47 (0.02) 1.96 (0.44) 2.55 (0.40) 5.70 (0.61) 46.29 (3.22) Hay 1.49 (0.01) 2.21 (0.08) 0.47 (0.01) 1.66 (0.08) 2.12 (0.07) 1.83 (0.33) 6.16 (0.47) 45.04 (2.53) HT 1.61 (0.02) 2.10 (0.03) 0.50 (0.01) 1.56 (0.03) 1.91 (0.18) 5.96 (1.0) 6.44 (0.62) 52.99 (2.14) 2004 FA-C 1.53 (0.02) 2.61 (0.12) 0.70 (0.06) 1.75 (0.08) 1.67 (0.10) 6.76 (0.72) 10.57 (0.80) 26.64 (3.69) FA-NC 1.54 (0.01) 1.91 (0.06) 0.44 (0.04) 1.38 (0.06) 1.31 (0.04) 5.35 (0.49) 7.56 (0.41) 18.92 (2.63) FV-C 1.45 (0.02) 2.44 (0.10) 0.64 (0.04) 1.80 (0.10) 1.40 (0.08) 7.40 (0.91) 11.21 (1.29) 29.08 (5.46) 74 FV-NC 1.56 (0.01) 1.95 (0.09) 0.46 (0.02) 1.42 (0.09) 1.26 (0.07) 5.73 (0.39) 8.01 (0.80) 22.53 (2.56) HA-C 1.56 (0.01) 2.39 (0.11) 0.53 (0.03) 1.75 (0.14) 1.71 (0.10) 8.34 (0.57) 10.65 (0.83) 32.95 (4.02) HA-NC 1.56 (0.02) 2.08 (0.09) 0.48 (0.03) 1.49 (0.11) 1.63 (0.11) 4.52 (0.66) 7.66 (0.44) 30.93 (2.83) HT-C 1.38 (0.02) 2.75 (0.08) 0.76 (0.06) 1.83 (0.15) 1.80 (0.25) 12.99 (3.79) 22.60 (3.67) 37.39 (6.13) HT-NC 1.43 (0.01) 1.96 (0.12) 0.57 (0.03) 1.31 (0.10) 1.27 (0.05) 10.01 (3.18) 7.49 (0.71) 25.51 (4.06)

aSoil sampling before the establishments of organic strategies.

Table 3.5: Mean (SE) of soil properties [bulk density (BD), organic matter % (TOM), % mineral associated organic matter + - -1 (MAOM) (<0.05 mm), particulate organic matter (0.05-2 mm) (POM), mineral-N (NH4 -N and NO3 -N (µg g dry soil), and microbial biomass-N (MB-N) (µg g-1)] in various strategies.

+ - BD OM POM MAOM NH4 -N NO3 -N DON MB-N

2005 FA-C 1.48 (0.02) 2.65 (0.14) 0.88 (0.03) 1.62 (0.10) 1.25 (0.09) 2.67 (0.32) 7.43 (0.39) 26.97 (3.05)

FA-NC 1.54 (0.02) 1.83 (0.08) 0.44 (0.01) 1.38 (0.07) 0.97 (0.05) 1.25 (0.42) 4.42 (0.74) 14.91 (1.68)

FV-C 1.41 (0.02) 3.09 (0.15) 1.18 (0.04) 1.58 (0.07) 1.40 (0.10) 4.25 (0.35) 7.55 (0.37) 37.73 (2.47)

FV-NC 1.52 (0.03) 1.81 (0.07) 0.50 (0.03) 1.29 (0.04) 1.18 (0.13) 2.31 (0.43) 3.73 (0.34) 20.54 (1.86)

HA-C 1.46 (0.01) 3.30 (0.14) 1.18 (0.06) 1.86 (0.08) 1.89 (0.16) 5.17 (0.62) 8.11 (0.80) 46.06 (2.34)

HA-NC 1.56 (0.01) 2.01 (0.08) 0.52 (0.01) 1.44 (0.07) 2.16 (0.18) 0.87 (0.24) 4.99 (0.24) 28.01 (2.48) 75 HT-C 1.38 (0.02) 2.88 (0.10) 1.13 (0.08) 1.39 (0.07) 1.58 (0.21) 7.23 (3.9) 13.79 (2.77) 43.07 (9.91)

HT-NC 1.41 (0.01) 1.94 (0.04) 0.60 (0.03) 1.31 (0.01) 1.42 (0.13) 3.69 (0.99) 11.05 (3.54) 25.70 (9.21)

2006* FA-C 1.42 (0.03) 3.83 (0.63) 1.61 (0.01) 1.76 (0.06) 1.41 (0.26) 9.76 (1.06) 6.20 (2.18) 24.26 (4.54)

FA-NC 1.49 (0.02) 3.47 (0.39) 0.37 (0.01) 1.56 (0.07) 1.26 (0.18) 11.35 (2.61) 3.31 (1.04) 20.82 (4.18)

FV-C 1.44 (0.04) 4.32 (0.82) 1.58 (0.01) 1.78 (0.05) 1.69 (0.26) 13.81 (2.19) 8.38 (2.44) 24.69 (5.67)

FV-NC 1.57 (0.03) 3.51 (0.46) 0.39 (0.02) 1.46 (0.05) 1.29 (0.12) 14.81 (3.00) 5.10 (1.17) 15.46 (3.32)

HA-C 1.41 (0.03) 3.66 (0.52) 2.65 (0.26) 1.88 (0.13) 1.36 (0.30) 9.45 (1.30) 6.56 (2.26) 21.89 (4.79)

HA-NC 1.45 (0.02) 3.07 (0.54) 0.58 (0.01) 1.73 (0.00) 1.41 (0.24) 17.79 (6.99) 3.12 (1.17) 20.25 (3.85)

HT-C 1.40 (0.05) 5.29 (1.61) 2.18 (0.07) 2.05 (0.13) 1.60 (0.24) 12.64 (2.20) 8.10 (2.74) 26.53 (4.97)

HT-NC 1.54 (0.01) 3.56 (0.47) 0.47 (0.01) 1.56 (0.02) 1.18 (0.14) 15.52 (3.11) 3.10 (0.75) 25.97 (5.62) Table 3.5: continued. *Soil sampling after a uniform crop tomato was grown in all strategies in 2006.

FA-C FA-NC Strategy (P = 0.10) FV-C C vs NC (P = 0.05) 20 FV-NC HA-C HA-NC HT-C 15 HT-NC

Strategy (P = 0.01) C vs NC (P = 0.04) 10 Nematode population

0 Pratylenchus Tylenchus + Filenchus

Fig. 3.1: Effect of various strategies on abundance of root lesion nematode Pratylenchus and root hair feeders (Tylenchus + Filenchus). Bars represent the mean population data in 2006. FA-C: Fallow composted, FA-NC: Fallow non-composted, FV-C: Field vegetable composted, FV-NC: Field vegetable non-composted, HA-C: Hay composted, HA-NC: Hay non-composted, HT-C: High tunnels composted, HT-NC: High tunnels non-composted.

100 100 2003 2004

75 AB 75 A B

50 50 Fallow-C Fallow-NC FV-C D C D C 25 FV-NC 25 HAY-C HAY-NC HT-C HT-NC 0 0 2005 2006

A B A Enrichment index B 75 Enrichment index 75

50 50

D C 25 25 D C

0 0 0 2550751000255075100 Structure index Structure index

Fig. 3.2: Food webs indicated by nematode faunal analysis in 8 sub plot management strategies in 2003-2006. FA-C: Fallow composted, FA-NC: Fallow non-composted, FV- C: Field vegetable composted, FV-NC: Field vegetable non-composted, HA-C: Hay composted, HA-NC: Hay non-composted, HT-C: High tunnels composted, HT-NC: High tunnels non-composted.

1.5 (A)

POM 1.0

Bf1 Bf2 0.5

Bf4 BD 0.0 NO3 NH4 MAOM Pp2 Pp5 Pr4 Ff2 -0.5 DONOm4 Pp3 Canonical variate 2 Canonical variate MB-N

-1.0

-1.5 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 Canonical variate 1

1.5 (B)

NO3 1.0 Ff2

BD Bf2 0.5 MAOM

DON Pp5 Bf4 MB-N 0.0 Pr4 Om4 POM NH4 Bf1 -0.5 Pp2 Canonical variate 2

Pp3 -1.0

-1.5 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 Canonical variate 1

Figure 3.3: Canonical correlation analysis of soil factors (Mineral nitrogen: NH4 and NO3, Microbial biomass-N: MB-N, Mineral associated organic matter: MAOM, particulate organic matter: POM, Dissolved organic nitrogen: DON, and Bulk density: BD) vs nematode c-p guilds (Bacterivores in c-p groups 1, 2 and 4: Bf1, Bf2, Bf4; fungal feeding in c-p 2: Ff2; Omnivores in c-p 4: Om4; Predators in c-p 4 Pr4; and Plant feeding in c-p 2, 3, and 5: Pp2, Pp3, Pp5). Figure 3.3A 2003 soil sampling. Figure 3.3B 2006 soil sampling.

Fig. 3.4: Bars represent monthly mean soil temperature from January to December. Each month average temperature is further average of three years (2003-2006) in that month.

CHAPTER 4

AN ASSESSMENT OF THE INDICATIVE VALUE OF NEMATODE TROPHIC GROUP ABUNDANCE AND FOOD WEB INDICES IN DIFFERENTIATING HABITATS WITH A GRADIENT OF ANTHROPOGENIC IMPACT

4.1. INTRODUCTION

Nematodes exist as a diverse and highly speciated group in the soil environment.

They occupy a central position in the soil food web, occurring at multiple trophic levels

(Yeates, 1979; Moore and de Ruiter, 1991). Therefore, nematodes have the potential to provide useful insights into the structure and function of the soil food web (Ferris et al.,

1999; Ritz and Trudgill, 1999; Neher et al., 2001). Nematodes can be grouped into five major trophic groups; bacterivores, fungivores, plant parasites, predators and omnivores

(Yeates et al., 1993). Plant-parasitic nematodes are considered as primary consumers and they affect food web resources through direct herbivory (Ferris and Bongers, 2006).

Bacterivore and fungivore nematodes graze on decomposer microbes, bacteria and fungi, and thus significantly contribute to nutrient mineralization (Ferris and Matute, 2003;

Ferris et al., 1996; Ingham et al., 1985). Bacterivore nematodes also promote rhizosphere colonization of beneficial bacteria (Kimpinski and Sturz, 1996; Knox et al., 2003).

Predatory nematodes regulate the food web by preying on other nematodes and invertebrates in the soil (Zimmerman and Cranshaw, 1990; Grewal et al., 2005).

The development of the maturity index (MI) based on the colonizer-persister (c-p) values of nematodes has helped in interpreting the biological and trophic status of the soil food web in different habitats (Bongers 1990). MI is routinely used as an ecological measure for assessing the status of soil food webs in terrestrial habitats (Nahar et al.,

2006; Neher et al., 2005; Yardim and Edwards, 1998; Yeates et al., 1999; Sohlenius and

Wasilewska, 1984; Sohlenius, 1990; Sohlenius and Boström, 1986; Nethi et al., 2002).

In addition, the plant parasitic index (PPI) (based on c-p value of plant-parasitic nematodes), combined maturity index (CMI, that includes both free-living and plant- parasitic nematodes), diversity (species richness and evenness), ratio of bacterivore to fungivore nematodes, and trophic diversity have been used for the assessment of the soil food web condition. Ferris et al. (2001) developed enrichment (EI), structure (SI), and channel (CI) indices based on the relative weighted abundance of nematode c-p guilds.

EI provides an indication of the response of primary decomposers to the available resources in the soil food web while SI suggests trophic linkages in a food web as indicated by the presence of higher c-p value nematodes particularly predatory and omnivores (Ferris et al., 2001). Therefore, plotting EI and SI provides a graphic representation of nematode faunal profile depicting the likely condition of the soil food web in a given habitat (Ferris et al., 2001). As CI provides information about the decomposition channels, a high CI (> 50 %) indicates fungal decomposition channels whereas low CI (< 50 %) suggests bacterial decomposition channels (Ferris et al., 2001).

In the present study, we compared different habitats located in two counties in

Ohio under different intensities of anthropogenic disturbances using the most widely used nematode community measures (indices and trophic groups). Although quantification of

human impact on a habitat and characterizing habitats into intensively managed or

unmanaged, largely depends on the type of human intervention, a broad classification due

to physical, chemical or biological disturbances is feasible (Neher et al., 2005).

Depending on the nature (physical, chemical or biological) and frequency (magnitude) of disturbance, croplands and forests can be considered as two extremes on the management intensity continuum. We designated croplands cultivated for the production of vegetables and field crops to represent intensively managed habitats, and the forests and shrublands to represent unmanaged habitats. Turfgrass lawns and grassy borders alongside croplands represented intermediate levels of management. Nematodes play crucial roles in many soil processes, so analysis of the soil nematode community is expected to reflect differences between habitats (Yeates, 1996; Neher et al., 2005). Further, response to disturbances can be integrated through nematode community measures such as trophic groups and various indices described above (Ferris et al., 2001; Yeates et al., 1999,

Bongers, 1990). A better understanding of nematode community measures to characterize different habitats would further advance the indicative capability of nematodes at global scales (Neher et al., 2005). Therefore, the specific objectives of this study were (1) to compare different habitats using a composite of nematode community indices with multivariate analysis of variance (MANOVA) and (2) to identify the relative importance and interrelationships among nematode community indices and trophic group abundance to discriminate habitats using principal components analysis (PCA).

As the northern Ohio landscapes contain a number of pockets of muck soils, we

intentionally included habitats located in muck and mineral soils for comparison. Muck soils have low bulk density and pH, and unlike mineral soils, they have high organic

matter content. These soils are well suited for the production of vegetables such as celery

and carrots (Ernst and Martin, 1994). However, muck soils are prone to wind erosion and

oxidation and are therefore becoming shallower than the other land tracts. These soils

often catch fire and burn underground for months. Although, some muck soils have also

been reclaimed for wildlife preserves, the ecology of these soils is poorly understood.

Therefore, we addressed this issue by including different habitats from a predominantly

muck soil landscape in this study.

4.2. MATERIAL AND METHODS

4.2.1 Selection of habitats

This research was conducted at two sites. Multiple locations were selected for

collection of soil samples representing each habitat. In Huron County, Ohio (41° 47' N-

82°' 55' W), five habitats: croplands, turfgrass lawns, grassy borders, shrublands and

forests, were sampled in 12, 7, 7, 6 and 4 different locations, respectively. The soil was

mainly sandy loam muck with 21-48% sand, 36-39% silt and 14-37% clay with an

organic matter content ranging from 9.4-19.5% (Table 4.1). In Wayne County, Ohio (40°

47' N-81° 55' W), two habitats, croplands and turfgrass lawns, were sampled in 5 and 8 different locations, respectively. The soil at this location was silt loam mineral with organic matter ranging between 2-5% (Table 4.1).

4.2.2. Soil sampling

A total of 604 soil samples from five habitats located in Huron County, and 242 samples from the two habitats located in Wayne County were collected. Each soil sample was a composite of three soil cores, collected using a soil auger (2.5 cm diameter

and 15 cm deep). Root pieces and stone pebbles were removed from the samples by

passing them through a soil sieve (6 mm mesh) and soil samples were stored in the dark

at 5oC until processed for nematode extraction. A portion of soil samples collected for nematode analysis was also analyzed for percent organic matter and pH at the Field Crop

Ecology Laboratory, Ohio Agricultural Research and Development Center (OARDC),

Wooster using standard methods (Sims et al., 1995).

4.2.3 Nematodes

4.2.3.1 Extraction, identification, counting and assigning functional groups

Nematodes were extracted from 10 g sub sample taken from each composite soil

sample using the Baermann funnel technique (Flegg and Hooper, 1970). Nematodes

were collected every 24 h intervals for 72 h, pooled, heat killed, and fixed with

triethanolamine formaldehyde (TAF) solution (Shepherd, 1970). Nematodes were identified to the genus level using an inverted microscope following diagnostic keys by

Goodey (1963), Siddiqui (1986), Jairajpuri and Ahmed (1992). All identified nematode

genera were assigned to a functional group (plant parasitic, fungal feeder, bacterial

feeder, omnivore, or predatory) and a colonizer-persister value (c-p value) was assigned

according to Yeates et al. (1993) and Bongers (1990) respectively.

4.2.3.2 Calculation of nematode indices

Shannon diversity (H’), was calculated for nematode diversity, using the following formulae: Shannon-Weiner Index H’ = ∑Pi (ln Pi), where Pi is the proportion

of genera ni in the nematode community n (Pielou, 1977). Maturity index (MI) for free-

living nematodes (all nematodes except plant parasitic nematodes) was calculated using

the formula MI = (∑vifi) /n, where vi is the c-p value for the nematode genera i, fi is the

frequency of nematode genera i, and n is the total number of individual nematodes of the genera i in the sample (Bongers, 1990). Plant parasitic index (PPI) was calculated considering only plant parasitic nematodes (Bongers, 1990) as PPI = (∑vifi) /n where vi is the c-p value for the plant-parasitic nematodes genera i, and fi is the frequency of plant- parasitic nematodes genera i, and n is the total number of individual nematodes of the genera i in the sample. Enrichment (EI), and structure indices (SI) were calculated according to Ferris et al. (2001), with basal components (b) of the food web (fungal and bacterial feeders in the c-p 2 guild) calculated as b = ∑kbnb where kb is the weighted constant for the guild, and n is the number of nematodes in that guild. Enrichment (e) and structure (s) components were similarly calculated, using nematode guilds indicative of enrichment (bacterivores in c-p 1, and fungivores of c-p 2), and guilds supporting structure (bacterivores in c-p 3-5, fungivores c-p 3-5, omnivores of c-p 3-5, and predatory nematodes of c-p =2-5). Finally the EI is calculated as 100 x e/(e + b), and the SI as 100 x s/(s + b). Channel Index (CI), which provides an index of nature of decomposition, was calculated as 100 x (0.8 fungivores c-p 2/(3.2 bacterivores c-p 1 + 0.8 fungivores c-p 2) where the coefficients are the ke enrichment weightings for the respective guilds (Ferris et al., 2001). SI and EI provide information about the structure and enrichment of the soil food web, respectively and CI provides an index of the bacterial and fungal driven decomposition channels in the soil food web.

4.2.4. Statistical analysis

All the analyses were performed using general linear models procedure in SAS

Version 9.1, SAS Institute, Inc. (Cary, NC). Multivariate analysis of variance

(MANOVA) using composite of various nematode community indices or trophic groups

was performed to obtain F and P-values to determine overall significance of the model

for comparison between the habitats. Locations were considered random nested within the habitats. Contrasts were included in the MANOVA model statement for habitat group comparisons. Among the various multivariate test statistics obtained in the SAS output, “Pillai’s trace”, was considered for F and P-values. An alpha level of 0.05 was

used for all tests. Statistical analysis to determine the combination of nematode indices

that contributed to the differences between the habitats was performed with principal

component analysis (PCA) based on the correlation matrix using Statistica (Version

2002) for Windows package (Stat soft Inc., Tulsa, USA). PCA was also performed on

nematode trophic groups abundance separately using the same statistical software.

Nematode abundance data were transformed by ln (x+1) prior to stabilize the variance

and achieve normality in the data . Raw means and standard errors were calculated using

Minitab Version 13.31 (Minitab, Inc., State College, PA).

4.3. RESULTS

A total of 43 nematode genera were identified. In the muck soils, 38, 32, 32, 34

and 33 nematode genera were detected in forests, shrublands, grassy borders, turfgrass

lawns and croplands, respectively. In the mineral soil, 30 nematode genera were detected

in turfgrass lawns and 32 in croplands. Among the free-living nematode genera,

Acrobeloides was the most abundant in all the habitats at both study sites. Labronema,

Teratocephalus, Discolaimus, Mylonchulus, Hemicriconemoides and Rotylenchus were

only observed in muck soils. Juveniles of the cyst nematodes Heterodera spp. were

observed in low numbers from croplands in the mineral soils but not from the croplands

in muck soils. Among the plant parasites, Tylenchus was dominant in all the habitats but maximum population of this genus was observed in turfgrass lawns followed by

shrublands. The population of Pratylenchus was least in forests and highest in turfgrass

lawns and grassy borders. Hemicriconemoides was only observed in forest soils.

4.3.1 Comparison between the habitats using multivariate analysis of variance

(MANOVA)

MI was observed to be the highest in shrublands and forests compared to the other

habitats (Table 2). Croplands at both the study sites had the lowest Shannon diversity

index (H’) as compared to the other habitats. Shrublands and forests also showed the

highest structure index (SI) followed by the turfgrass lawns and grassy borders in muck

soils and croplands and turfgrass lawns in mineral soils, respectively (Table 4.2). EI

ranged between 62 and 78 in all the habitats. Croplands in the muck soil had the lowest

PPI compared to the other habitats (Table 4.2).

Results of MANOVA showed overall significance of the model for comparison

among the habitats (F = 2.25, df = 120, P = 0.002) using composite of various nematode

indices (MI, PPI, EI, SI, CI and H’). Contrasts revealed that croplands differed significantly from forests (F = 4.29, df = 27, P = 0.003), shrubs (F = 6.36, df = 27, P =

0.0003), grassy borders (F = 4.08, df = 27, P = 0.0004) and turfgrass lawns (F = 4.85, df

= 27, P = 0.001) (Table 4.3). Although no differences were observed between shrublands

and forests (F = 1.15, df = 27, P = 0.36), turfgrass lawns differed significantly from

shrublands (F = 2.43, df = 27, P = 0.005) but not from forests (F = 0.54, df = 27, P =

0.77) and grassy borders (F = 0.33, df = 27, P = 0.91).

Although croplands in muck soil were significantly different from croplands in the mineral soils (F = 2.85, df = 23, P = 0.03), no significant differences were observed between turfgrass lawns in the muck soils and mineral soil (F = 1.36, df = 23, P = 0.27).

Further, no significant differences were detected between the muck and mineral soil types when data from different habitats were lumped (F = 1.74 df = 23, P = 15). Turfgrass lawns in general were significantly different from croplands when averaged over the soil type (F = 7.56, df = 23, P = 0.0001) (Table 4).

MANOVA was also performed separately using composite of various nematode trophic groups and the results showed similar differentiation between the habitats as observed using indices (results not presented).

4.3.2 Principal components analysis (PCA)

The eigenanalysis of the nematode indices composite showed that 78% of the variation in the data is accounted for by the first three components (Table 4.5). Loading variables (nematode indices) that explained maximum variation along PC1, PC2 or PC3 were identified by their PC loadings. MI, EI and CI explained the spread along the PC1 while SI, EI and CI influenced habitat differences along the PC2 axes. Along the PC3 axis only PPI explained the variation in the data.

Eigenanalysis of the nematode trophic group abundance shows that 81% of the variation in the data is accounted for by the first three components (Table 4.6). Along the

PC1 axis, variation is partly explained by all the trophic groups but plant parasites explins the maximum variation among all the trophic groups. Along the PC2 axis only bacterivores and fungivores explained majority of the variation. Predatory nematodes explained most of the variation between the habitats along the PC3 axis.

Mean scores of PC1 and PC2 were calculated for comparison between the habitats using nematode indices or trophic groups for each location sampled within a habitat.

These mean scores were then plotted on X and Y-axes (X-axis as PC1 as and Y-axis as

PC2) for nematode indices or trophic groups (Figure 4.1and 4.2). Both PC1 and PC2

discriminated among the habitats using nematode indices and trophic group abundance

scores, respectively. However, the resolution of discrimination among the habitats

differed between the two plots. Nematode indices scores revealed that highly disturbed

habitats (croplands in muck and mineral soils) grouped on the lower left side of the plot

whereas undisturbed habitats (forests and shrubs) ordinated on the right side of both the

PC axes. Turfgrass lawns in mineral and muck soils and grassy borders showed an

intermediate position between the undisturbed and disturbed habitats (Figure 4.1).

Nematode trophic group abundance scores showed that most of the cropland locations

were observed on the right side of the PC1 and PC2 axes. Turfgrass lawns in mineral

soils ordinated on the left side of plot and turfgrass lawns in muck soils and shrublands

and forests are aligned on the lower negative side of the PC2. Grassy borders are

scattered but in general had an intermediary position along the two PC axes (Figure 4.2).

4.4. DISCUSSION

In the present study we used MANOVA to test the utility of the composite of

various nematode measures to compare different habitats representing a gradient of

anthropogenic disturbance. The nematode data showed that the croplands were

significantly different from all other habitats including forests, shrublands, turfgrass

lawns, and grassy borders. Turfgrass lawns were not significantly different from forests

but showed significant differences from shrublands. PCA analysis using composite of

various nematode indices revealed the relative relationship among the indices and the

contribution of the indices to habitat ordination.

We observed maximum contribution of MI, CI and EI for the differences between

the habitats in the PC1 while SI CI and EI significantly influenced PC2 axes. By

measuring the relative proportion of colonizers and persisters in the soil, the MI assesses

the impact of disturbance in any habitat (Bongers, 1990; Bongers and Bongers, 1998;

Neher, 1999). Greater abundance of high c-p value nematodes results in higher MI value,

which means higher maturity of the soil food web in a given habitat (Neher, 1999;

Porazinska et al., 1999; Yeates and Bongers, 1999). SI value provides information about

the levels of trophic links indicated by the abundance of high c-p value nematodes mainly omnivores and predatory nematodes, while EI provides an indication of the response of primary decomposers or enrichment opportunists like rhabditids towards the labile sources of organic material (Ferris et al, 2001; Ferris and Matute, 2003). CI indicates predominant decomposition channels in the soil food web (Ferris et al., 2001). We observed that all the habitats had moderately enriched food webs with EI ranging from 69 to 78%. However, SI and MI showed maximum differences among the habitats: forests, shrublands and turfgrass lawns in muck soils had high MI and SI while grassy borders and turfgrass lawns in mineral soils had moderate and the croplands in both muck and mineral soils had the lowest MI and SI values. MI has the highest weight in the first component axis followed by EI and SI with moderate weights while PC2 was mainly represented by SI. The PCA results also revealed that the croplands in both muck and mineral soils lie on the negative side of the PC axes 1 and 2, suggesting low maturity,

structure and diversity but high enrichment conditions as expected in these habitats

(Neher et al., 2005). Turfgrass lawns in muck soils also had moderately enriched but highly mature and structured food web compared to mineral soil turfgrass lawns in mineral soil. Position of forests and shrublands on the first two PC axes suggests mature and structured soil food webs that are moderately enriched. Increase in enrichment of the soil may lead to decrease in the maturity of the system (Villenave et al., 2003). Relative weights of the various indices as observed in the PCA analysis in our study might suggest

that highly structured and mature food webs might not have highly enriched food webs.

Since all the habitats showed enriched food webs in the present study, the opposite

relationship of SI and MI with EI cannot be clearly established. However, structure and

maturity of the system seems to be more prone to disturbances as indicated by the clear

differences of croplands from forests and shrublands. Our study also revealed that both

SI and MI are independent of the food web enrichment, at least at moderate to high levels

of enrichment conditions. Our results also indicate that SI and MI are more sensitive than

EI in detecting the differences among the habitats.

Forests did not show higher SI or MI compared with the shrublands. Our results

contrast with general observations of Ferris et al. (2001) that forest and shrublands are

considered to be poorly enriched and highly structured. We took soil samples at 15 cm

depth for nematode analysis in all habitats but this may not be sufficient to capture all the

nematodes in the lower layers of soil especially in the forests. In spite of this, forests

were at par with shrublands in terms of MI and both the habitats showed higher MI, SI

and diversity compared to all other habitats except for turfgrass lawns in muck soils. The

high level of enrichment of the food web in the unmanaged forests and shrublands may

be unique to the muck soils.

PPI is generally considered to be higher in the nutrient enriched conditions

because it is affected by the host conditions (Bongers et al., 1997). Several nematode

community indices are insensitive to the changes in the agroecosystem due to the fact that

plant parasitic nematodes, which represent a large portion of the total nematode

community, are not included in the calculation of community indices including SI, EI and

MI (Berkelmans et al., 2003). Although overall contribution of PPI is less as compared

to MI, SI, EI and CI in differentiating the habitats, PPI was the only index, that explained

the variations along the PC3 axis. Most of the plant-parasitic nematodes in our study

were ectoparasites, which are generally polyphagous (Cadet et al., 2005). For example,

Tylenchus and Filenchus are epidermal root hair feeders (Siddiqi, 1986; Yeates et al.,

1993). Therefore, results of our research suggest the importance of plant parasitic

nematodes, especially ectoparasites in determining the impact of human activities in

various habitats (Freckman and Ettema, 1993; Urzelai et al., 2000).

Ordination of habitats using both nematode indices and trophic group abundance

showed differences among the habitats however, resolution of discrimination differed between the two approaches. Nematode indices scores arranged and grouped the habitats much as we hypothesized along a gradient of anthropogenic disturbance. On the other hand ordination of habitats based on the scores calculated for nematode trophic group abundance showed different grouping of the habitats. Forests, shrublands and turfgrass lawns in muck soils were grouped together and mineral turfgrass lawns placed on the left

side of the PC1 axis while croplands aligned on the right side of the plot. Total

abundance of bacterivores was lower in forests, shrublands, and turfgrass lawns in muck

soils because general opportunists (c-p 2 bacterivores) (mainly cephalobids) were not

found in higher numbers in these habitats compared to the croplands. However, populations of enrichment opportunists (c-p 1 bacterivores) (mainly rhabditids) were observed at levels comparable to the croplands. Therefore, enriched conditions in these habitats resulted from the low abundance of c-p 2 bacterivores. Abundance of trophic

groups may not show an agreement with food web indices, because trophic groups are

further subdivided as functional guilds (indicating distinct food web conditions) in the

calculation of food web indices. Therefore, nematode c-p guilds may be a more useful

tool for habitat differentiation than total abundance of trophic groups. However, total

abundance of different trophic groups of nematodes addresses other ecological questions

such as effect of agricultural inputs (Neher, 1999, 2001; Bulluck et al., 2002; Yardim and

Edwards 1998; Nahar et al., 2005).

Using a combination of nematode indices in our study we were able to detect

differences between the habitats. PCA provided an exploratory approach to resolve the

differences among the habitats. Croplands were clearly different from the unmanaged forests and shrublands and even from moderately managed turfgrass lawns and grassy borders in the ordination plot. Turfgrass lawns and grassy borders also lay in the intermediary position of the plot indicating medium level of disturbance on the disturbance continuum. However, forests and shrublands do not follow a gradient as expected in our hypothesis. Using multivariate approaches, we revealed the relationship between various nematode indices and their relative importance in the differentiation of

habitats. Our study also highlighted the importance of nematode trophic groups and PPI

while comparing different habitats with varying degrees of human impact.

4.5. SUMMARY

Indicative value of nematode food web indices and trophic group abundance was

assessed for differentiating habitats in two soil types. Five habitats located in muck and two in mineral soils were compared using trophic group abundance and composite of soil

nematode community indices by multivariate analysis. A total of 604 soil samples were collected from croplands, grassy borders, turfgrass lawns, shrublands and forests in

Huron County, Ohio characterized by muck soils and a total of 242 soil samples were collected from two habitats, turfgrass lawns and croplands in Wayne County, Ohio characterized by mineral soils. Multiple locations were selected for collection of soil samples for each habitat. Croplands represented intensively managed habitats whereas forests and shrublands were considered unmanaged habitats. Turfgrass lawns in urban landscapes and grassy borders alongside croplands represented intermediate levels of management due to lack of tillage disturbance. All nematodes extracted from soil samples were identified to genus level, categorized to trophic groups and various nematode community indices were calculated. Multivariate analysis of variance revealed that croplands were significantly different from forests, shrublands, turfgrass lawns and

grassy borders in the muck soils. Turfgrass lawns in muck soils were not different from

forests but were different from shrublands. No differences were observed between

forests and shrublands. Turfgrass lawns in mineral soils differed significantly from

croplands in the mineral soil. No differences were observed between the soil types when

nematode data from croplands and turfgrass lawns from each county were lumped together. All habitats showed moderately enriched food webs as indicated by the mean enrichment index (EI) values (69-78%) in all the habitats at both the locations. Forests, shrublands and turfgrass lawns in muck soils had high maturity (MI) and structure index

(SI) whereas grassy borders and turfgrass lawns in mineral soils had intermediate and croplands in both muck and mineral soils had the lowest MI and SI values. PCA analysis indicated that MI and EI contributed the most to differences between habitats in the first principal component (PC1) while SI and CI significantly influenced PC2. Forests and shrublands had highly structured food webs as expected but their high enrichment may be unique to the muck soils. Ordination of habitats using nematode indices revealed that grassy borders and turfgrass lawns hold an intermediary position between undisturbed habitats (forests and shrublands) and highly disturbed habitats (croplands). Although overall contribution of plant-parasitic nematode index (PPI) was less compared to the other indices, PPI explained the variation among the habitats in the PC3 axis. Ordination of habitats using trophic group abundance may not show complete agreement with food web indices, because trophic groups are further subdivided as functional guilds while calculating food web indices.

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100

County Soil type Habitats Annual disturbance OM Soil pH (%) Insecticides Fertilizers Tillage Huron Muck Croplands High High High 19.53 6.2 Muck Turfgrass lawns Low-medium Moderate-high None 10.00 6.9

101 Muck Grassy borders* Low None None 15.44 7.0 Muck Shrublands None None None 9.40 6.1 Muck Forests None None None 12.40 5.2 Wayne Mineral Mineral Croplands High High High 2.20 6.3 Mineral Turfgrass lawns Moderate Low-high Moderate 5.20 6.3 *Grassy borders although do not receive direct fertilizer application, they may receive fertilizer nutrients due to runoff from the croplands.

Table 4.1: Description of different habitats at Huron and Wayne Counties in Ohio.

Habitats and soil types Croplands Croplands Grassy borders Turfgrass lawns Turfgrass Shrublands Forests muck mineral muck muck lawns mineral muck muck Indices MI 1.84 ± 0.02 1.93 ± 0.02 1.83 ± 0.03 2.09 ± 0.03 1.93 ± 0.02 2.33 ± 0.03 2.14 ± 0.03 PPI 2.02 ± 0.06 2.83 ± 0.02 2.68 ± 0.03 2.67 ± 0.02 2.62 ± 0.03 2.52 ± 0.02 2.44 ± 0.03 EI 71.57 ± 1.11 72.59 ± 1.08 78.88 ± 1.00 74.07 ± 1.05 69.51 ± 1.54 71.95 ± 1.07 72.22 ± 1.61

102 SI 33.95 ± 1.69 34.61 ± 1.68 48.55 ± 1.85 59.95 ± 1.63 45.11 ± 1.77 70.26 ± 1.37 61.55 ± 2.07 CI 21.81 ± 1.41 14.57 ± 1.19 9.20 ± 0.695 15.31 ± 1.15 9.37 ± 0.833 20.56 ± 1.68 17.03 ± 1.42 H’ 1.73 ± 0.03 1.49 ± 0.06 2.22 ± 0.03 2.30 ± 0.02 2.27 ± 0.02 2.20 ± 0.03 2.12 ± 0.03 Trophic gp. Bacterivores 61.82 ± 4.53 67.67 ± 4.12 89.63 ± 7.90 44.76 ± 2.96 92.04 ± 8.43 38.17 ± 3.61 47.78 ± 5.41 Fungivores 26.07 ± 2.65 15.77 ± 1.14 15.87 ± 1.68 13.49 ± 1.01 18.21 ± 1.70 16.63 ± 3.15 13.67 ± 1.71 Omnivores 3.82 ± 0.27 4.82 ± 0.65 7.42 ± 0.51 10.76 ± 0.93 9.73 ± 0.69 15.80 ± 0.48 8.08 ± 0.58 Predatory 0.95 ± 0.13 2.52 ± 0.29 1.56 ± 0.24 1.69 ± 0.16 2.01 ± 0.24 1.62 ± 0.17 1.81 ± 0.27 Plant-parasitic 29.7 ± 3.40 47.69 ± 3.18 64.00 ± 5.89 92.50 ± 6.50 79.59 ± 7.39 83.03 ± 5.83 39.70 ± 3.34

Table 4.2: Mean (± SE) values of different nematode indices including maturity (MI), plant parasitic (PPI), enrichment (EI), structure (SI), channel (CI) and Shannon diversity indices (H’) and trophic groups abundances used in the multivariate analysis.

Habitat F df P Overall habitat effect 2.25 24, 120 0.002

Croplands vs. forests 4.29 6, 27 0.003

Croplands vs. shrublands 6.36 6, 27 0.0003

Croplands vs. grassy borders 4.08 6, 27 0.004

Croplands vs. turfgrass lawns 4.85 6, 27 0.001

103 Turfgrass lawns vs. shrublands 2.43 6, 27 0.005

Turfgrass lawns vs. forests 0.54 6, 27 0.77

Turfgrass lawns vs. grassy borders 0.33 6, 27 0.91

Forests vs. shrublands 1.15 6, 27 0.36

Table 4.3: Multivariate test (Pillai’s trace) based on different nematode indices including MI, PPI, EI, SI, CI and H’ comparing the habitats within muck soils.

Habitat F df P

Turfgrass lawns mineral vs. croplands in 5.06 6, 23 0.001 mineral soils Turfgrass lawns in muck soil vs. turfgrass lawns 1.36 6, 23 0.27 in mineral soils Muck soil vs. mineral soilsa 1.74 6, 23 0.15

Turfgrass lawns vs. croplandsb 7.56 6, 23 0.0001

Croplands in muck soils vs. croplands in mineral 2.85 6, 23 0.03 soils 104 aAverage of turfgrass lawns and croplands in muck soils compared with average of turfgrass lawns and croplands mineral soils, bAverage of turfgrass lawns in muck and mineral soils compared with average of croplands in the muck and mineral soils.

Table 4.4: Multivariate test (Pillai’s trace) based on different nematode indices including MI, PPI, EI, SI, CI and H’ comparing turfgrass lawns vs croplands habitats within mineral soils and with muck soils turfgrass lawns and croplands.

Component Eigen value Variation explained (%) Cumulative variation (%) 1 2.125 35.421 35.421 2 1.677 27.963 63.384 3 0.885 14.755 78.140 4 0.812 13.535 91.676 5 0.367 6.129 97.805 6 0.131 2.194 100.00 Attribute loading for eigenvectors a Variable 1 2 3

105 MI 0.606 0.146 -0.173 PPI 0.149 0.342 0.921 EI -0.495 0.429 -0.160 SI 0.3466 0.562 -0.257 CI 0.445 -0.459 0.0276 H ’ 0.213 0.386 -0.165 aEigen vector loading for components 1, 2 and 3 only are shown. These three components account for 78 % of the total variation in the data sets.

Table 4.5: Eigenanalysis of the correlation matrix for nematode indices including MI, PPI, EI, SI, CI and H’ for all habitats.

Component Eigen value Variation explained (%) Cumulative variation (%) 1 1.985 39.711 39.711 2 1.276 25.522 65.233 3 0.816 16.335 81.569 4 0.465 9.311 90.880 5 0.455 9.119 100.000 Attribute loading for eigenvectors a Variable 1 2 3 Bacterivores -0.437 0.548 0.101 Fungivores -0.422 0.572 -0.058 106 Omnivores -0.477 -0.371 -0.465 Predatory -0.361 -0.315 0.853 Plant-parasites -0.520 -0.366 -0.201 aEigen vector loading for components 1, 2 and 3 only are shown. These three components account for 78 % of the total variation in the data sets.

Table 4.6: Eigenanalysis of the correlation matrix for nematode trophic groups for all habitats.

Fig. 4.1: Principal components analysis of nematode indices in different habitats. The plot shows the distribution of the habitats on the first two principal components. Each point represents the mean score of a location within the habitat including muck soil croplands (■), mineral soil croplands (□), turfgrass lawns muck (▲), turfgrass lawns mineral (∆), grassy borders (▲), forests (♦) and shrublands (●).

107

Fig. 4.2. Principal components analysis of nematode trophic group abundance in all habitats. The plot shows the distribution of the habitats on the first two principal components. Each point represents the mean score of a location within the habitat including muck soil croplands (■), mineral soil croplands (□), turfgrass lawns muck (▲), turfgrass lawns mineral (∆), grassy borders (▲), forests (♦) and shrublands (●).

108

CHAPTER 5

SYNTHESIS AND FUTURE DIRECTIONS

OVERALL SYNTHESIS

The results of this study supported my thesis of the value of using nematode

communities to quantify soil health. I focused mainly on understanding the effect of

different farming strategies transitioning from conventional to organic management on

nematode communities. Further, comparison of various habitats across a gradient of

anthropogenic impact also revealed the indicative value and relative contribution of

nematode community measures to habitat differences.

Application of composted manures increased the total abundance of bacterivore

nematodes in organic compared with conventional farming systems (Chapters 2 and 3).

Nematode faunal profile estimates showed that the food webs were highly enriched but poorly structured in both organic and conventional farming systems. Even long-term application of organic amendments failed to increase the structure index (SI) in organically managed farms, probably due to the excessive tillage. Further, research findings in chapter 4 showed that forests and shrublands had higher SI compared with the managed croplands. Therefore, our research findings suggest that reduction in the intensity of tillage in organic farming systems may produce more structured soil food webs.

109

The research conducted also pointed out some of the problems associated with the

use of food web indices such as EI, SI and CI (Chapter 3). Calculations of food web indices require numerator and denominator and therefore, increase in the value of an index does not necessarily reflect that the numerator would be higher (Ferris and Matute,

2003). Application of organic amendments showed greater abundance of bacterivores dominated by c-p 1 (enrichment opportunists) and c-p 2 (general opportunists) but EI was

not higher in the composted than the non-composted management strategies. Increase in

the abundance of general opportunists along with enrichment opportunist bacterivores in

the composted strategies reduced the overall level of EI. However, higher abundance of

c-p 2 bacterivores in the composted strategies may indicate some level of succession in

the soil food web (Ferris and Matute, 2003). Therefore, caution should be exercised

while interpreting the results based on values of EI alone. EI could be a suitable measure of enrichment in the food web immediately after the application of external inputs but not

later.

Among the four different management transition strategies (high tunnels vegetable, mixed species hay crop, tilled fallow and open field vegetables), high tunnels in general had the highest total abundance of bacterivore and fungivore nematodes. The average increase in soil temperature of 2-5 o C might have enhanced the decomposition

processes thereby making the soil food web biologically more active in high tunnels

compared with the other strategies. These findings suggested that even a marginal manipulation of soil temperature could affect the soil biology as indicated by increase in free-living nematodes and supported by measured soil characteristics.

110

STATUS OF PLANT PARASITIC NEMATODES IN SOIL FOOD WEBS

Free-living nematodes are found in larger numbers in the total soil nematode

community and most studies have relied only on the use of free-living nematodes in

understanding the biological functioning of soil processes (Cadet et. al., 2003). Bongers

(1990) developed an index of maturity (MI) by dividing nematode taxa into five groups based on the c-p value of 1-5, each representing different life strategies and ecological

requirements, but the plant parasitic nematodes were excluded from the calculation of

MI. Bongers (1990) also developed an index based on plant parasitic taxa only, known

as plant parasitic index (PPI) analogous to MI and scaled 2-5 based on c-p values.

However, it was observed that MI and PPI showed an inverse relationship under nutrient

rich conditions (Bongers et al., 1997; Bongers and Kortahls 1995; Freckman and Ettema,

1993).

Yeates (1994) proposed another index of maturity including both plant parasites

and free-living nematodes known as combined maturity index (CMI). Some studies have

used CMI as an indicator of maturity. For example, Neher (1999) observed higher CMI in

organic farming systems compared to the conventional farming systems and claimed a

better maturity in organic farming systems.

In addition to other nematode community measures discussed above, Ferris et al.

(2001) developed enrichment (EI), structure (SI), and channel (CI) indices based on the relative weighted abundance of nematode c-p guilds. Calculation of these indices is also

based strictly on free-living nematodes. Therefore, plant-parasitic nematodes have been

excluded from various community measures and may provide useful information that

would improve them.

111

Plant parasitic nematodes possess different feeding habits. They can be broadly

classified into two main categories: ectoparasites and endoparasites. Ectoparasites are

those that do not enter the host roots, while endoparasites feed in the roots. The two

groups of plant parasitic nematodes also appear to compete against each other. For

example, Brinkman et al. (2004) noted that high densities of endoparasitic nematodes

suppress the development of the ectoparasitic nematode Tylenchorynchus ventralis.

Cadet and Spaull (1985) observed that increase in abundance of the ectoparasitic

Helicotylenchus dihystera was associated with a reduction in the pathogenic impact of

other plant-parasitic nematodes. Since endoparasitic nematodes are considered as major

pests of host plants, it would be important to understand the relationship between

ectoparasitic and endoparasitic nematodes. I explored their relationship in my study.

Table 5.1 shows the list of plant-parasitic nematode genera observed in the

present study. Ectoparasitic nematodes were dominated by two genera, Tylenchus and

Filenchus, while the root lesion nematode Pratylenchus was the dominant migratory

endoparasite (Chapter 2, 3 and 4). Percentage of ectoparasites ranged from 20-44% in the total number of nematodes, with the highest percentage in unmanaged habitats including shrubs and forests followed by urban landscapes (turfgrass lawns) and the

lowest in vegetable crops (Figure 5.1). This clearly demonstrates that while calculating

various nematode indices a large portion of the nematode community is left out.

The endoparasitic nematode Pratylenchus crenatus was favored by corn followed

by soybeans and oats but not by hay. Cultivation of the host crop, corn, in the rotation

might have contributed to the build up of high levels of this endoparasitic nematode in

the soil (Chapter 2). However, ectoparasitic nematodes did not show any such response

112

in our study (Figures 5.2). This could be due to polyphagous nature of ectoparasites

(Cadet et al., 2005). Moreover Tylenchus and Filenchus were the dominant genera but

they are presumed to be algal feeders and weak phytoparasites (Yeates et. al., 1993).

Our research (Chapter 3) indicated that compost application might not have the

same effect on all the plant-parasitic nematode guilds especially when the biology and

host range of nematode guilds are contrasting (Zunke, 1990; Okada et al 2002;

Vestegard, 2004). The soil population of Pratylenchus crenatus was significantly lower

in the composted than the non-composted transition strategies, while that of the Pp2

guild, which mainly consisted of Tylenchus and Filenchus, was higher in the former

(Figure 5.3). Migratory endoparasites like Pratylenchus are considered harmful to the host plant, as they cause cell death during feeding and migration through root tissue, and

also predispose the cortex to attack by other plant-pathogens (Zunke, 1990). On the other hand ectoparasites like Tylenchus, Filenchus and Psilenchus primarily feed on root hairs

(Yeates et al., 1993) and fungi (Okada et al., 2002).

Correlation of c-p 2 plant-parasitic nematodes with MB-N further suggests that

they are functionally more active than other guilds (Chapter 3). Root hair feeders have

been shown to contribute significantly to the increase in microbial activity by eliciting

leakage of metabolites from the roots (Verschoor, 2002; Bardgett et al., 1999; Yeates et

al., 1999).

FUTURE DIRECTIONS

Although, the current research suggested that frequent tillage operations required

in the organic systems was the potential reason for the low SI, further research is needed

113

to test this hypothesis that a low tilled organic farming system would possibly have

higher abundance of omnivores and predatory nematodes resulting in high structured

food webs.

Compost suppressed the population of Pratylenchus sp. but favored ectoparasites

Tylenchus, Filenchus and Psilenchus. Further studies to determine the mechanism of

differential effects of compost on these plant-parasites can fill some of the major gaps in

our understanding of their biology and contribution to soil ecosystem function.

Correlation between c-p 2 plant-parasites and microbial activity indicated that this guild

of plant parasites is functionally important at least in our soil and climatic conditions.

However, our research was not aimed at quantifying the feeding habits of this group of nematodes. Further research in this direction is required to ascertain their feeding habits and enhancing our understanding of their role in soil biology. Studies to quantify the relative contribution of the ectoparasites to nitrogen pools may also be considered.

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117

Genus C-p value Nature of feeding Family Tylenchus a 2 Ectoparasites Tylenchidae Filenchus a 2 Ectoparasites Tylenchidae Boleodorus 2 Ectoparasites Tylenchidae Psilenchus 2 Ectoparasites Psilenchidae Tylenchorynchus 3 Ectoparasites Dolichodoridae Criconemella 3 Ectoparasites Criconematidae Paratylenchus 2 Ectoparasites Criconematidae Trichodorus 4 Ectoparasites Trichodoridae Longidorus 5 Ectoparasites Longidoridae Xiphinema 5 Ectoparasites Longidoridae Helicotylenchus 3 Ectoparasites Hoplolaimidae Hoplolaimus 3 Ectoparasites Hoplolaimidae Pratylenchus b 3 Endoparasite Pratylenchidae Heterodera 3 Endoparasite Heteroderidae Meloidogyne 3 Endoparasite Heteroderidae adominant ectoparasites, bdominant endoparasite. C-p: colonizer persister

Table 5.1: List of genera of plant-parasitic nematodes.

118

Ectoparasites Ectoparasites 44% 33%

Endoparasites 8% Free-living Endopar as it es 64% 3% Free-living 48% Forest lands Shrub lands

Ectoparasites 31%

Ectoparasites Endoparasites 6% Free-living %age:10% -- Free-living 59% Endoparasites 93% 1%

Field crops Vegetable crops

Ectoparasites Ec t opar as it es 41% 27%

Endoparasites Endoparasites 13% 10% Free-living 63% Free-living 46% Turf lawns Grassy borders

Figure 5.1: Percent ectoparasites, free-living and endoparasites in total nematode community in different habitats

119

g soil population/10 Ectoparasite

0 Corn Soybean Oats Hay

Crop

Figure 5.2: Comparison of the abundance of ectoparasitic nematodes in corn, soybeans, oats and hay.

120

Composted Non-composted 20 a P = 0.05

15 b

10 P = 0.04 a

b 5 Nematode population / 10 g soil Nematode

0 Pratylenchus PP2

Figure 5.3: Effect of composted and non-composted farming strategies on Pratylenchus sp. and plant-parasitic nematode c-p guild 2 consisting mainly of Tylenchus and Filenchus.

121

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