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ARBUSCULAR MYCORRHIZAL FUNGI ALTER PRODUCTIVITY AND
MORPHOLOGY AND DECREASE STORAGE ROT IN SOLANUM TUBEROSUM
UNDER HIGH INPUT CONDITIONS

By

Brendan Anthony N iemira

A DISSERTATION

Submitted to Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1996

ABSTRACT
ARBUSCULAR MYCORRHIZAL FUNGI ALTER PRODUCTIVITY AND
MORPHOLOGY AND DECREASE STORAGE ROT IN SOLANUM TUBEROSUM
UNDER HIGH INPUT CONDITIONS

By

Brendan Anthony Niemira

Arbuscular mycorrhizal (AM) fungi are ubiquitous soil fungi that form beneﬁcial
associations with the roots of most crop species, including potato (Solanum tuberosum).
The AM spore population structure in agricultural soils devoted to potato production is
inﬂuenced by the type of management employed (low-input vs. high-input), and, to a
lesser extent, by the speciﬁc rotation used. Low-input management and complex
rotations tend to foster a greater complexity and resiliency in the AM spore population
than high-input management and simple rotations. In a greenhouse setting, the
reproductive physiology of potato plants grown in the presence of AM inoculum is
altered, resulting in increased production of minitubers, and a production shift in favor
of smaller minitubers. Plants exposed to AM had longer stolons, increased total stolon
length per plant, more uniform stolon development, and more tuber initiates per plant
than plants grown in sterile medium. Minitubers produced in the presence of AM
inoculum showed signiﬁcantly increased resistance to F usarium sambucinum, an
important potato storage dry rot. Four week old potato roots colonized by AM showed
signiﬁcantly reduced total activity of peroxidase, a defense related enzyme. Collectively,
these data suggest that AM induce a systemic resistance response in potato during the

earliest stages of their association. A given species’ propensity to establish AM

associations is related to its capability to produce border cells (BC, formerly known as
"sloughed root cap cells"). It is observed that strongly mycorrhizal families (e.g.
Poaceae, Fabaceae) produce thousands of BC per root tip, moderately mycorrhizal
families (e.g. Solanaceae, Amaranthaceae) produce dozens or hundreds of BC per root
tip, and minimally or non-mycorrhizal families (e. g. Brassicaceae, Chenopodiaceae) do
not produce BC. The mechanism of the role of BC and BC-produced compounds in the

development of AM associations has not been determined.

This dissertation is dedicated to my wife Meegan, whose constant and undiminishing

love and support made this work possible.

iv

ACKNOWLEDGMENTS

I would like to gratefully acknowledge Dr. Gene Saﬁr, my major professor and
friend, for his counsel and advice, and for his support and conﬁdence;

The other members of my committee, Dr. George Bird, Dr. Raymond
Hammerschmidt and Dr. Muralee Nair, and also Dr. Richard Harwood, for their
individual and collective assistance and expertise;

The College of Natural Science and the Department of Botany and Plant
Pathology at Michigan State University for their assistance and ﬁnancial support;

Don and Mary Kay Sklarczyk of Sklarczyk Seed Farms, Johannesburg, M1, for
their extraordinary generosity of time and resources in support of my research; Butler
Potato Farm, Crystal Falls, MI and Kitchen Potato Farm, Alba, M1 for their cooperative
efforts;

Dr. Mike Birney, Dept. Entomology, for his collaboration in the ﬁeld, and Eric
Netzloff, Sean O’Donnell, Sarah Toms, Todd Smith and Michelle Wisniewski for
technical assistance;

All of my friends (and debating partners), including, but not limited to, Dr. Steve
Stephenson, Dr. Leadir Fries, Dr. Ayse Ozan, Laura Arriola, Virginia Baker, Sigrid

Ewers, Richard Kaitany and Venu and Madhuri Yagallah. Particular thanks go to John

and Olivia Alford, both of whom hold a special place in my heart, for their friendship
and support;

Finally, I would like to gratefully acknowledge my families, Thadeus and Helen
Niemira, and Charles and Judi Stock, for their love and support, and most especially my

wife, Meegan, to whom this dissertation is dedicated.

vi

TABLE OF CONTENTS

List of Tables ............................................................ Page viii
List of Figures ............................................................ Page ix
Introduction . .............................................................. Page 1
Mycorrhizae ........................................................... Page 1
Germination . ....................................................... Page 2
Colonization ....................................................... Page 2

Host responses ....................................................... Page 3
Sustainable agriculture . ................................................ Page 4
Literature cited . ....................................................... Page 8

Chapter 1: Arbuscular mycorrhizal fungus spore populations and colonization of
potato: crop rotation and input level as major factors.

Abstract . ............................................................ Page 12
Introduction .......................................................... Page 13
Materials and Methods . .............................................. Page 15
High-input system ................................................. Page 15
Low-input system ................................................. Page 16
AM root colonization assay ....................................... Page 16
AM spore isolation and census ..................................... Page 16
Statistical analysis ................................................. Page 17
Results .............................................................. Page 17
High-input system ................................................. Page 17
Low-input system ................................................. Page 20
Discussion ............................................................ Page 30
Literature cited . ..................................................... Page 37

Chapter 2: Production of pre-nuclear minitubers of potato with peat-based
arbuscular mycorrhizal fungal inoculum.

Abstract . ............................................................ Page 41
Introduction .......................................................... Page 42
Materials and Methods . .............................................. Page 43
Experiment 1: minituber production ................................. Page 44
Experiment 2: stolon development and minituber initiation ............ Page 47
AM root colonization assay ....................................... Page 47

vii

Statistical analysis ................................................. Page 47

Results .............................................................. Page 48
Experiment 1: tuber production .................................... Page 48
Experiment 2: stolon production, tuber initiation . ................... Page 53

Discussion ............................................................ Page 60

Literature cited . ..................................................... Page 65

Chapter 3: Post-harvest suppression of potato dry rot (Fusarium sambucinum) in
prenuclear minitubers by arbuscular mycorrhizal fungal inoculum.

Abstract . ............................................................ Page 68
Introduction .......................................................... Page 69
Materials and Methods . .............................................. Page 70
Storage rot: Research greenhouse ................................. Page 70
Storage rot: Commercial greenhouse ................................ Page 71
Root protein extraction and sample preparation ...................... Page 72
Total peroxidase assay ............................................. Page 72
AM root colonization assay ....................................... Page 73
Fusarium sambucinum challenge ................................... Page 73
Statistical analysis ................................................. Page 73
Results .............................................................. Page 74
Storage rot: Research greenhouse . ................................. Page 74
Storage rot: Commercial greenhouse ............................... Page 74
Root protein total peroxidase ........................................ Page 74
Discussion ............................................................ Page 77
Literature cited . ..................................................... Page 82
Chapter 4: Summary and Conclusions ..................................... Page 85

Appendix A: A Theoretical Study of the Association of Arbuscular Mycorrhizal
Colonization with Border Cell Production.

Abstract . ............................................................ Page 88
Introduction .......................................................... Page 88
Materials and Methods . .............................................. Page 90
Reference list collection ............................................ Page 90
Statistical analysis ................................................. Page 91
Results .............................................................. Page 91
AM colonization, BC production ................................... Page 91
Cluster analysis .................................................... Page 91
Discussion ............................................................ Page 91
Literature cited . .................................................... Page 101

viii

LIST OF TABLES

Table 1.1. Yield and AM colonization at the high-input farm .............. Page 18
Table 1.2. AM spore populations at the high-input farm ................... Page 23
Table 1.3. Yield and AM colonization at the low-input farm ............... Page 25
Table 1.4. AM spore populations at the low-input farm ................... Page 28

Table 2.1. Table 1. Application rate and timing of fungicides used in a greenhouse
study of potato minituber production (Exp’t. 1) ........................... Page 45

Table 2.2. Prenuclear minituber yield of potato grown in the greenhouse with and
without AM (Exp’t. 1) . .............................................. Page 49

Table 2.3. Phosphorus concentration of stem + leaf tissue and AM colonization level
of potato roots grown in the greenhouse (Exp’t. 1) . . ................... Page 51

Table 2.4. Stolon and tuber initiate development of potato grown in the greenhouse
with and without AM (Exp’t.2) ....................................... Page 54

Table 2.5. Phosphorus concentration of stem plus leaf tissue and colonization level of
potato roots grown in the greenhouse with and without AM (Exp’t. 2) Page 56

Table 3.1. Progression of fusarium dry rot in potato minitubers grown in
nonmycorrhizal (CTRL) and mycorrhizal (INOC) growing media ........ Page 75

Table A. 1. AM colonization and BC production .......................... Page 92

LIST OF FIGURES

Figure 1.1. Population of AM spores in ﬁeld soil samples taken from three different

rotations within two input systems. Individual points are the mean of four samples,
bars indicate standard error. ........................................... Page 21

Figure 2.1. Total length of stolon material (cm) produced per potato plant, by ranges
of production, (Exp’t.2, Apr.-July 1994, at Johannesburg, MI). Each of the six
samples per treatment represents the average of three plants. The data from each
of the six inoculated (INOC) and the six control (CTRL) beds are expressed as total
stolon production per plant (cm). Weeks post planting .................. Page 45

Figure A.1. Cluster analysis of the major families represented in the data set
(Fabaceae, Poaceae, Amaranthaceae, Solanaceae, Brassicaceae, Chenopodiaceae).
Shapes indicate family, patterns indicate cluster ......................... Page 96

INTRODUCTION

Mycorrhizae

Mycorrhizal fungi are ubiquitous soil fungi that form associations with the roots
of most plant species on Earth; these associations are generally symbiotic and beneﬁcial
to the health and growth of the plant (Pﬂeger and Linderman, 1994). There are ﬁve
major categories of mycorrhizal associations, each having distinct morphology anatomy
and host range: arbuscular mycorrhizae, ectomycorrhizae, ecetendomycorrhizae,
ericaceous mycorrhizae and orchidaceous mycorrhizae (Harley and Smith, 1983). Of
these, arbuscular mycorrhizae (AM), previously known as vesicular-arbuscular
mycorrhizae (V AM), are the most predominant (Harley and Smith, 1983). These fungi
form associations in most of the world’s biomes with most of the world’s plant species;
these species include most of the major agricultural and horticultural plants, including
com (Zea mays) , wheat (Triticum aestivum), oat (Avena sativa), cucumber (Cucumis
sativus), clover (Trifolium repens), soybean (Glycine max), bean (Phaseolus vulgaris),
coffee (Coﬁea arabica) and citrus (Citrus spp.) (Saﬁr, 1994). Given the cosmopolitan
nature of AM fungi, plants that do not form these associations are considered the
exception (Gerdemann, 1968; Tester et al. , 1987). Within Zygomycetes, the order
Glomales contains two sub-orders, Glomineae and Gigasporineae. There are ﬁve genera

of AM fungi, containing approximately 150 species: Glomus, Acaulospora,

2

Entrophospora (all within Glomineae), Gigaspora and Scutellospora (both within
Gigasporineae) (Morton et a1. , 1995).
Germination

AM fungal spores can germinate and initiate hyphal growth in the soil
independently. The infective hyphae grow through the soil, and attach to the surface of
nearby plant roots. The hyphae form appresoria, penetrate the root, and initiate the
association. If there are no plant roots in the vicinity, hyphal growth ceases and the
fungus re-enters a dormant state (Bowen, 1987). Continued hyphal growth through the
soil and sporulation can only occur in the presence of host plant roots, although actual
physical contact between hyphae and root is not required (Becard and Piche, 1989).
Plant exudates stimulate hyphal growth, branching and differentiation, and also stimulate
host penetration and arbuscule development (Becard and Piche, 1989). AM spores will
germinate axenically in vitro, but hyphal growth ceases after a short time upon depletion
of spore carbon reserves. Growth of the hyphae in the presence of the root may be
separated into two phases: an initial slow hyphal growth phase, relying on the AM spore
carbon reserves, and a more rapid hyphal growth phase , relying on carbon obtained
from the plant root via arbuscules. The initial growth phase may be independent,
stimulated by signals from the root; the second phase is entirely root dependent (Becard
and Piche, 1989).
Colonization

AM fungal hyphae form appresoria, penetrate the root and spread through the
outer layers of root tissue. The fungal hyphae invades both inter- and intracellularly

through the periderm; the meristems and central root cylinder are not colonized

3

(Bonfante-Fasolo, 1984; Jacquelinet-Jeanmougin et a1. 1988). In the deeper layers of the
root cortex, intercellular and intracellular hyphae branch to form vesicles, which are used
for storage of fungal lipids (Cooper, 1984). Intercellular hyphae also differentiate into
arbuscules, transitory structures with large surface area, which are the site of the
majority of nutrient exchange between the fungus and the plant (Jakobsen, 1995).
Individual arbuscules have a functional life span of 4-10 days, after which they are
degraded by the plant; the host cell returns to normal. The remainder of the mycelium
is unaffected by this degradation and continues to grow and form other arbuscules.
Host responses

AM fungi enhance nutrient uptake and translocation to the host plant, while the
host provides high-energy carbon compounds to the fungus (Hampp and Schaeffer,
1995). In nutrient limiting soils, mycorrhizal plants have higher rates of growth than
non-mycorrhizal plants. Growth responses may have a lag time of weeks after the start
of colonization; this may be a result of competition for photosynthate between the fungus
and the host sinks (Smith and Gianinazzi-Pearson, 1988).

AM fungi enhance the assimilation of a number of mineral nutrients, including
zinc (Zn), sulfur (S), calcium (Ca) and, most signiﬁcantly, phosphorus (P). Nutrient
uptake is the major contribution of the fungus to the symbiosis. The fungal hyphae are
able to exploit the soil proﬁle more extensively than roots and root hairs, extending
signiﬁcantly beyond the zone of depletion without mycorrhizae (Hayman, 1983). AM
fungal hyphae are able to penetrate soil microaggregates that roots are unable to penetrate
(Miller and Jastrow, 1992). Mycorrhizal uptake and importation of soil nutrients

depends on development of extramatn'cal hyphae in soil, hyphal absorption of the

4

relatively immobile phosphates, translocation of P through the hyphae and transfer of P
from the fungus to the plant (Rhodes and Gerdemann, 1975). Initial soil P availability
will determine the extent to which AM fungi will form a functioning association with the
plant roots. Under high P availability, the advantage gained from luxury P contributions
from the fungus to the plant tend not to offset the metabolic cost of maintaining the
association, and the extent of mycorrhizal infection is therefore reduced (Hepper, 1983).

In addition to enhanced nutrition, mycorrhizal plants can have increased resistance
to diseases, including phytonematodes (Rosendahl and Rosendahl, 1990) and pathogenic
fungi (Newsham et al., 1993), increased drought tolerance (Nelsen and Saﬁr, 1982),
tolerance of salt stress (Sylvia and Williams, 1992) and heavy metal toxicity (Sylvia and
Williams, 1992).
Sustainable agriculture

Unintended ancillary effects of high-input agriculture are only now coming to light
(Harwood, 1990). Long—term use of pesticides has resulted in resistant insect, fungal and
bacterial pathogens, requiring the production of new and more toxic agrochemicals
(W intersteen and Higley, 1993). Pesticide runoff from agricultural soils has been shown
to contaminate groundwater. while excess fertilizer runoff contributes to algal blooms in
surface water, harming ﬁsh populations and disrupting aquatic ecosystems (Logan, 1990;
Wintersteen and Higley, 1993). Extensive plowing and intensive monocropping can
increase the rate of degradation of topsoil through loss of mineral nutrients and depletion
of soil organic matter (Okigogo, 1990; Cuperus et al., 1993). This loss of organic
matter decreases the soil’s ability to retain water and mineral nutrients, as well as its

ability to resist erosion by wind and water (Sposito, 1989). Techniques developed in

5

organic and low—input production systems can be adapted to reduce the impact of the
more damaging aspects of conventional high-input agriculture (Brady, 1990;
Bethlenfalvay, 1992).

Crop rotations that include nitrogen ﬁxing legumes can increase crop productivity
and soil quality directly by incorporation of nitrogen-rich organic residues, and indirectly
by inﬂuencing other components of the agroecosystem (Johnson and Pﬂeger, 1992;
Francis and Clegg, 1990). Rotations have historically been essential to proﬁtable crop
production (Harwood, 1990), but the introduction of relatively inexpensive nitrogen
fertilizer after World War II encouraged farmers to focus on continuous cash cropping,
and to rely more heavily on chemical inputs for fertility (Francis and Clegg, 1990;
Bethlenfalvay, 1992). Incorporation of detritus into the soil contributes to soil organic
matter content, which aides in maintaining good soil structure, nutrient exchange capacity
and water holding capacity (Miller and Iarson, 1990; Sposito, 1989). Crops in a well
designed rotation sequence can reduce populations of plant pathogenic insects,
nematodes, fungi and bacteria (Francis and Clegg, 1990; Hendrix et al., 1990). As
pathogens often associate themselves with particular plant hosts, switching between plants
that differ greatly (long-season vs. short-season, perennial vs. annuals, etc.) can interrupt
the pests’ reproductive cycles. This principle is one of the cornerstones of the modern
concept of integrated pest management (Brown, 1993). More complex plant communities
tend to foster more diverse soil microbial populations that serve to reduce the likelihood
of explosive growth of any one species (Linderman, 1992).

The most common form of multiple cropping, crop rotations, involve the

sequential planting and growth of different plants on a given site. Planting systems that

6

incorporate different plants simultaneously in a given location is more correctly called
intercropping (Stinner and Blair, 1990). In intercropped systems, the lack of an
intervening time between crops as in a crop rotation results in a greater degree of
synergy between the plants. This synergy serves to promote the health and productivity
of the crop plants in the absence of conventional chemical inputs (Francis and Clegg,
1990; Stinner and Blair, 1990). Concurrently grown non-crop plants may exert direct
inﬂuences on the crop plant, such as increasing nitrogen availability (Lal, 1987; Brown
and Thomas, 1990). The inﬂuences may also be more indirect, such as the suppression
of pathogens (Brust et al., 1986) and/ or stimulation of predators (Stinner and Blair, 1990)

Under appropriate conditions, intercropping can be an effective system, with
multiple food producing plants growing together. A useful example is a polyculture of
corn, beans and squash, a type of intercropping practiced in Central America since
prehispanic times (Pinchinat et a1. , 1976). Given sufﬁcient water, the synergy between
the three plants stimulates corn productivity over that of monoculture yields, and allows
the production of a larger total amount of food per unit area than could be expected from
a monocropping system of any of the three plants alone (Gliessman, 1990). The corn
stalk provides support for the beans, the beans ﬁx nitrogen into the soil, and the wide,
low leaves of the squash shade out weed competitors (Gliessman, 1983; Gliessman 1990).
As legumes are known to create a variety of compounds that stimulate AM fungi (Nair
et al., 1991; Siqueira et al., 1991; Saﬁr, 1994), it may be that AM associations increase
in the intercropped system. AM fungi are known to connect the roots of adjacent plants,
facilitating nutrient transfer (Francis and Read, 1984). In tropical and subtropical

climates, the three plants are seeded simultaneously; in temperate climates, this results

7

in the beans overwhelming the corn, and the synergy instead becomes competition. This
can be addressed by allowing the corn to germinate and develop a stalk before the other
seeds are planted. The modiﬁcations and adaptations necessary to make this particular
production system effective are strongly emblematic of one of the central premises of
sustainable agriculture, that care must be taken to design a production system that is
appropriate for the particular climate, edaphic and biological inﬂuences at the particular

growing site.

8

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Becard, G. and Y. Piche. 1989 New aspects on the acquisition of biotrophic status by
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Siqueira, J .O., G.R. Saﬁr and MG. Nair. 1991. Stimulation of vesicular-arbuscular
mycorrhiza formation and growth of white clover by ﬂavonoid compounds. New
Phytol. 118287-93

Smith, SE. and V. Gianinazzi Pearson. 1988. Physiological interactions between
symbionts in vesicular-arbuscular mycorrhizal plants. Ann. Rev. Plant Phys. and Plant
Mol. Biol. 39:221-244

Stinner, BR. and J .M. Blair. 1990. Ecological and agronomic characteristics of
innovative cropping systems. pp. 123-140 in CA. Edwards, R. Lal, P. Madden, R.H.
Miller, and G. House. Sustainable agricultural systems. Soil and Water Conservation
Soc., Ankeny, IA

Sposito, G. 1989. Soil organic matter. pp. 42-66 In The chemistry of soils. Oxford
University Press, New York, NY.

Sylvia, D.M. and SE. Williams. 1992. Vesicular-arbuscular mycorrhiza and
environmental stress. pp. 101-124 In G]. Bethlenfalvay and R.G. Linderman. (eds.)
Mycorrhizae in sustainable agriculture, American Society of Agronomy, Inc. Madison,
WI

Tester, M., Smith, SE, and Smith, FA. 1987. The phenomenon of "nonmycorrhizal"
plants. Can. J. Bot. 65:419-431

Wintersteen, W.K. and LG. Higley. 1993. Advancing IPM systems in corn and
soybeans. pp. 9-32 In A.R. Leslie and G.W. Cuperus. Successful implementation of
integrated pest management for agricultural crops.

Chapter 1. Arbuscular mycorrhizal fungus spore populations and colonization of

potato: crop rotation and input level as major factors

Abstract

Arbuscular mycorrhizal (AM) fungal colonization of a crop can be inﬂuenced by
crop rotation sequence and by the overall management regimen. This study was
conducted to examine potato yield, colonization of potato (AM) fungi, and AM spore
populations under different rotations within different management regimens. Two types
of management schemes were used: a high-input (high fertility and irrigation) potato-
alfalfa rotation in which potato crops were interrupted by varying years of alfalfa, and
a low-input (low fertility and zero irrigation) system that employed several types of
intervening crops in rotations with varying yield potential (Low, Medium and High).
Field soil AM spore populations, AM root colonization at harvest, and total yield (by
size class) were measured. The reproductive capacity of AM spore populations was
assessed with sorghum trap cultures. For the high-input rotation system, potato
following potato (PP) was clearly inferior to potato following one (AP) or two (AAP)
years of alfalfa, in terms of total yield, size distribution and AM colonization. There
were no signiﬁcant total yield or proportional differences between AP and AAP, although
AM colonization tended to be higher in AP. For the low-input system, there were no
signiﬁcant differences in total yield and AM colonization between rotations. During the
growing season, differences in AM spore populations between rotations were more
pronounced in the high-input system, although overall spore numbers tended to be higher

in ﬁeld soil from the low-input system. Trap culturing and identiﬁcation of AM spores

12

13

showed that Glomus was the predominant genus in soil samples from both systems. In
the high-input system, total spore numbers followed the pattern APP < AAP < PAP;
proportional distributions within and between genera showed little difference between
rotations. The AM population in the low-input system showed greater differences
between rotations in spore numbers and in the proportional distributions within and
between genera. These results suggest that lower input management systems result in
more diverse mycorrhizal communities that are better able to adapt to perturbations

caused by changes in the rotation.

Introduction

Arbuscular mycorrhizae (AM) are ubiquitous soil fungi that form symbiotic
associations with the root systems of many crop plants (Bagyaraj, 1984). These
associations can increase growth and crop yield of many plants, in many cases by
enhancing nutrient uptake (particularly phosphorus), and improving tolerance to disease,
pathogens and water stress (Nelsen and Saﬁr, 1982; Powell, 1984; Linderman, 1992).
High-input management techniques, i.e. incorporate extensive use of chemical inputs,
irrigation and extensive tillage, have been shown to reduce AM spore populations and
colonization of crop plants, while low-input management regimens, i.e. reduced chemical
inputs and organic cultivation, increased the abundance of AM fungi (Douds et a1. , 1993;
McGonigle and Miller, 1993; Kurle and Pﬂeger, 1994).

Crop rotations have long been known to inﬂuence crop yield, water availability
and disease progression (Francis and Clegg, 1990; Luna and House, 1990). Crop

rotations, usually incorporating at least one legume component, are especially important

14

in potato production, and are standard practice in most potato-growing regions of the
US. (Rowe, 1983). The legume crop ﬁxes atmospheric nitrogen into organic matter.
This is incorporated into the soil: shoot and root material in the case of green manure or
mop crops seeded post-harvest, or root material alone in the case of hayﬁeld or pasture
production. The increased soil organic matter provides numerous well understood
beneﬁts, including increased water retention, improved soil aggregation and soil
structure, resistance to erosion, season-speciﬁc increase in available nitrogen and
phosphorus (due to increased microbial degradation and nutrient release with rising soil
temperatures in spring), increased populations of soil macrofauna (e. g. earthworms,
micro- and macroarthropods) (Francis and Clegg, 1990; Luna and House, 1990).
Different rotation schemes can have distinctly different effects on soil quality and
soil microbiota, including AM fungi (Francis and Clegg, 1990; Johnson et al., 1991).
Different kinds of rotations, as a component of the larger management philosophy, can
inﬂuence AM spore populations, and mycorrhizal colonization of the crop plants
(Johnson et a1. , 1992; Kurle and Pﬂeger, 1996). AM populations in the soil can be
manipulated directly by inoculation with speciﬁc AM cultures, or indirectly by altering
the rotation design and structure. AM cannot yet be cultured axenically, which suggests
that the increased input costs associated with AM inoculum would limit it’s applicability
to relatively high-value cr0ps (Niemira et a1. 1995). Indirect manipulation via cropping
or management practices may therefore be the most practical approach to manipulation
of AM populations in the ﬁeld (Hendrix et al. , 1990; Thompson, 1994; Kurle and

Pﬂeger, 1996).

15

This study was conducted in collaboration with Mike Birney (Dept. of
Entomology, Michigan State University) to determine the impact of different rotations
(simple and complex) within different management schemes (high-input, low-input) on
potato yield, mycorrhizal colonization of potato roots, AM spore populations in ﬁeld

soils, and the relative reproductive capacities of the AM fungal populations.

Materials and Methods
High-input system

Kitchen Potato Farm, Alba, MI, is a high-input center pivot irrigated farm on
sandy loam soil. This farm uses a conventional management philosophy, including high
levels of irrigation and fertility, with fertilizer applications to the ﬁeld at planting and at
3 weeks post-planting. Potatoes were grown in simple multi-year rotations, with
intervening alfalfa used as green manure. Pesticides, including the biocide Vapam
(metham sodium), were used as needed during the course of the season. At the Kitchen
Potato Farm, three rotation types were used: alfalfa-alfalfa—potato (AAP, two year gap),
potato-alfalfa-potato (PAP, one year gap), and alfalfa-potato-potato (APP, zero year gap).
Plots consisted of multiple rows 6.2m (20’) long. Potato root samples used for AM
colonization were collected 7-10 days prior to harvest. Soil samples were collected for
AM spore analysis during the course of the season. Harvest of the experimental plots
was done by hand. Potatoes were sorted by size and weighed. This study was repeated

in an additional year.

16

Low-input system

Butler Potato Farm, Crystal Falls, MI, is a low-input farm using a dry land potato
production system on sandy loam soil. The farm uses organic procedures and extensive
IPM monitoring; pesticides and fungicides are used sparingly. Potato production on this
farm involved a variety of complex multi-year rotation types, based on a range of yield
expectancies: red clover-annual rye-red clover-annual rye-potato rotation (a four year
gap, the highest yield expectation), red clover-red clover/winter rye-potato rotation (a
medium yield expectation), red clover-red clover-potato rotation (the least complex, with
the lowest yield expectation). Plots consisted of multiple rows 6.2m (20’) long. Potato
root samples used for AM colonization were collected 7-10 days prior to harvest. Soil
samples were collected for AM spore analysis during the course of the season. Harvest
of the experimental plots was done by hand. Potatoes were sorted by size and weighed.
This study was repeated in an additional year.
AM root colonization assay

The collected roots were cleared and stained with Trypan blue according to
Phillips and Hayman (1970). Root colonization by AM fungi was determined by the
line-intersect method (Kormanik and McGraw, 1982).
AM spore isolation and census

Soil samples were collected to survey the population of AM spores. Soil samples
at one week, four weeks and 16 weeks post planting during the 1992 growing season
were assessed directly for number of AM spores per gram using sucrose centrifugation
(Neslen and Saﬁr, 1982; Daniels and Skipper, 1982). The early season samples can

yield information about the inoculum potential of the soil primarily due to the previous

l7

season’s AM spores, while the later season samples yield information primarily about the
AM spores produced during the season.

Soil samples from the 1993 season were collected at harvest. The structure and
reproductive capacity of the AM spore populations was determined according to Morton
et al. (1993), with the following modiﬁcations. Soil samples were stored at 4°C for
approx. 8 months, then used to inoculate sorghum trap cultures. These trap cultures
were grown for 24 weeks, and allowed to rest for approx. 12 months. AM spores were
isolated from the soil/ sand matrix (Daniels and Skipper, 1982), and sorted by genus using
phenotypic characters (Morton, 1988). Trap culturing provides information about the
relative reproductive capacity of the AM spore populations that cannot be obtained from
isolation from ﬁeld soils directly. Trap cultures also yield AM spores that can be more
easily separated taxonomically.

Statistical analysis
Data were analyzed with multivariate one-way ANOVA (analysis of variance)

using the statistical package Minitab.

Results
High-input system

The zero year rotation clearly resulted in the poorest yields and lowest AM
colonization (Table 1.1). In general, 1993 was a better production year than was 1992.
In 1992 and 1993, the APP rotation resulted in a consistently higher percentage of overall
crop in the form of small tubers. The AAP rotation tended to have a slightly higher

overall yield than the PAP rotation, but this was not a statistically signiﬁcant difference.

18

Table 1.1. High-input system ﬁeld results, 1992-93

a Data from 1992 are mean of four samples, data from 1993 are mean of
nine samples. Within years, data followed by different letters are
signiﬁcantly different (P s 0.05).

b "Year Diff." indicates difference between growing seasons. Treatments
marked with a "*" are signiﬁcantly different (P5005).

c APP = alfalfa-potato-potato; PAP = potato-alfalfa-potato; AAP =
alfalfa-alfalfa-potato

 

 

 

 

 

Factor Rotation 1992a 19933 Pb
Small (Kg/row) APPc 3.3a 2.0a *
PAP 2.0b 3.5b *
AAP 1.2b 4.0b *
Large (Kg/row) APP 6.5a 2.0a nd
PAP 22.7b 26.0b nd
AAP 24.1b 28.5b nd
Total (Kg/row) APP 9.8a 4.0a *
PAP 24.8b 29.5b nd
AAP 25.4b 32.5b *
% Small APP 34.1a 57.0a *
PAP 8.3b 12.2b nd
AAP 4.9b 12.4b nd
AM colonization % APP 3.2a 5.9a nd
PAP 11.0b 17.3b *
AAP 4.8b 11.2c *

 

20

In the 1993 season, the PAP rotation had signiﬁcantly higher AM colonization than the
AAP rotation; in 1992, this difference was not statistically signiﬁcant at P 50.05.

AM spore populations in soil samples taken during the growing season from the
high-input farm showed signiﬁcant (P S 0.05) differences between rotations early in the
season (Figure 1.1B), with APP having more spores per gram of soil than either PAP
or AAP, which were not different from each other. There were no signiﬁcant
differences later in the season.

Spore population distributions after trap culturing soil samples from the high-input
system showed signiﬁcant (P $0.05) differences in total spore population between
rotations (Table 1.2). These differences were due to signiﬁcant differences in the
population of spores from the predominant genus, Glomus, and were speciﬁcally due to
differences in the populations of a single one of the three phenotypic sub-groups within
Glomus that were present in the trap cultures. There were no signiﬁcant differences
between rotations in the number of spores from the other AM genera in the population,
Gigaspora and Acaulospora, each of which were represented by two phenotypic sub-
groups. The proportional representations of the three genera were not different between
rotations. The predominant phenotypic sub-group within Glomus had a lower
proportional representation in the APP rotation than in either PAP or AAP.
Low-input system

Changes in the rotations had little signiﬁcant effect on absolute yield, proportional
yield or AM colonization (Table 1.3). For these plots, 1992 was a better production year
than 1993. In 1992, the rotation with the highest yield expectation tended to have higher

production than other rotations, but these differences were not statistically signiﬁcant.

21

Figure 1.1. Population of arbuscular mycorrhizal (AM) fungal spores in
ﬁeld soil samples taken from three different rotations within two input
systems. Individual points are the mean of four samples, bars indicate
standard error.

AM spores/g soil

AM spores/g soil

22

 

 
 
 
 

 

Butler (Low-input), 1992

0 Low

El Medium
A ngh

 

 

 

Kitchen (High-input), 1992

O APP
[I] PAP
A AAP

 

 

 

 

“’1

I I I I f I
6 8 1O 12 14 16

Weeks post planting

18

23

Table 1.2. AM spore population distribution in trap cultures inoculated with
soil from the high-input system.

a APP = alfalfa-potato-potato; PAP = potato-alfalfa—potato; AAP =
alfalfa-alfalfa-potato

b number of spores per 20 g sample, mean of four samples

c numbers in a given row with different letters are signiﬁcantly different
(P s 0.05)

(1 number of spores per 20 g sample as % of total spores, mean of four
samples

24

 

 

APP“ PAP AAP
All generab 118.5ac 269.3b 203.8c
Total Glomus 104.3a 247.5b 170.3c
brown, clear 84.5a 222.0b 170.3c
white, opaque 12.5a 13.0a 11.5a
white, clear 7.3a 12.5a 5.8a
Total Gigaspora 8.5a 7.5a 7.5a
white, opaque 2.0a 2.0a 4.0a
brown, clear 6.5a 5.5a 3.5a
Total Acaulospora 5.8a 14.3a 8.8a
brown, clear 2.5a 8.0a 5.8a
white, clear 3.3a 6.3a 3.0a
All generad 100.0a 100.0a 100.0a
Total Glomus 87.4a 91.3a 91.3a
brown, clear 70.1a 81.4b 82.3b
white, opaque 11.0a 5.1a 6.0a
white, clear 6.3a 4.8a 3.0a
Total Gigaspora 7.5a 3.0a 4.0a
white, opaque 1.8a 0.8a 2.2a
brown, clear 5.7a 2.2a 1.8a
Total Acaulospora 5.0a 5.7a 4.7a
brown, clear 2.2a 3.1a 3.1a
white, clear 2.8a 2.6a 1.6a

 

25

Table 1.3. Low-input system ﬁeld results, 1992-93

a Data from 1992 are mean of 4 samples, data from 1993 are mean of 12
samples. Within years, data followed by different letters are signiﬁcantly
different (P5005).

b " Year Diff." indicates difference between growing seasons. Treatments
marked with a "*" are signiﬁcantly different (P3005).

26

 

 

 

 

 

Factor Yield 1992a 19938 Pb
potential

Small (Kg/row) Low 2.7a 2.9a ND
Medium 3.5a 3.2a ND
High 2.8a 3.7a ND

Large (Kg/row) Low 21.8a 16.5a *
Medium 23.0a 16.7a *
High 26.0a 13.8b *

Total (Kg/row) Low 24.5a 19.5a *
Medium 26.4a 19.9a *
High 28.9a 17.5a *

% Small Low 11.1a 15.6a ND
Medium 13.1a 16.3a ND
High 9.5a 21.0b *

AM colonization % Low 12.0a 4.4a *
Medium 13.2a 4.5a *
High 12.7a 5.2a *

 

27

In 1993, the rotation with the highest expectation yielded fewer large tubers, and a higher
percentage of small tubers. There were no differences between rotations in overall yield
in either year. Mycorrhizal colonization for each year was not statistically different
between rotations, but AM colonization was clearly higher for all rotations in 1992 than
in 1993.

There were no signiﬁcant differences in spore number in ﬁeld soil samples taken
during the growing season from the different rotations used at the low-input farm at any
of the sampling dates (Figure 1.1A).

Spore population distributions after trap culturing soil samples from the low-input
system showed signiﬁcant (P 50.05) differences in total spore population between
rotations (Table 1.4). These differences were due to signiﬁcant differences in the
population of spores from the predominant genus, Glomus. Unlike the trap cultures of
the soils from the high-input system, there was no single dominant sub-group. The
differences were due to differences in the populations of three of the four phenotypic sub-
groups within Glomus that were present in the trap cultures, including one sub-group
which was not present in trap cultures of soils from the high-input system. The genera
Gigaspora and Acaulospora were each represented by two phenotypic sub-groups.
Gigaspora was not represented at all in trap cultures of soils from the high expectation
rotation, while Acaulospora was not represented at all in trap cultures of soils from the
medium expectation rotation. The proportional representations of Glomus and Gigaspora
were signiﬁcantly (P s 0.05) different between rotations. The proportional
representations of three of the four phenotypic sub-groups within Glomus were different

between rotations.

28

Table 1.4. AM spore population distribution in trap cultures inoculated with
soil from the low-input system.

a APP = alfalfa-potato-potato; PAP = potato-alfalfa-potato; AAP =
alfalfa-alfalfa-potato

b number of spores per 20 g sample, mean of four samples

c numbers in a given row with different letters are signiﬁcantly different
(P s 0.05)

d number of spores per 20 g sample as % of total spores, mean of four
samples

29

 

 

Lowa Medium High
All generab 468.0a° 114.3b 194%
Total Glomus 465.8a 107.0b 193.8c
brown, clear 125.8ab 70.3a 188.8b
white, opaque 52.5a 10.3b 1.5b
white, clear 286.3a 21.5b 1.8c
red, opaque 1.3a 5.0a 1.8a
Total Gigaspora 0.8a 7.3a 0.0a
white, opaque 0.0a 4.5a 0.0a
brown, clear 0.8a 2.8a 0.0a
Total Acaulospora 1.5a 0.0a 0.3a
brown, clear 0.8a 0.0a 0.3a
white, clear 0.8a 0.0a 0.0a
All genera‘l 100.0a 100.0a 100.0a
Total Glomus 99.5ab 94.1a 99.9b
brown, clear 28.2a 62.2b 95.8c
white, opaque 11.4a 9.6a 1.3b
white, clear 59.7a 18.1b 1.4c
red, opaque 0.3a 4.2a 1.4a
Total Gigaspora 0.2a 5.9b 0.0a
white, opaque 0.0a 3.7a 0.0a
brown, clear 0.2a 2.1a 0.0a
Total Acaulospora 0.3a 0.0a 0. la
brown, clear 013 0.0a 0.1a
white, clear 013 0.0a 0.0a

 

30
Discussion

Our study suggests that the extent to which speciﬁc rotations can inﬂuence potato
yield, AM spore density, AM spore population reproductive capacity and AM
colonization of potato is determined by the overall management scheme. Across
rotations, degree of AM colonization is associated with yield in both the high-input and
the low-input systems. Signiﬁcant differences in spore reproductive capacity in trap
culture from the high-input system are associated with differences in AM colonization
in the ﬁeld, whereas the even more dramatic differences in spore reproductive capacity
and diversity from the low-input system did not signiﬁcantly alter the AM colonization
of potatoes in the ﬁeld. This suggests that the more diverse AM fungal populations from
the low—input system are more responsive to changes in rotation than that from the high-
input system.

On the high-input farm, the APP rotation is inferior to either the PAP or AAP
rotation, with regard to AM colonization and yield. The pattern of AM colonization
follows the pattern of spore reproductive capacity shown in trap culture, APP < AAP
< PAP. Potato yields differed from this pattern in that yields were not signiﬁcantly
different between PAP and AAP rotations, both of which were higher than the APP
rotation. Spore numbers in the ﬁeld in the early part of the season were higher in the
APP ﬁeld than in the PAP or AAP ﬁeld. This does not correlate with the levels of AM
colonization in potato roots taken at the end of the season, with colonization in the APP
ﬁeld lower than in the PAP or AAP ﬁelds.

On the low-input farm, there were no differences in AM colonization or yield

between the rotations. There were no signiﬁcant differences in total yields for 1992 or

31

1993, although in 1993, the rotation with the highest yield expectation had signiﬁcantly
fewer large tubers, and a higher proportion of small tubers than other rotations. The
marked difference in tendencies between the years suggests that, for the low-input
system, the differences in yield caused by the different rotations are less critical than
climatological factors.

The different rotations on the low-input farm show no differences in AM spore
densities in ﬁeld soils at any point during the growing season. Trap culturing of these
soils shows greater variation in the reproductive capacity of the AM spore populations
from the low-input system vs. the high-input system. This suggests a legacy "bank" of
AM spores in the low-input system that is more extensive and more responsive than that
of the high-input system. The complexity of the low-input farm’s agroecology may
provide a greater ﬂexibility in response to changes in rotation (Stinner and Blair, 1990),
although the relationship between a system’s complexity and its stability is not well
deﬁned (Crawley, 1986). The AM spore population distribution in the trap cultures of
the soils from the high-input system showed little signiﬁcant difference in AM population
structure between rotations, with a single phenotypic sub-group of Glomus dominating
in all rotation types. There were signiﬁcant differences in population density, with the
shorter rotation (PAP) having the highest spore density in trap culture. The AM spore
population distribution in trap cultures of the soils from the low-input system showed
marked differences in AM population structure. Although Glomus was the predominant
genus, the representation of the phenotypic sub-groups, including a sub-group not found
in trap cultures of the high-input system soils, was variable between rotations, with no

single dominant phenotypic sub-group.

32

Notable previous studies of the effect of rotation and management level on AM
population and colonization of crop plants have been conducted with studies of
continuous monocropping of corn (Zea mays) and soybean (Glycine max) (Johnson et al. ,
1991; McGonigle and Miller, 1993), different designs of corn - soybean rotations
(Johnson et al., 1992; Douds et al., 1993; Kurle and Pﬂeger, 1994; Kurle and Pﬂeger,
1996), and long term asparagus (Asparagus oﬁicinalis L.) plantings (W acker et al. ,
1989). These studies show that AM fungi in the soil respond to the biotic and abiotic
changes that occur after extensive monocropping, and during a multi-year corn - soybean
rotation. Johnson et a1. (1992) hypothesized that continuous monoculture caused shifts
in the AM population resulted in the predominance of AM species that are less beneﬁcial
(or detrimental) to the crop plant. This hypothetical shift may thus contribute to the
observed yield decline in a monoculture. The AM spore population data from trap
cultures of the high-input system in this study show a slight, though statistically
signiﬁcant, shift in spore distribution within Glomus. While recognizing the agreement
in results, comparisons between the results of this part of this study, with extremes of
two years of continuous potato or potato following two years of alfalfa, with the work
of Johnson et al. (1992), which used corn or soybeans following ﬁve years of continuous
corn or soybeans, should be made with caution. However, the logical conclusion from
that previous work, that an interrupted polyculture would prevent the buildup of AM
species less beneﬁcial (or detrimental) to the crop, is also supported by the AM spore
population data presented herein from trap cultures of the low-input system.

These studies of the effect of monocropping/ rotations on AM fungi do not fully

address the nature of the mechanisms underlying the effects observed (Johnson et al. ,

33

1991; Johnson et al. 1992). Crop residues, particularly leguminous residues, are known
to be rich in a complex mixture of compounds, many classes and examples of which are
known to be biologically active (Siqueira et al. , 1991a). Of the compounds derived from
leguminous organic material, phenolic compounds such as ﬂavonoids and isoﬂavonoids
have been shown to be particularly active in inﬂuencing the behavior of soil fungi (Nair
et al., 1991; Siqueira et al., 1991a; Siqueira et al., 1991b). Phenolic compounds, such
as p-coumeric acid, p-hydroxybenzoic acid, as well as the isoﬂavones formononetin,
biochanin-A and quercitin, have been shown to inﬂuence AM colonization in clover
(Siqueira et al., 1991b; Fries, 1995) and sorghum (Fries, 1995). Pederson et al. (1991)
showed inhibition of AM colonization of asparagus plants with increasing concentrations
of the phenolic compounds caffeic acid and ferulic acid. Isoﬂavones are known to
stimulate rapid encystment in parasitic Oomycetes (Carlile, 1983; Morris and Ward,
1992; Deacon and Donaldson, 1993). Vedenyapina et al. (1996) showed that micromolar
concentrations of the isoﬂavone genistein can alter the in vitro growth and reproductive
pattern of Phytophthora sojae. The extent to which one crop in a rotation could
chemically inﬂuence fungal behavior, and particularly AM colonization, of the
succeeding crop through phenolic—rich detritus would necessarily be dependent on the
extent to which bioactive compounds remain resident in the soil from season to season.
Soil organic matter that is carried over winter from one growing season to the next is not
degraded until soil temperatures rise in the spring (Francis and Clegg, 1990). Associated
nutrients and bioactive compounds are therefore released into the soil environment as
temperatures rise and days lengthen; this is the period of most rapid growth of root

tissue, and the period of most extensive colonization by AM fungi (Spanu and Bonfante-

34

Fasolo, 1988; Fries et al. , 1996). In consideration of the multi-year persistence of
organic detritus, and the timing of the release of the potentially bioactive compounds
contained therein, it has been suggested that organic residues from leguminous
components of crop rotations constitute an important chemical, as well as physical,
contribution (Siqueira et al. , 1991a). Although not well developed, techniques for the
chemical management of soil microbiota, including AM fungi, through exogenous
applications and selective rotation with crops rich in bioactive phenolics, is an area with
important economic possibilities (Siqueira et al. , 1991a).

Early season AM spores, produced during the previous crop, can potentially show
the types of population effects suggested to occur during a monoculture (Johnson et al. ,
1991; Johnson et al. , 1992). In the high-input system, the APP rotation had signiﬁcantly
higher spore numbers in the ﬁeld than did PAP or AAP, but resulted in lower AM
colonization at the end of the season. Despite higher numbers in the early season, the
AM population in the APP ﬁeld was, overall, less effective at colonizing potato than the
AM population in either the PAP or AAP ﬁeld. In the low-input system, where a potato
following potato treatment was not used, there were no differences in early season spore
numbers, and there were no differences in AM colonization at the end of the season.
This suggests that a single season of potato is sufﬁcient to produce the types of changes
to the AM populations hypothesized by Johnson et al. (1992). The results presented
herein suggest that AM fungi in soils long devoted to potato production respond to crop
rotations in a similar fashion to AM fungi in soils long devoted to corn and soybean
production, despite the differences inherent in potato production vs. corn/ soybean

production.

35

Abbot and Gazey (1994) hypothesized that a diverse AM fungal population may
be more beneﬁcial to a crop than a single predominant AM species, since a diverse
population may allow greater adaptability to changing cultural and environmental factors.
Low-input management has been shown to promote greater AM spore populations than
high-input management (Douds et al. , 1993). The low-input nature of the management
used on the Butler farm may have allowed the AM population to attain a diversity and
concomitant ﬂexibility that was lacking in the high-input farm. Therefore, the speciﬁc
inﬂuences of the different rotations may have been somewhat buffered by the legacy of
AM population structure. By extension, a high-input system has a reduced inherent
ﬂexibility, and may be therefore more susceptible to the perturbations caused by the
different rotations. It should be noted, however, that greenhouse studies have indicated
that AM spore production is decreased under conditions of drought stress (Nelsen and
Saﬁr, 1982). It may be expected that drought stress may induce a similar reduction in
AM spore populations under ﬁeld conditions. Thus, a non-irrigated system would be
more susceptible to diminution in spore number because of drought stress than an
irrigated system; the dominance of the effect of water availability on AM spore
production may serve to balance the impact of the other inputs of the system.

The complex interactions of host plants and AM fungi in potato rotation systems
inﬂuence crop yield, the AM spore populations in the ﬁeld, the AM spore reproductive
capacity and the resulting colonization of the potato roots. These interactions, and their
control mechanisms are not well understood. Careful design of the rotation system as

a component of the entire production system (fertility, chemical and water inputs) is

36

therefore a precursor to management of the AM fungal population in agricultural soils

devoted to potato production.

37

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mycorrhizas. Plant Soil 159269-78

Carlile, M.J. 1983. Motility, taxis and tropism in Phytophthora. pp. 95-107 in Erwin,
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ecology and pathology. American Phytopathological Society, St. Paul, MN

Crawley, M.J. 1986. Structure of plant communities. pp.l-50 in M.J. Crawley. Plant
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Daniels, B.A. and H.D. Skipper. 1982. pp. 29-35 in: Schenck, N .C. (ed) Methods and
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Deacon, J .W. and Donaldson, SP. 1993. Molecular recognition in the homing responses
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Douds, D.D., Jr., R.R. Janke, and SE. Peters. 1993. VAM fungus spore populations
and colonization of roots of maize and soybean under conventional and low-input
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Francis, CA. and Clegg, MD. 1990. Crop rotations in sustainable agriculture. pp.107-
122 in CA. Edwards, R. Lal, P. Madden, R.H. Miller, and G. House. Sustainable
agricultural systems. Soil and Water Conservation Soc. , Ankeny, IA

Fries, L.L.M., R.S. Pacovsky, and GR. Saﬁr. 1993. Expression of isozymes altered
by Glomus intraradix or formononetin application in corn roots. p. 130 In Peterson,
L. and M. Schelkle Abstracts of the 9th North American Conference on
Mycorrhizae, Guelph, Ontario, Canada August 8-12, 1993 University of Guelph

Fries, L.L.M. 1995. Plant growth and arbuscular mycorrhizal (Glomus intraradicies)
colonization affected by exogenously applied phenolic compounds. pp. 94-130 in
Fries, L.L.M. Physiological alterations in mycorrhizal plants as affected by phenolic
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Hendrix, P.F., D.A. Crossley, J.M. Blair, and DC. Coleman. 1990. Soil biota as
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P. Madden, R.H. Miller, and G. House. Sustainable agricultural systems. Soil and
Water Conservation Soc. , Ankeny, IA

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Johnson, N.C., F.L. Pﬂeger, R.K. Crookston, S.R. Simmons and P.J. Copeland. 1991.

Vesicular-arbuscular mycorrhizas respond to corn and soybean cropping history. New
Phytol. 117:657-663

Johnson, N.C., P.J. Copeland, R.K. Crookston, and FL. Pﬂeger. 1992. Mycorrhizae:

possible explanation for yield decline with continuous corn and soybean. Agron. J.
84:387-390

Korrnanik, PP. and AC. McGraw. 1982. Quantiﬁcation of vesicular-arbuscular
mycorrhizae in plant roots, p. 37-45. in: N .C. Schenck (ed.) Methods and
principles of mycorrhizal research. APS Press, St. Paul, MN.

Kurle, J .E. and FL. Pﬂeger. 1994 Arbuscular mycorrhizal fungus spore populations
respond to conversions between low-input and conventional management practices in
a com-soybean rotation. Agron. J. 86:467-475

Kurle, J .E. and FL. Pﬂeger. 1996. Management inﬂuences on arbuscular mycorrhizal
fungal species composition in a corn-soybean rotation. Agron. J. 88:155-161

Linderman, R.G. 1992. Vesicular-arbuscular mycorrhizae and soil microbial interactions,
pp. 45-70 in: GI. Bethlenfalvay and R.G. Linderman (eds.). Mycorrhizae in
sustainable agriculture. Amer. Soc. Agron., Madison, WI.

Luna, J .M. , and GI. House. 1990. Pest management in sustainable agricultural systems.
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Sustainable agricultural systems. Soil and Water Conservation Soc. , Ankeny, IA

McGonigle, T.P. and M.H. Miller. 1993. Mycorrhizal development and phosphorus
absorption in maize under conventional and reduced tillage. Soil Sci. Soc. Am. J.
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Morris, PF. and Ward, E.W.B. 1992. Chemoattraction of zoospores of the soybean
pathogen, Phytophthora sojae, by isoﬂavones. Physiol. Mol. Plant Pathol. 40: 17-22

Morton, LB. 1988. Taxonomy of VA mycorrhizal fungi: classiﬁcation, nomenclature,
and identiﬁcation. Mycotaxon 32:267-324

Morton, J .B., S.P. Bentivenga and W.W. Wheeler. 1993. Germplasm in the
international collection of arbuscular and vesicular-arbuscular mycorrhizal fungi

(INVAM) and procedures for culture development, documentation and storage.
Mycotaxon 48:491-528

Nair, M.G., Saﬁr, GR. and Siqueira, J .O. 1991. Isolation and identiﬁcation of
vesicular-arbuscular mycorrhiza-stimulatory compounds from clover (T rifolium repens)
roots. Appl. and Envir. Microbiology 57:434-439

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Nelsen, CE. and GR. Saﬁr. 1982. Increased drought tolerance of mycorrhizal onion
plants caused by improved phosphorus nutrition. Planta 154:407-413.

Newsham, K.K. , Fitter, AH. and Watkinson, AR. 1993 Arbuscular mycorrhiza protect
an annual grass from root pathogenic fungi in the ﬁeld. J Ecology 83:991-1000.

Niemira, B.A., Saﬁr, G.R., Hammerschmidt, R. and Bird, G.W. 1995 Production of
prenuclear minitubers of potato with peat-based arbuscular mycorrhizal fungal
inoculum. Agron. J. 87:942-946

Pederson, C.T., Saﬁr, G.R., Siqueira, JD. and Parent, S. 1991. Effect of phenolic
compounds on asparagus mycorrhiza. Soil Biology and Biochemistry 5:491-494

Phillips, J .M. and D.S. Hayman. 1970. Improved procedures for clearing roots and
staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment
of infection. Trans. Brit. Mycol. Soc. 55:158-161.

Ponton, F., Y. Piché, S. Parent and M. Caron. 1990. Use of vesicular-arbuscular
mycorrhizae in Boston fern production: 11. Evaluation of four inocula. HortScience
25:416-419.

Powell, CL. 1984. Field inoculation with VA mycorrhizal fungi, p. 205-222. in:
C.L. Powell and DJ. Bagyaraj (eds.). VA mycorrhiza. CRC Press, Boca Raton.

Rowe, RC. 1993. Ch.2., pp.ll—18, Potato health management. APS Press, St. Paul,
Minn.

Siqueira, J .O, Nair, M.G., Hammerschmidt, R., and Saﬁr, G.R. 1991a. Signiﬁcance
of phenolic compounds in plant-soil-microbial systems. Crit. Rev. in Plant Sci. 10
(1):63-121

Siqueira, J .O, Saﬁr, GR. and Nair, M.G. 1991b. Stimulation of vesicular-arbuscular
mycorrhizae formation by ﬂavonoid compounds. New Phytol. 118: 87-93

Spanu, P. and P. Bonfante-Fasolo. 1988. Cell-wall-bound peroxidase activity in roots of
mycorrhizal Allium porrum. New Phytologist 92:75-87

Stinner, BR. and J .M. Blair. 1990. Ecological and agronomic characteristics of
innovative cropping systems. pp. 123-140 in C.A. Edwards, R. Lal, P. Madden, R.H.
Miller, and G. House. Sustainable agricultural systems. Soil and Water Conservation
Soc., Ankeny, IA

Thompson, J .P. 1994. what is the potential for management of mycorrhizas in
agriculture? pp. 191-200 in AD. Robson et al. (ed.) Management of mycorrhizas in
agriculture , horticulture, and forestry. Kluwer Acad. Publ. , Dordrecht, Netherlands.

40

Vedenyapina, E.G., Saﬁr, G.R., Niemira, B.A., and Chase, T.E. 1996. Low
concentrations of the isoﬂavone genistein inﬂuence in vitro asexual reproduction of
Phytophthora sojae. Phytopathology 86: 144-148

Wacker, T.L., Saﬁr, GR. and Stephenson, SN. 1989. Evidence for succession of
mycorrhizal fungi in Michigan asparagus ﬁelds. Acta Hort. 271:273-278

Chapter 2. Production of prenuclear minitubers of potato with peat-based arbuscular

mycorrhizal fungal inoculuml

Abstract

Prenuclear minitubers of potato (Solanum tuberosum L.) are the source material
used to produce ﬁeld-grown seed potatoes. Seed potatoes are in turn planted by
commercial growers to produce potatoes for fresh packing and processing. Arbuscular
mycorrhizae (AM) have been demonstrated to increase yield in low-input systems. This
study was conducted to determine whether and how a commercial AM inoculum
inﬂuences prenuclear minituber production under high-input commercial conditions. A
peat-based medium containing the mycorrhizal fungus Glomus intraradix was tested in
a commercial minituber greenhouse production facility. This medium increased yields
of the most valuable sizes of prenuclear minitubers by 84% , and increased total
prenuclear minituber yield by 49% when compared with conventional peat-vermiculite
media under commercial growing conditions. Potato plants grown in this mycorrhizal
medium had more uniform stolon development as well as stolons 39% longer than plants
grown in the conventional medium. These yield increases and morphological changes
occur in the presence of very low levels of mycorrhizal colonization, and there was no
evidence of enhanced plant P nutrition generally associated with mycorrhizal symbiosis.

These effects may indicate the presence of a hormonally mediated plant response to the

 

1 This chapter appeared in slightly modiﬁed form as Niemira, B.A., Saﬁr, G.R.,
Hammerschmidt, R. and Bird, G.W. Production of prenuclear minitubers of potato with
peat—based arbuscular mycorrhizal fungal inoculum. Agron. J. 87:942-946 (1995)

41

42

presence of the mycorrhizal fungi that results in more uniform stolon growth and an

increase in initiation of minitubers.

Introduction

Arbuscular mycorrhizae are ubiquitous soil fungi that form symbiotic associations
with many crop plants (Bagyaraj, 1984). The AM associations can increase plant
growth; in many cases by enhancing P uptake from soils with low to moderate P
availability (Powell, 1984). Attempts to increase yields under low-P growing conditions
through inoculation with fungal propagules have improved yields and nutrition of many
ﬁeld and horticultural crops (Nelsen and Saﬁr, 1982; Johnson & Pﬂeger, 1992),
including potato (Black and Tinker, 1977). Previous studies involving mist-culture
inoculation of potato showed a positive effect on vine growth and tuber production
(Graham et al. , 1976). The effects of peat-based AM inoculum have been investigated
in non-soil substrates commonly used in the production of many horticultural crops
(Ponton et al., 1990; Pedersen, 1991). Mycori-Mix, (Premier Peat Moss, Ltd., Riviére-
du Loup, Québec, Canada) a commercial peat-based AM inoculum, has been tested on
asparagus (Asparagus oﬁ‘icinalis L.) (Pedersen et al. , 1991) and leek (Allium porrum;
syn. A. Ampeloprasum L. Porrum group) (Caron and Parent, 1988), and shown to
enhance AM colonization and increase yield.

While inoculation with AM can be beneﬁcial in low-input systems, additions of
P fertilizer are known to reduce AM colonization of roots (Kiernan et al. , 1983; Abbott
and Robson, 1984). Under ample P fertilization, the additional potential P uptake

capacity provided by a mycorrhizal association is usually less advantageous (Marschner,

43

1986; Johnson and Pﬂeger, 1992). There have been no previous reports of signiﬁcant
positive impact of AM inoculum on the production of prenuclear minitubers in a high-
fertility system. The relatively high value of prenuclear seed minitubers prompted a
study was conducted to determine whether AM inoculum inﬂuences prenuclear minituber
production under high-input commercial conditions. These conditions, which include
high fertility, high water availability and extensive pesticide application, have been
previously shown to minimize or eliminate the beneﬁts of AM inoculation (Trappe, 1984;
Hayman, 1987). The amount of mycorrhizal colonization of the potato root system was
assessed as a measure of the degree of association. Potato stem and leaf P concentration
was determined for use as a possible indicator of the degree of beneﬁt derived from the
mycorrhizal association. We measured the absolute yields of different sizes of
minitubers, which have accordingly different economic values. We also calculated the
proportional yields of the most valuable tubers as a fraction of total production. In
addition, levels of stolon production and tuber initiation by inoculated and noninoculated

plants were examined.

Materials and methods

The experiments were conducted at Sklarczyk Seed Farms, Johannesburg, NII in
commercial minituber production greenhouses from July 1992 through November 1994.
The potato cultivar Atlantic was used. Plantlets were micropropagated on-site in agar
using sterile tissue culture techniques. Tuber production beds, 120 by 230 by 10 cm,
were lined with polyethylene to control drainage, and ﬁlled with the growing media

(described below). Depressions, approximately 7 cm deep, were made in the medium.

44

The plantlets were split from their micropropagation agar block and placed in the
depressions. The beds were thoroughly wetted with a 90-180-90 g L‘1 (N-P-K) nutrient
mix before placement in the greenhouse.

The greenhouse was open-framed, with a polypropylene barrier. The conditions
in the greenhouse were standardized for commercial production. Air temperature was
maintained at approximately 24°/ 10°C day/night. Mist-fans were used to maintain
relative humidity of 90%. Plants were sprayed weekly with overhead sprinklers
throughout the ﬁrst 6 wks of the growing season using a 90-180-90 g L’1 (N-P-K) nutrient
solution. The watering was discontinued at 14 wks and the plants were allowed to
senesce. Tubers were harvested at week 16.

The normal growing practices of the tuber production facility incorporate the
application of a number of different fungicides at different times in the growing period.
Timing and dosage of these applications were determined by the grower speciﬁcally to
eliminate pathogenic fungi. Fungicides were used as 600-ml stock solution diluted and
applied with a hand sprayer as described in Table 2.1.

Experiment 1: minituber production

This study was conducted from July through November 1993 with two types of
media in the experimental beds: a sterilized Sphagnum peat (SB Mix, Premier Peat Moss,
Ltd.) and a 50:50 (v/v) mixture of noninoculated and inoculated (Glomus intraradix, 1
propagule g“) Sphagnum peat (50:50 SB Mix/Mycori-Mix, Premier Peat Moss, Ltd.).
The control beds were ﬁlled with the commercial peat-vermiculite mixture (Alpar Peat

Co., Ovid, MI) regularly used in these greenhouses. Each type of medium was

45

Table 2.1. Application rate and timing of fungicides used in a greenhouse
study of potato minituber production (Exp. 1, July-Nov. 1993, at
Johannesburg, MI).

1‘ Weeks post planting.
1: An ornamental fungicide, product of WA Cleary Chemical, Somerset, NJ.

46

 

 

 

Application
Fungicide Rate Time
g a.i. rn'2 wk PPT
metalaxyl + chlorothalonil 0.29 3

[N-(2,6-dimethylphenyl)-N-(methoxyacetyl) alanine methyl ester +
tetrachloroisophthalonitrile]

carbofuran 0.32 3
[2-3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate]
methamidophos 0.58 5, 6

[O,S-dimethylphosphoramideothioate]

3336 WP: 1.09 5, 6, 10
[dimethyl 4,4’-o-phenylenebis-(3-thioallophanate)]

iprodione 0.61 7, 10, 11

[3-(3,5-dichlorophenyl)-N-(1-methylethyl)-2,4-dioxo-l-
imidazolidinecarboxamide]

maneb 1.16 8, 9, 10
[manganese ethylenebisdithiocarbamate]

metalaxyl 0.84 9
[N -(2,6-dimethylphenyl)-N-(methoxyacetyl) alanine methyl ester]

 

47

replicated three times in separate beds. The beds were arranged in a random block design.

Experiment 2: stolon development and minituber initiation

In April to July 1994, a separate study was conducted at the same tuber
production facility to evaluate the effect of AM inoculation on stolon development and
tuber initiation. Six beds were ﬁlled with the inoculated medium mix, and another six
beds ﬁlled with the conventional peat-vermiculite medium. Plants were removed from
the beds once each week during the ﬁrst 4 wk, and analyzed for length of individual
stolons, total length of stolon material per plant, number of stolons per plant, and number
of tuber initiates per stolon.

At the end of the growing periods, the minitubers were harvested and sorted by
size. Small tubers have a diameter less than 2.2 cm, medium tubers have a diameter
between 2.2 and 3.3 cm, and large tubers have a diameter greater than 3.3 cm. Small
and medium tubers are the sizes most valued for commercial use. These sizes
correspond to the relative economic value of the tubers, and results from the agricultural
machinery currently in wide use in the United States potato industry (D. Sklarczyk,
Sklarczyk Seed Farms, personal communication, 1993).

AM root colonization assay

Plant roots were cleared and stained with Trypan blue according to Phillips and
Hayman (1970). Root colonization by AM was determined by the line-intersect method
(Kormanik and McGraw, 1982). Phosphorus concentration of stem + leaf tissue was
determined using the method of Nelsen and Saﬁr, 1982.

Statistical analysis

48

Data were analyzed with multivariate one-way AN OVA (analysis of variance)

using the statistical package Minitab.

Results
Experiment 1: minituber production

Potato plants grown in inoculated peat have total minituber yields signiﬁcantly (P
s 0.05) greater than those produced from the conventional commercial medium (Table
2.2). The difference in yield represents a 49% increase in total tuber production and an
84% increase in the production of small and medium minitubers.

Potato vines grown in the inoculated peat produced a proportion of total yield
comprised of the most valuable tubers that was signiﬁcantly (P s 0.05) greater than that
produced using the conventional commercial medium (Table 2.2). The proportion of
small and medium tubers from inoculated peat was 69.0% of the total tuber mass
produced, compared with 55.5 % of the total tuber mass produced from the bed ﬁlled
with the conventional medium. Sampling of commercial beds from the same greenhouse
conﬁrmed that the yields from the control beds in our experiment were equivalent to the
normal commercial yields from the rest of the same greenhouse. The SB Mix growing
medium did not signiﬁcantly increase yields in this experiment, nor in three subsequent
experiments.

The P levels of the plants showed no signiﬁcant difference among the treatments
(Table 2.3). The AM colonization levels in subsequent samplings were uniformly low.
No colonization was found in plants from noninoculated treatments, and no colonization

was observed after eight wks post-planting. This experiment was repeated three

49

Table 2.2. Prenuclear minituber yield of potato grown in the greenhouse
with and without arbuscular mycorrhizae (Exp. 1, July-Nov. 1993,
Johannesburg, MI; mean of three samples).

1' Control: standard commercial peat-vermiculite mix. Peat: sterilized
Sphagnum peat. Peat + AM, sterilized Sphagnum peat + arbuscular
mycorrhize.

1 Within rows, means followed by the same letter are not different (P s 0.05).

50

 

 

 

Yield Growing mediumT
Control Peat Peat+A
M
Small + medium tubers, kg rn’2 1.46azl: 2.09ab 2.70b
Total yield, kg m'2 2.63a 3.20ab 3.91b

Small + medium as % of total 55.5a 65.6ab 69.9b

 

51

Table 2.3. Phosphorus concentration of stem + leaf tissue and arbuscular
mycorrhizal (AM) colonization level of potato roots grown in the
greenhouse (Exp.1, July-Nov. 1993, Johannesburg, MI; mean of three
samples).

1' Control: commercial peat-vermiculite. Peat: sterilized Sphagnum peat.
AM: arbuscular mycorrhizae.
1' ND: colonization not detected

52

 

 

 

Week post Growing media‘i'
Planting Control Peat Peat P s 0.05
+AM
P concentration, pg P mg" dry wt.
5 2.71 1.78 2.40 NS
8 8.71 6.18 6.22 NS
10 11.68 8.49 7.63 NS
Colonization %
5 0.3 0.5 1.5 NS
8 ND: ND ND -

14 (harvest) ND ND ND -

 

53

additional times at the same tuber production facility, with similar yield, P concentration

and colonization results.

Experiment 2: stolon development and minituber initiation

The study conducted from April through July 1994 demonstrated that AM
inoculation caused dramatic effects on stolon development and tuber initiation, as shown
in Table 2.4. Plants from the inoculated beds had stolons 39% longer (P S 0.05) than
those from the control beds. The total length of stolons per inoculated plant was
increased by 31% (P s 0.05). The inoculated plants’ production of longer individual
stolons did affect the number of stolons supported per plant. Inoculated plants tended
to have more tuber initiates per stolon.

The plants showed no signiﬁcant differences in mycorrhizal colonization and
tissue P levels (Table 2.5).

The data obtained from the inoculated beds showed dramatically less variability
than that from the control beds. Although the means do not differ signiﬁcantly until the
fourth week, the entire data set is of interest because the standard deviations derived
from the control data were larger than those derived from inoculation data by two-,
three- or even ﬁvefold (Table 2.4). This suggests that inoculated plants had more
uniform stolon development. This was visible as early as three wks after planting. To
better demonstrate this reduced variability, the total length of stolon material produced
per plant is shown as a histogram (Figure 2.1). At 2 wk, the inoculated and the control
plants had a similar total stolon production per plant. At 3 wk, total stolon production

by the control plants ranged widely, in contrast with that of the inoculated plants. At 4

54

Table 2.4. Stolon and tuber initiate development of potato grown in the
greenhouse with and without arbuscular mycorrhizae (Exp.2, Apr.-July
1994, Johannesburg, MI; means of six beds).

T Control: commercial peat-vermiculite. Peat sterilized Sphagnum peat. AM:
arbuscular mycorrhizae.
i No stolons were present at 1 wk post planting.

§ Within rows, means followed by the same letter are not signiﬁcantly
different (P s. 0.05).

55

 

Growing mediaT

 

 

Factor examined Commercial Peat + AM
Week 2:1: Mean SD Mean SD
avg. stolon length, cm 3.72a§ 0.79 3.63a 0.61
total stolon length, cm 5.19a 2.91 4.80a 2.66
stolons, no. plant" 1.33a 0.67 1.22a 0.58
initiates, no. stolon" 0.07a 0.16 0.03a 0.08
Week 3

avg. stolon length, cm 7.77a 1.98 8.66a 0.76
total stolon length, cm 31.96a 17.19 30.94a 3.20
stolons, no. plant" 4.06a 2.02 3.39a 0.57
initiates, no. stolon" 1.11a 0.28 1.34a 0.18
Week 4

avg. stolon length, cm 11.68a 1.89 16.22b 1.46
total stolon length, cm 58.99a 18.48 77 .12b 9.05
stolons, no. plant" 5.11a 1.20 4.78a 0.27
initiates, no. stolon" 1.59a 0.57 1.98a 0.65

 

56

Table 2.5. Phosphorus concentration of stem plus leaf tissue and
colonization level of potato roots grown in the greenhouse with and
without arbuscular mycorrhizae (Exp. 2, Apr.-July 1994, Johannesburg,
MI; means of six beds).

1‘ Control: commercial peat-vermiculite. Peat sterilized Sphagnum peat. AM:
arbuscular mycorrhizae.
1; ND: colonization not detected

57

 

 

 

Week post Growing media'i'

planting Control Peat + AM P S 0.05
P concentration, pg P mg" dry wt.

4 11.34 10.48 NS

8 10.61 10.54 NS
Colonization %

4 NDi 0.4 -

8 0.1 0.2 NS

 

58

Figure 2.1. Total length of stolon material (cm) produced per potato plant,
by ranges of production, (Exp.2, Apr.-July 1994, at Johannesburg, MI).
Each of the six samples per treatment represents the average of three
plants. The data from each of the six inoculated (INOC) and the six
control (CTRL) beds are expressed as total stolon production per plant
(cm). Weeks post planting.

Samples

Samples

Samples

59

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6" a
5 x 2weeks
— \ :Control
4 _ § Inoculated
3- s

s
2“ \

\
1* s
o_ s
5_ 3weeks
4_
3_
2“ \

\
1— x
\ § V

0 DH s s s 88
5_ 4weeks
4-
3-
1‘ s
0 . . . H. . is . .

 

 

 

 

 

 

 

<10 <20 <30 <40 <50 <60 <70 <80 <90 <100

Total stolon length per plant (cm)

60

wk, the inoculated plants had a greater total stolon production in addition to reduced
variability in length. The average length of individual stolons and the number of stolons
per plant showed similar distributions (data not shown). This suggests more uniform
growth of stolons under inoculation. This experiment was repeated at the same tuber

production facility, with similar results.

Discussion

Beneﬁts from AM inoculation are generally associated with nutritional effects
resulting from a mature symbiosis (Hayman, 1987). However, we found the peat-based
AM inoculum beneﬁcial even in the absence of a mature association. Inoculation
increased the total production of prenuclear minitubers grown in a greenhouse. By
increasing the proportion of the total production represented by the most valuable sizes,
the AM inoculum increased the value of the tubers produced. Inoculation with AM also
results in an increase in average stolon length and total stolon production per plant; these
increases are associated with dramatically reduced variability of stolon growth and
development (Table 2.4). The greater uniformity in the inoculated treatments suggests
that AM may inﬂuence stolon growth associated with AM inoculation. The effect of
Glomus fasciculatus and G. mosseae on potato grown in mist-culture has been previously
shown to increase the vine dry weight (Graham et al. , 1976). In addition, G.
fasciculatus increased the number of tubers produced, which may have been the result
of an increased rate of tuber initiation after planting (Graham et al. , 1976).

The extremely low AM colonization levels we observed were probably due to the

high nutritional growing conditions, and also to the application of a combination of

61

fungicides beginning after the third wk of the growing period (Table 2.1). The effects
of many commercial fungicides, singly and in combinations, on AM activity are not fully
understood (Menge et al. , 1979; Johnson and Pﬂeger, 1992; Kurle and Pﬂeger, 1994).
However, several of the fungicides used in our study (e.g. maneb, metalaxyl, and the
metalaxyl + chlorothanlonil combination) have each been shown to be highly detrimental
to AM activity at higher concentrations (Trappe, 1984). These concentrations correspond
to the application rates used in our study, and those used in many commercial settings.

In 1977, Black and Tinker (1977) demonstrated the effects of ﬁeld—scale AM
inoculation in the ﬁeld production of potatoes, and the inﬂuence of P fertilization. Potato
plants were grown in a non-irrigated ﬁeld with high superphosphate applications.
Experimental ﬁelds were treated with highly AM-infective topsoil from a barley
(Hordeum vulgare L.) ﬁeld, while control ﬁelds received less infective topsoil from a
fallow ﬁeld. The infective topsoil contained a mixed-culture of AM, with G.
macrocarpus being the dominant species. The plant tissue samples from these two
treatments did not differ in P concentration and showed uniformly low early AM
colonization, ranging from 0.9% to 4.0%. This study conﬁrms the results of Black and
Tinker (1977), and shows similarly low early AM colonization levels (Tables 3 and 5).
Although Black and Tinker (1977) reported no difference in ﬁnal potato yield between
the inoculated and non-inoculated treatments, the results of this study showed a
signiﬁcant increase in yield of the inoculated vs. the non-inoculated treatments under
identical fertilization regimes. In addition to the differences between the above ﬁeld
study and our greenhouse study, the mixed-culture soil inoculum source Black and Tinker

(1977) used was primarily G. macrocarpus as opposed to the peat-based pure-culture G.

62

intraradix inoculum used in our study. This difference in inoculum composition is
signiﬁcant, as host responses are known to differ with different fungal species, and with
mixed vs. pure culture inoculum (Bethlenfalvay, 1992).

Establishment of mycorrhizal symbiosis induces early biochemical changes in the
plant (Dumas et al., 1989; Hilbert et al., 1991) and in the fungus (Fries et al., 1993;
Ozan et al. , 1993). In the early stages of the AM colonization, the plant symbiont elicits
a transitory, weak defense response comparable to the strong response elicited by
pathogen infection (Gianinazzi and Gianinazzi-Pearson, 1992; Volpin et al., 1994).
These plant responses may be hormonally regulated, as is carbon allocation to roots, to
reproductive structures and to the fungal symbiont (Marschner, 1986; Spanu et al. ,
1989). Evidence of AM colonization causing biochemical changes is well established
(Allen et al., 1982; Marschner, 1986), but hormonal responses of host plants to AM
colonization are not conclusive (Bonfante-Fasolo and Spanu, 1992; Hock et al. , 1992).
Foliar application of cytokinin has been demonstrated to increase tuber yield of some
potato varieties (Lang and Langille, 1984). In this study, AM inoculation causes
increased stolon production and increased tuber production. These may be the result of
a hormonally-mediated regulation of the processes leading to these physiological effects.

Our results also suggest that the increased tuber production may be due to a shift
in carbon allocation induced in the plant during the early stages of AM colonization.
This shift may have increased the rate of tuber initiation by altering the pattern of stolon
development. The developmental changes (e.g. stolon morphology) that persist in the

absence of a continually developing AM association suggest a hormonally-mediated

63

response. In addition, there was no evidence of early senescence of stolons, which could
lead to a later proliferation of stolons and tuber initiates.

Growth promotion associated with low early AM colonization levels was also
demonstrated by O’Keefe and Sylvia (1992) using sweet potato (Ipomoea batatas L.)
Enhanced P inﬂow in that sweet potato study preceded extensive AM hyphal
proliferation. In contrast, the AM beds in this study showed signiﬁcantly increased
yields without an associated increase in plant P concentration. Similar growth increases
in the absence of P concentration effects were reported in sweetgum (Liquidambar
styraciﬂua) (Schultz et al. , 1979), and in asparagus using the same AM inoculum as we
have used here (Pedersen et al. , 1991). In a 1994 report, Volpin et al. showed an AM-
induced increase in isoﬂavonoid production in alfalfa (Medicago sativa L.) under very
low levels of hyphal colonization in the root tissue. Thus, they suggested that the
presence of AM in the rhizosphere was sufﬁcient to induce biochemical changes in the
root tissue, even in the absence of a mature AM association. Volpin et al. were using
alfalfa grown under high P levels in sterile sand inoculated with the same AM species
used in our study, G. intraradix. The increased uniformity of stolon development and
the increased formation of minitubers observed in our study may thus result from a S.
tuberosum response to the rhizosphere proximity of G. intraradix propagules similar to
the response of alfalfa demonstrated by Volpin et al. (1994).

Our results also indicate that AM inoculation can be effective for increasing
prenuclear minituber production. To emphasize the signiﬁcance of these results, consider
a hypothetical prenuclear minituber greenhouse production facility of 100ml. At a

market value of $55/kg for the small and medium minitubers and $19/kg for large

64

minitubers (R. Chase, Michigan State University, personal communication, 1994), a
100m2 greenhouse facility using the commercial practices described herein will yield
approximately $10 300 worth of minitubers. All other inputs held constant, the same
facility using the AM inoculum regime described herein would yield approximately $17
100 worth of minitubers. Although use of this inoculum may have a similar effect on
ﬁeld-grown seed tubers, the increased input cost of the inoculum suggests that its most
practical application would be for higher value, greenhouse-grown seed tuber crops.
The positive effects presented in this study were unrelated to high AM
colonization or enhanced plant P nutrition, and occurred despite applications of speciﬁc
fungicides previously shown to be inimical to AM colonization. The presence of AM
inoculum in the rhizosphere is beneﬁcial, even in the absence of a mature AM
association. These beneﬁts may derive from a transient stimulation of the plants’ defense
response resulting in a more uniform rate of development of stolon tissue. Further work

is necessary to fully understand the mechanisms of this rhizosphere proximity interaction.

65

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growth p. 113-130 In Powell, CL. and DJ. Bagyaraj (ed) VA Mycorrhiza, CRC
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Allen, M.F., T.S. Moore, and M. Christensen. 1982. Phytohormone changes in
Bouteloua gracilis infected by vesicular-arbuscular mycorrhiza. II. Altered levels

of gibberellin-like substances and abscisic acid in the host plant. Can. J. Bot.
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Bagyaraj, DJ. 1984. VA Mycorrhizae: Why all the interest? p. 1-4 In Powell, CL.
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Bethlenfalvay, G]. 1992. Mycorrhizae and crop productivity p. 1-28 In
Bethlenfalvay, G.J . and R.G. Linderman (ed) Mycorrhizae in sustainable
agriculture, American Society of Agronomy, Inc. Madison, WI

Black, R.L.B., and PB. Tinker. 1977. Interaction between the effects of vesicular-
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Bonfante-Fasolo, P. , and P. Spanu. 1992. Pathogenic and endomycorrhizal
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Caron, M., and S. Parent. 1988. Deﬁnition of a peat-lite medium for the use of
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Dumas, E. , V. Gianinazzi-Pearson, and S. Gianinazzi. 1989. Production of new
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Fries, L.L.M., R.S. Pacovsky, and GR. Saﬁr. 1993. Expression of isozymes altered
by Glomus intraradix or formononetin application in corn roots. p. 130 In Peterson,
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Mycorrhizae, Guelph, Ontario, Canada August 8-12, 1993 University of Guelph

Gianinazzi, S. , and V. Gianinazzi-Pearson. 1992. Cytology, histochemistry and
immunocytochemistry as tools for studying structure and function in
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Graham, 8.0., N .E. Green, and J. W. Hendrix. 1976. The inﬂuence of vesicular-
arbuscular mycorrhizae on growth and tuberization of potatoes. Mycologia 68:925-929

Hayman, D.S. 1987. VA Mycorrhizae in ﬁeld crop systems p.171-192 In Saﬁr, G.R.
(ed) Ecophysiology of VA Mycorrhizal plants, CRC Press, INC. Boca Raton, FL

Hilbert, J .L., G. Costa, and F. Martin. 1991. Ectomycorrhizin synthesis and

polypeptide changes during the early stage of eucalypt mycorrhiza development
Plant Physiol. 97:977-984

Hock, B., S. Liebmann, H. Beyrle, and K. Dressel. 1992. Phytohormone analysis by
enzyme immunoassays p.249-274 In Norris, J.R. , D.J. Read and A.K. Varma (ed)
Methods in microbiology Vol. 24 Academic Press Limited, New York, NY

Johnson, N.C. , and F .L. Pﬂeger. 1992. Vesicular-arbuscular mycorrhizae and cultural
stresses p. 71-99 In Bethlenfalvay, G.J. and R.G. Linderman (ed) Mycorrhizae in
sustainable agriculture, American Society of Agronomy, Inc. Madison, WI

Kiernan, J .M., J.W. Hendrix, and D.M. Maronek. 1983. Fertilizer-induced
pathogenicity of mycorrhizal fungi to sweetgum seedlings Soil Biol. Biochem.
Vol. 15, No. 3, 257-262

Kormanik, P.P. , and AC. McGraw. 1982. Quantiﬁcation of vesicular-arbuscular
mycorrhizae in plant roots. p.37-45 In Schenck, N .C. (ed) Methods and principles
of mycorrhizal research APS Press, St. Paul, MN

Kurle, J .E. , and EL. Pﬂeger. 1994. The effects of cultural practices and pesticides
on VAM fungi p. 101-132 In Pﬂeger, EL. and R.G. Linderman (ed) Mycorrhizae
and plant health APS Press, St. Paul, MN

Lang, D.J. , and AR. Langille. 1984. Inﬂuence of plant growth and concentration of

cytex and kinetin applications on tuber yields of two potato cultivars HortScience
19(4), 582-583

Marschner, H. 1986. Mineral nutrition of higher plants p.469-475 Academic Press
INC. San Diego, CA

Menge, J .A., E.L.V. Johnson, and V. Minassian. 1979. Effect of heat treatment and
three pesticides upon the growth and reproduction of the mycorrhizal fungus
Glomus fasciculatus New Phytol. 82:473-480

Nelsen, C.E. , and GR. Saﬁr. 1982. Increased drought tolerance of mycorrhizal onion
plants caused by improved phosphorus nutrition. Planta 154:407-413

O’Keefe, D.M. , and D.M. Sylvia. 1992. Chronology and mechanisms of P uptake by
mycorrhizal sweet potato plants New Phytol. 122:651-659

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Ozan, A.G., R.S. Pacovsky, and GR. Saﬁr. 1993. A unique malate dehydrogenase
isozyme of endomycorrhizal white clover roots and the associated effects of the
isoﬂavone formononetin on isozyme activities. p.133 In Peterson, L. and M.
Schelkle Abstracts of the 9th North American Conference on Mycorrhizae,
Guelph, Ontario, Canada August 8-12, 1993 University of Guelph

Pedersen, C.T., G.R. Saﬁr, S. Parent, and M. Caron. 1991. Growth of asparagus in
a commercial peat mix containing vesicular-arbuscular mycorrhizal (VAM) fungi
and the effects of applied phosphorus. Plant and Soil 135:75-82

Phillips, J.M. , and D.S. Hayman. 1970. Improved procedures for clearing roots and
staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment
of infection. Trans. Brit. Myc. Soc. 55:158-161

Ponton, F ., Y. Piché, S. Parent, and M. Caron. 1990. Use of vesicular-arbuscular
mycorrhizae in Boston fern production: 11. Evaluation of four inocula. HortScience
25:416-419

Powell, C .L. 1984. Field inoculation with VA mycorrhizal fungi p. 205-222 In
Powell, CL. and DJ. Bagyaraj (ed) VA Mycorrhiza, CRC Press, Inc. Boca
Raton, FL

Schultz, R.C., P.P Kormanik, W.C. Bryan, and G.H. Brister. 1979. Vesicular-
arbuscular mycorrhiza inﬂuence growth but not mineral concentrations in seedlings
of eight sweetgum families Can. J. For. Res. 9, 218

Spanu, P., T. Boller, A. Ludwig, A. Wiemken, A. Faccio, and P. Bonfante-Fasolo.
1989. Chitinase in roots of mycorrhizal Allium porrum: regulation and localization
Planta 177:447—455

Trappe, J.M., R. Molina, and M. Castellano. 1984. Reactions of mycorrhizal fungi
and mycorrhiza formation to pesticides Ann.Rev.Phytopathol. 22:331-59

Volpin, H., Y. Elkind, Y. Okon, and Y. Kapulnik. 1994. A vesicular arbuscular
mycorrhizal fungus (Glomus intraradix) induces a defense response in alfalfa roots
Plant Physiol 104:683-689

Chapter 3. Postharvest Suppression of Potato Dry Rot (Fusarium sambucinum) in

Prenuclear Minitubers by Arbuscular Mycorrhizal Fungal Inoculum1

Abstract

Arbuscular mycorrhizae (AM) have been shown to increase plant resistance to
root-rotting pathogens. This study was conducted to determine whether a commercial
peat-based medium containing the AM fungus Glomus intraradix (Schenck & Smith)
could inﬂuence a) postharvest progression of tuber dry rot in prenuclear minitubers of
potato (Solanum tuberosum), and b) activity of the defense related enzyme peroxidase
(POX). Minitubers grown in this medium had signiﬁcantly less tuber dry rot (20-90%
reduction) when later inoculated with the dry rot fungus Fusarium sambucinum relative
to minitubers grown in an identical peat-based medium without the AM fungus. This
disease suppression was also demonstrated in a high-input commercial greenhouse, and
occurred despite only trace levels of AM colonization of the parent plants, and with no
evidence of enhanced plant P nutrition or differences in minituber mineral content.
Protein extracts from four week old potato roots colonized by AM show a signiﬁcantly
reduced peroxidase activity vs. noncolonized roots. These results suggest that the AM

fungal inoculum has potential for use in suppression of tuber dry rot of potato.

 

1 This chapter was submitted in modiﬁed form to the American Potato Journal as
Niemira, B.A., Hammerschmidt, R., and Saﬁr, G.R. Postharvest suppression of potato
dry rot (Fusarium sambucinum) in prenuclear minitubers by arbuscular mycorrhizal
fungal inoculum.

68

69

Introduction

The F usarium dry rots of potato (Solanum tuberosum L.) are among the most
important postharvest pathogens worldwide (Rowe, 1993). Current methods for control
of F usarium dry rot rely on cultural practices and chemical treatments (Kawchuk et al. ,
1994; Rowe, 1993), rather than biological control (Jarvis, 1992).

Associations between plant roots and arbuscular mycorrhizal (AM) fungi can
increase plant growth, in many cases by enhancing P uptake from soils with low to
moderate P availability (Powell, 1984). While inoculation with AM fungi can be
beneﬁcial in low-input systems, conditions that limit AM fungal colonization of roots and
associated beneﬁts include high P availability (Abbott and Robson, 1984), high water
availability (Hayman, 1987), and extensive fungicide application (Trappe et al., 1984).
Peat-based commercial AM fungus inocula tested in the production systems of many
horticultural crops have enhanced AM fungi colonization and increased yield (Ponton et
al., 1990; Pedersen et al., 1991).

AM fungi can also reduce the severity of damage caused by plant pathogenic
nematodes and fungi (Linderman, 1992). Newsham et al.,(1993) suggest that in Vulpia
ciliata ssp. ambigua (Le Gall), the main beneﬁt of AM fungi is protection from
pathogenic fungi, rather than from improved P uptake. Investigations of disease
suppression promoted by AM fungi have focused on pathogens of the host plant
(Newsham et al. ,1993; Linderman, 1992). AM-induced suppression of storage pathogens
on the resulting produce in a post-harvest setting is, to our knowledge, unexplored.

A seed potato minituber grower (D. Sklarczyk, Sklarczyk Seed Farms,

Johannesburg, MI.) noted that minitubers produced by plants exposed to a commercial

70

AM fungal inoculum had reduced storage rot levels. The purpose of the research
reported herein was to determine the effect of AM fungal inoculum on the postharvest
progression of tuber dry rot caused by Fusarium sambucinum (Gibberella pulicaris) in
potato minitubers, and on the activity levels of peroxidase (POX), a defense-related

enzyme involved in lignin synthesis.

Materials and methods
Storage rot: research greenhouse

Plants were grown from prenuclear (produced from tissue cultured plants)
minitubers of potato (cv. Atlantic) in research greenhouses at Michigan State University,
with daily watering and fertilization with 1 L 90-180-90 g L" (N-P-K) at 3 and 6 wk.
The inoculated experimental medium consisted of Mycori-Mix (Premier Peat Moss, Ltd. ,
Riviére-du Loup, Québec, Canada), a Sphagnum peat-based medium inoculated with
approx. one propagule of the AM fungi fungus Glomus intraradix per gram. The non-
inoculated medium consisted of the identical Sphagnum peat-based medium without AM
(SB Mix, Premier Peat Moss, Ltd., Riviére-du Loup, Quebec, Canada).

Each media was replicated four times in separate growing beds (18" by 14" by
4", 45 cm by 35 by 10 cm). Plants were grown under sodium vapor lights (14h/10h
light/dark) for 10 weeks. After 10 weeks, minitubers were harvested, and potato root
samples were collected for assay of AM colonization. Minitubers harvested from each
of the four beds from each treatment were combined and stored at Michigan State

University at 4°C for 5 months, and subsequently challenged with F. sambucinum. This

71

experiment was repeated in four additional replications, and the data from these ﬁve
experiments were pooled (ﬁnal population size, n=49).
Storage rot: commercial greenhouse

Tissue cultured plantlets of potato cv. Atlantic were grown in a commercial
minituber production greenhouse at Sklarczyk Seed Farms, Johannesburg, MI under high-
input conditions, including frequent watering, and frequent foliar fertilization with 90-
180-90 g L" (N-P-K). The commercial practices used at this facility included soil
drenches with pesticides following the third week after planting. Pesticides used of
particular signiﬁcance included the fungicides metalaxyl + chlorothalonil (0.29 g a. i./m2)
and maneb (1.16 g a.i./m2), and the insecticide carbofuran (0.32 g a.i./m2).

The inoculated experimental medium consisted of a 50:50 (vzv) mixture of sterile
SB Mix and the AM containing Mycori-Mix, the mixture used in a previous study
(Niemira, et al., 1995). The noninoculated medium consisted of a similar steam-
pasteurized peat-based mixture (Alpar Peat Co. , Ovid, Mich.) used in these greenhouses.
The treatments were replicated six times in separate growing beds (4’ by 8’ by 4", 120
cm by 240 cm by 10 cm). Plants were grown for 14 weeks. Samples of root, stem and
leaf tissue were collected at 4 weeks and 8 weeks after planting. After the 14 week
growing period, the minitubers were harvested from each of the six beds from each
treatment. These subsamples were combined and stored at Michigan State University at
4°C for 5 months, and subsequently challenged with F. sambucinum. Tuber pieces were
dried and analyzed for mineral content by the Michigan State University soil and plant
nutrient laboratory. This experiment was repeated at the same tuber production facility,

and the data from the two experiments were pooled (ﬁnal population size, n=16).

72

Root protein extraction and sample preparation

Potatoes were grown in the same type of media as for the storage rot studies
conducted in the research greenhouse. At four weeks after germination, ﬁve plants
grown in sterile and AM inoculated media were harvested. The root system was
subsampled for AM colonization. The remaining root system was washed in water at
0°C, then immediately wrapped in foil and frozen in liquid nitrogen. The root samples
were stored at -80°C until use. The roots were ground to a ﬁne powder in a stainless
steel pestle using liquid nitrogen. To extract root protein contents, powdered root tissue
were mixed with grinding buffer (0.75 ml g" root powder) containing 50 mM Tris-HCl
pH 7.0, 3.0 mM ethylenediamine tetraacetic acid, 2.5 mM dithiothreitol, 250 mM
sucrose, 50 mM NaCl, 2 mM phenylrnethylsulfonyl ﬂuoride, and 2 mM N-ethyl-
maleirnide (Pacovsky, 1989). The chilled samples were homogenized with a tissue
grinder (Tekrnar, Cincinnati, OH). The liquiﬁed extract was centrifuged for 10 minutes
at 12K rpm at 4°C (Marathon 21K/BR, Fisher Scientiﬁc, Chicago, IL). The supernatant
containing the root proteins was transferred to rnicrocentrifuge tubes and frozen at -80°C.
Protein concentrations of the samples (pg protein/p1 extract) was determined using the
Bradford dye-binding assay, with bovine y-globulin as a standard (Biorad, Richmond,
CA).
Total peroxidase assay

Measurement of total peroxidase activity was determined using a modiﬁed version
of Ridge and Osborn (1970). For each sample, 2 pg of protein were added to 5 ml of
a pH 5.5 phosphate buffer solution containing 10mM H202 and 20 mM guiacol. Optical

density at 480 nm was recorded every 30 seconds for 10 minutes. The slope of the

73

linear portion of the data set (0.5 min. to 3.5 min.) for each replicate was determined,
and expressed as 00D“;0 min" mg protein ". Five replicates from each treatment were
assayed.
AM root colonization assay

Plant roots were cleared and stained with Trypan blue according to Phillips and
Hayman (1970). Root colonization by AM was determined by the line-intersect method
(Kormanik and McGraw, 1982). P concentrations of stem + leaf tissue (Exp’t. 2) were
determined using the method of Nelsen and Saﬁr (1982).
Fusarium sambucinum challenge

Stored minitubers of each treatment were randomly selected and allowed to
equilibrate in the dark at room temperature (z23°C) for 24 h. A circular wound z4
mm, was made through the tuber periderm. A 5-mm plug of PDA taken from the edge
of a day-old culture of F. sambucinum colony growing on PDA was placed top down
onto the wound (culture provided by R. Hammerschmidt, Dept. Botany and Plant
Pathology, Michigan State University). The inoculated tubers were stored in the dark
for 14 days at room temperature (z23°C) and then bisected. The depth, and width of
the rotted tissue was measured. The volume of rotted tissue was estimated with a conic
approximation using these measurements.
Statistical analysis

Storage rot data and 00D480 min" mg protein " of the total POX activity were
analyzed with one-way AN OVA (analysis of variance) using the statistical package

Minitab.

74
Results

Storage rot: research greenhouses

Severity of tuber dry rot caused by F. sambucinum is reduced by 30-74% in
tubers that were produced in medium containing AM fungi (Table 3.1). Minitubers
grown in AM inoculum had signiﬁcantly less (P s 0.01) rotted tissue by all measurements
(depth, width and volume). AM colonization of the roots of inoculated plants was
moderate (15.4%), while colonization of non-inoculated plants was negligible (5 1%).
Storage rot: commercial greenhouses

Severity of tuber dry rot caused by F. sambucinum is reduced by 21-58% in
tubers that were produced in medium containing AM fungi under high-input commercial
growing conditions (Table 3.1). The pattern of disease reduction is similar to that shown
by tubers grown in research greenhouses. The depth, width and volume of rotted tissue
are signiﬁcantly reduced (P $0.05). There were no signiﬁcant differences in stem +
leaf tissue P levels between treatments. Analysis of tuber mineral content, including
micronutrients known to affect progression of diseases (Graham and Webb, 1991), e.g.
B, Ca, Cu, Fe, Mn and Zn, showed no signiﬁcant differences between treatments. In
this experiment, unlike that conducted in the research greenhouses, levels of root
colonization by the mycorrhizal fungus were negligible (S 1%) in plants taken from
inoculated and non-inoculated treatments.
Root protein total POX activity

The average oOD480 min" mg protein " of control potato roots not colonized by

AM was 12.288, while the average aOD480 min" mg protein " of potato roots that

75

Table 3.1. Progression of fusarium dry rot2 in potato minitubers grown in
nonmycorrhizal (CTRL) and mycorrhizal (INOC) growing media.

2 Reductions are statistically signiﬁcant at P5005 (*) and P5001 (**).
’ Data are followed in parenthesis by percent of control.

" Volume is calculated as a conical approximation with the following
equation: Volume = «(breadth/2)2 *1r) *depth)/ 3

76

 

 

 

Medium Depth Breadth Volume
(mm)y (mm (mm3)"

Research greenhouse

CTRL 6.59 13.33 694.8

INOC 4.02 (60.9)“ 9.27 (69.5)“ 183.3 (26.3)**
Commercial greenhouse

CTRL 4.69 12.06 203.4

INOC 2.94 (62.7)** 9.56 (79.3)" 84.8 (41.8)**

 

77

colonized by AM was 9.23. This reduction is suggestive (P 50.10), although not signiﬁcant.

Discussion

The results presented herein represent what may be the ﬁrst demonstration of
postharvest disease suppression caused by the presence of AM fungi inoculum during
minituber production. Increased disease resistance to plant pathogens resulting from
inoculation with AM fungi had previously been associated primarily with root-infecting
pathogens (Newsham et al., 1993; Linderman, 1992; Benhamou et al., 1994). AM
related disease reduction effects often are associated with enhanced nutrition resulting
from a mature AM symbiosis (Hayman, 1987). The results from experiments conducted
in the commercial greenhouses show that peat-based AM fungal inoculum can enhance
postharvest disease resistance in the absence of nutritional differences.

The progression of a variety of rot pathogens in potato tubers can be inﬂuenced
by the concentrations of mineral nutrients within the tissues (Huber, 1991; Graham and
Webb, 1991). As there were no differences in the mineral content of the tubers grown
in the different media in our study, it seems unlikely that the effects observed arise from
a purely nutritional effect. These effects are shown in tubers, which are modiﬁed stem
tissue, rather than in true seed (see Koide and Lu, 1992). The negligible levels of
mycorrhizal colonization observed in commercial greenhouses were probably due to the
high nutritional growing conditions, as well as application of pesticides after the third
week after planting (see Niemira et al. ,1995). The effects of many commercial
fungicides, singly and in combinations, on AM fungi activity are not understood fully

(Kurle and Pﬂeger, 1994). However, several of the pesticides applied in the commercial

78

greenhouse in the course of the experiments (e. g. , metalaxyl + chlorothalonil,
carbofuran, and maneb) are detrimental to AM fungi activity at concentrations
corresponding to the levels used (Trappe et al. , 1984).

Previous studies have shown that early AM inoculation can cause speciﬁc changes
in plant physiology, even in the absence of a mature association. These changes can
include alterations in tuber initiation patterns and stolon production in potato (Niemira
et al. , 1995), increased rooting of grape (Vitus vinifera) cuttings (G.R. Saﬁr, unpublished
data) and increased survivalship and rooting of umbrella pine (Sciadopitys verticillata)
cuttings (Douds et al. , 1995). Studies into the effect of AM inoculation on survivorship
and rooting of yew (T axus spp.) are ongoing (R. Schutski, unpublished data). The
absence of a developed root system in the case of woody cuttings precludes extensive
root colonization by AM fungi; that physiological alterations are still observed lends
credence to the suggestion that extremely low levels of AM colonization can signiﬁcantly
impact growth and plant vigor.

In the early stages of AM association, the fungus elicits a transitory, weak plant
defense response comparable to the strong response elicited by pathogen infection (V olpin
et al. , 1994). Under low to moderate fertility in the research greenhouses, AM fungal
inoculum caused moderate AM colonization of the parent plants. In contrast, the high-
input growing conditions in the commercial greenhouses prevented the development of
a mature AM symbiosis, and limited AM root colonization of the parent plants to
negligible levels. The signiﬁcantly reduced levels of Fusarium dry rot in the minituber
progeny in both series of experiments, even in the absence of a mature AM association

as in the commercial greenhouses, suggest that the plant’s transitory antagonistic response

79

was initiated within the ﬁrst few days or weeks of growth in the presence of AM
inoculum. At four weeks, the total root tissue POX activity of AM colonized potatoes
tended to be less than that of the noncolonized control. Reduction in total POX activity
after 3-4 weeks of exposure to the AM fungus has previously been observed in leek,
Allium porrum, (Spanu and Bonfante-Fasolo, 1988), corn, Zea mays, (Fries et al. , 1996),
and clover, T rifolium repens (Ozan, 1996). In these previous studies, POX activity
during the initial stage (1-2 weeks) of the symbiosis increased, and after the initial stages
of the AM symbiosis, the host apparently suppresses this particular defense response.
This suppression occurs during the period of the most rapid spread of the AM fungus
throughout the root system, and it is felt that this suppression facilitates root colonization
(Spanu and Bonfante-Fasolo, 1988; Fries et al., 1996).

The published information relating to POX activities associated with AM
colonization is therefore somewhat in contrast to POX activities associated with induced
resistance. Previous studies of induced systemic resistance in cucumber, Cucumis
sativus, and tobacco, Nicotiana tabacum, have reported increased POX activity after
exposure to eliciting agents such as tobacco mosaic virus on tobacco (Simons and Ross,
1970; van Loon, 1976), and pathogenic bacteria and heat shock on cucumber
(Hammerschmidt et al. , 1982; Stermer and Hammerschmidt, 1984). Increased POX
activity indicates an increased level of lignin synthesis, a deterrent modiﬁcation that
inhibits the spread of the pathogen, a condition that is in precise opposition to the
reduced POX activity levels in roots involved in AM associations. POX’s role in defense
related lignin synthesis has been proposed as a possible mechanism for induced systemic

resistance in cucumber (Hammerschmidt and Kuc, 1982). The assumption that POX is

80

the primary mechanism of induced resistance leads to the conclusion that the suppression
of POX activity in AM associations would tend to make AM-colonized plants more
susceptible to disease. Studies of disease susceptibility in mycorrhizal plants (Newsham
et al. , 1993; Linderman, 1992) have concluded that colonization by AM fungi tends to
decrease, rather than increase disease susceptibility. Stermer’s (1995) suggestion that
induced systemic resistance consists of several mechanisms rather than POX as the single
motive force resolves this question. The transitory increase in POX activity shown in
other mycorrhizal systems (Spanu and Bonfante-Fasolo, 1988; Fries et al. , 1996; Ozan,
1996) may serve to induce a response similar to systemic resistance.

Reduced POX activity levels of the root protein extracts clearly indicates
coordinated biochemical changes in the potato plant physiology, suggesting a systemic
response. AM fungi have been associated with induction of a variety of host metabolic
changes that enhance resistance to disease, such as induction of phenolic compounds
(Dehne, 1982; Krishna and Bagyaraj , 1983) and chitinases with potential antimicrobial
properties (Spanu et al. , 1989; Benhamou et al. , 1994). The reduction of Fusarium dry
rot severity presented herein may be a form of AM-induced systemic resistance
(Hammerschmidt and Kuc, 1995) that is maintained in the tuber progeny. Alternatively,
the reduction may be a result of some action of the AM fungi directly on the surface of
the minitubers, or a combination of these factors. The AM-induced reduction of
F usarium dry rot was apparently unrelated to high levels of AM fungal root colonization,
enhanced plant nutrition, or changes in the tuber mineral nutrient content. A detailed
explanation has not yet been formulated for the seemingly contradictory condition of an

AM-induced reduction at 4 weeks in total root POX activity that is coincident with an

81

increased resistance to the spread of F. sambucinum in tuber tissue. Based on the
response to F usarium oxysporum f. sp. chrysanthemi of mycorrhizal and non-mycorrhizal
Ri T-DNA-transforrned carrot roots, Benhamou et al. (1994) hypothesize that the
presence of a signal from the pathogen is necessary for full induction of the defense
response, and accumulation of defense gene products. Exposure to abiotic or biotic
elicitors is thought to sensitize the plant to respond more quickly to any subsequent
microbial attack without accumulation of metabolically expensive defense gene products
in the absence of pathogens (Benhamou et al. , 1994). The physiological interactions of
the early stages of AM symbiosis that result in the transitory increase in POX activity
may serve to sensitize the plant in this way, initiating other mechanisms that enhance the
mycorrhizal plant’s resistance to disease, despite the decrease in root POX activity after
3-4 weeks. This possibility, as it relates to the observations presented herein, could be
addressed more fully by an investigation into AM-related induction of other potential

mechanisms of induced resistances.

82

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Boca Raton.

Benhamou, N ., J.A. Fortin, C. Hamel, M. St-Amaud and A. Shatilla. 1994. Resistance
responses of mycorrhizal Ri T-DNA-transformed carrot roots to infection by F usan'um
oxysporum f. sp. chrysanthemi. Phytopathology 84:958-968

Bradford, M.M. 1976. A rapid and sensitive method for the quantiﬁcation of micrograrn
quantities of protein utilizing the principle of protein-dye binding. Analytical
biochemistry 72:248-254

Dehne, H.W. 1982. Interaction between vesicular-arbuscular mycorrhizal fungi and plant
pathogens. Phytopathology 72: 1115-1119

Fries, L.L.M., Pacovsky, RS. and Saﬁr, GR. 1996. Expression of isozymes altered by
both Glomus intraradicies colonization and formononetin application in corn (Zea mays
L.) roots. Soil Biology and Biochem. (in press)

Graham, RD. and Webb, M.J. 1991. Micronutrients and disease resistance and tolerance

in plants. pp. 331-368 in Micronutrients in agriculture, 2nd ed. Soil Science Society
of America, Madison, WI

Hammerschmidt, R. and J .Kuc. 1982. Ligniﬁcation as a mechanism for induced systemic
resistance in cucumber. Physiological Plant Pathology 20:61-71

Hammerschmidt, R., E.M. Nuckles and J. Kuc. 1982. Association of enhanced
peroxidase activity with induced systemic resistance of cucumber to Colletotrichum
lagenarium. Physiological Plant Pathology 20:73-82

Hammerschmidt, R. and J. Kuc. 1995. Induced resistance to disease in plants. Kluwer
Academic Publishers, Dordrecht, Netherlands.

Hayman, D.S. 1987 . VA mycorrhizae in ﬁeld crop systems, p. 171-192. in: GR. Saﬁr
(ed.). Ecophysiology of VA mycorrhizal plants. CRC Press, Boca Raton.

Huber, D. 1991. The use of fertilizers and organic amendments in the control of plant
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management in agriculture, 2nd edition, Vol. 1. CRC Press, Boca Raton, FL

Jarvis, W.R. 1992. Managing disease in greenhouse crops. APS Press, St. Paul, Minn.

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Kawchuk, L.M., J.D. Holley, D.R. Lynch and RM. Clear. 1994. Resistance to
thiabendazole and thiophanate-methyl in Canadian isolates of Fusarium sambucinum
and Helrninthosporium solani. Amer. Potato J 71(3):185-192

Koide, R.T. and Lu, X. 1992. Mycorrhizal infection of wild oats: maternal effects on
offspring growth and reproduction. Oecologia 90:218-226

Kormanik, RP. and AC. McGraw. 1982. Quantiﬁcation of vesicular-arbuscular
mycorrhizae in plant roots, p. 37-45. in: NC. Schenck (ed.) Methods and principles
of mycorrhizal research. APS Press, St. Paul, MN.

Krishna, K.R. and DJ. Bagyaraj. 1983. Interaction between Glomus fasiculatum and
Sclerotium rolfsii in peanut. Can. J. Bot. 61:2349-2351

Kurle, J .E. and EL. Pﬂeger. 1994. The effects of cultural practices and pesticides on
VAM fungi, p. 101-132 in: FL. Pﬂeger and R.G. Linderman (eds.). Mycorrhizae
and plant health. APS Press, St. Paul, Minn.

Linderman, R.G. 1992. Vesicular-arbuscular mycorrhizae and soil microbial interactions,
p. 45-70 in: GI. Bethlenfalvay and R.G. Linderman (eds.). Mycorrhizae in
sustainable agriculture. Amer. Soc. Agron., Madison, Wis.

Nelsen, CE. and GR. Saﬁr. 1982. Increased drought tolerance of mycorrhizal onion
plants caused by improved phosphorus nutrition. Planta 154:407-413.

Newsham, K.K. , Fitter, AH. and Watkinson, A.R. 1993 Arbuscular mycorrhiza protect
an annual grass from root pathogenic fungi in the ﬁeld. J Ecology 83:991-1000.

Niemira, B.A., Saﬁr, G.R., Hammerschmidt, R. and Bird, G.W. 1995 Production of
prenuclear minitubers of potato with peat-based arbuscular mycorrhizal fungal
inoculum. Agron. J. 87:942-946

Ozan, A. 1996. Isozyme activity of developing T nfolium-Gloum mycorrhiza and
associated effects of the isoﬂavone formononetin. Allelopathy J. 3(2):217-228

Pacovsky, RS. 1989. Carbohydrate, protein and amino acid status of Glycine-Glomus-
Bradyrhizobium symbiosis. Physiologia Plantarum 75:346-354

Pedersen, C.T., G.R. Saﬁr, 8. Parent and M. Caron. 1991. Growth of asparagus in
a commercial peat mix containing vesicular-arbuscular mycorrhizal (V AM) fungi
and the effects of applied phosphorus. Plant Soil 135175-82.

Phillips, J .M. and D.S. Hayman. 1970. Improved procedures for clearing roots and
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of infection. Trans. Brit. Mycol. Soc. 55:158-161.

WIT-

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Ponton, F. , Y. Piché, S. Parent and M. Caron. 1990. Use of vesicular-arbuscular

mycorrhizae in Boston fern production: H. Evaluation of four inocula. HortScience
25:416-419.

Powell, C.L. 1984. Field inoculation with VA mycorrhizal fungi, p. 205-222. in: C.L.
Powell and DJ. Bagyaraj (eds.). VA mycorrhiza. CRC Press, Boca Raton.

Rowe, RC. 1993. Potato health management. APS Press, St. Paul, Minn.

Sirnons, T]. and A.F. Ross. 1970. Enhanced peroxidase activity associated with
induction of resistance to tobacco mosaic virus in hypersensitive tobacco.
Phytopathology 60:383-384

Spanu, P. and P. Bonfante-Fasolo. 1988. Cell-wall-bound peroxidase activity in roots of
mycorrhizal Allium porrum. New Phytologist 92:75-87

Spanu, P., T. Boller, A. Ludwig, A. Weimken, A. Faccio and P. Bonfante-Fasolo.
1989. Chitinase in roots of mycorrhizal Allium porrum: regulation and localization.
Planta 177 :447-455

Stermer, BA. and R. Hammerschmidt. 1984. Heat shock induces resistance to

Cladosporum cucumerinum and enhances peroxidase activity in cucumber.
Physiological Plant Pathology 25:239-249

Stermer, BA. 1995. Molecular regulation of systemic induced resistance. pp. 111-140
In Hammerschmidt, R. and J. Kuc. 1995. Induced resistance to disease in plants.
Kluwer Academic Publishers, Dordrecht, Netherlands.

Trappe, J .M., R. Molina and M. Castellano. 1984. Reactions of mycorrhizal fungi
and mycorrhiza formation to pesticides. Ann. Rev. Phytopathol. 22:331-59.

van Loon, LC. 1976. Systemic acquired resistance, peroxidase activity and lesion size
in tobacco reacting hypersensitively to tobacco mosaic virus. Physiological Plant
Pathology 82231-242

Volpin, H. , Y. Elkind, Y. Okon and Y. Kapulnik. 1994. A vesicular-arbuscular
mycorrhizal fungus (Glomus intraradix) induces a defense response in alfalfa roots.
Plant Physiol. 104:683-689.

Chapter 4. Summary and Conclusions

AM fungi respond to crop rotations and input level, with input high chemical
inputs associated with irrigation being the more dominant of the two factors. On the
basis of the results of experiments presented in Chapter 1, I would recommend,
therefore, that in order to manage the population of AM fungi in the soil, the
management decisions that are most key are those that involve chemical and irrigation
inputs. Additional ﬁeld experimentation could be conducted to establish the impact of
varying chemical and irrigation levels for a given rotation, and the impact of other
rotations within the particular management framework. The extent to which speciﬁc
crops in the rotations (particularly legumes) contribute AM-stirnulatory bioactive
compounds to the soil, and the relative signiﬁcance and/ or duration of this stimulus, is
not fully known. Soil sampling could be conducted to test for the presence of known
plant produced compounds with bioactive properties. This sampling could be for
extracted residues from plant detritus from crops earlier in the rotation, or as percentage
recovery of a known amount of exogenously applied compound. These experiments
could be used in conjunction with efforts to design novel rotation structures, perhaps
incorporating unconventional crops such as canola, in order to identify crops that are
especially effective at encouraging healthy AM populations.

Inoculation of potato plants in a high chemical and water input greenhouse growth
environment with AM fungi results in signiﬁcantly increased yield and signiﬁcantly
increased percentage of small and medium prenuclear seed minitubers. These alterations

in the yield are of economic importance because of the high value (SSS/kg) of the

85

86

prenuclear seed minitubers in general, and the small and medium minitubers in
particular. AM inoculation also results in longer stolons, more stolon produced per plant
and reduced variability in stolon production. I would therefore recommend that AM
inoculum be incorporated into standard bedding material for prenuclear minituber
production facilities. The series of experiments presented in Chapter 2 was conducted
entirely with the cultivar Atlantic, which tends to produce smooth, regular tubers. Other
cultivars, such as Russett Burbank, produce tubers that are more irregular. Although
characters such as shape and regularity are less signiﬁcant in seed tubers, they are quite
important in determining the value of a crop of ﬁeld-grown tubers. Smooth, regular
tubers can be used for baking and chipping, while knobby or irregular tubers are used
for lower-value applications. In light of the observed reduction in variability of stolon
production, additional experiments could be conducted to determine the effect of
inoculation with AM fungi on the surface features of characteristically irregularly shaped
varieties such as Russett Burbank.

Inoculation of potato plants with AM fungi results in tubers that are more resistant
to Fusarium storage rot. Controls for Fusarium rot are primarily cultural in nature, as
there are few effective chemical controls for this pathogen. The results of the
experiments presented in Chapter 3 strengthen the recommendation that AM inoculum
be incorporated into standard bedding material for prenuclear minituber production
facilities. Although more smooth and regular, tubers of the cultivar Atlantic tend to be
more susceptible to storage rot than tubers of other varieties such as Russet Burbank.
Experiments could be conducted to evaluate the susceptibility of AM-inoculated tubers

of other varieties, either more susceptible or less susceptible than Atlantic, to Fusarium

87

storage rot. Also, tests with storage pathogens other than F usarium sambucinum should
be conducted. An application that controls a broad variety of pathogens would have
numerous potential applications for potato producers. Further experiments could be
conducted to determine if tubers from ﬁeld-grown potato plants exhibit a similar response
to AM inoculation. The disease susceptibility of tubers from a ﬁeld with high AM
colonization potential could be compared with tubers from a ﬁeld with low AM
colonization potential. The results of this experiment may allow for more speciﬁc
recommendations to ﬁeld tuber growers.

The physiology of potato plants grown in AM inoculum is signiﬁcantly altered,
even in the absence of a mature mycorrhizal association, and that AM fungi induce a
response in potato similar to systemic induced resistance. Experiments could be
conducted to explore this phenomenon. These could include attempts to more ﬁrmly
establish a time course of the response to a transitory AM stimulus, biochemical analysis
to identify speciﬁc alterations in the physiology of tuber periderrn tissue or leaf

intercellular ﬂuid isozyme activities.

APPENDIX

Appendix A

A Theoretical Study of the Association of Arbuscular Mycorrhizal Colonization with

Border Cell Production‘

Abstract

Border cells (BC, formerly referred to as sloughed root cap cells) are produced
at the root tip. Species in different families produce different numbers of BC per root
tip. A survey of the mycorrhizal literature suggests that BC production is positively
correlated with colonization by arbuscular mycorrhizal (AM) fungi; families which
produce thousands of BC per root tip (e. g. Fabaceae and Cucurbitaceae) tend to be
heavily colonized by AM, families which produce hundreds of BC per root tip (e.g.
Solanaceae) tend to be less heavily colonized by AM, while families which do not
produce BC (e.g. Brassicaceae and Chenopodiaceae) are not colonized by AM. This
previously unknown correlation suggests a causational role of BC and/or BC produced

signal molecules in the AM symbiosis.

Introduction
Arbuscular mycorrhizae (AM) are ubiquitous soil fungi that form symbiotic

associations with many plant species, including most signiﬁcant crop plants (Bagyaraj,

 

‘ This chapter appeared in slightly modiﬁed form as Niemira, B.A., Saﬁr, GR. and
Hawes, M.C. Arbuscular mycorrhizal colonization and border cell production: a possible
correlation. Phytopathology 86:563-565 1996.

88

89

1984; Walker, 1995). The AM associations can increase plant growth, in many cases
by enhancing P uptake from soils with low to moderate P availability (Powell, 1984).
A given plant species’ AM propensity, i.e. the extent to which the root system of an
individual plant is colonized by the AM symbiont, may vary under different conditions,
e.g. natural soils vs. disturbed agricultural soils. This variability can arise from
differences in AM inoculum composition, soil nutrient availability, and plant community
composition (Linderman, 1992). Plant families may be generally, although not perfectly,
categorized by the AM propensities of plant species within that family (Gerdemann,
1968; Tester et al.,1987). Many families in the order Caryophyllales are considered
minirnally- or non-mycorrhizal; of these, Brassicaceae and Chenopodiaceae have received
the most attention (Tester et al.,1987). Baylis (1975) hypothesized that a species’
mycorrhizal propensity is related to root morphology. Species with thick, unbranched
roots and few root hairs are more heavily dependent on mycorrhizal associations, either
endomycorrhizal or ectomycorrhizal, than are species with ﬁnely branched roots and
numerous root hairs. The primary exception to the Baylis hypothesis is the family
Ericaceae, the species of which have very ﬁnely branched roots in addition to large
numbers of hyphal connections with ericacious endomycorrhizal fungi (Harley and Smith,
1983). Despite this exception, Baylis’ connection between root anatomy and mycorrhizal
propensity is a widely accepted generalization (Manjunath and Habte, 1991).

Many plants release metabolically active border cells (BC) from the root cap into
the rhizosphere (Hawes, 1990; Hawes and Lin, 1990). These cells undergo
differentiation upon separation from the root, including distinct patterns of gene

expression (Brigham et al. , 1995b). Border cells (previously referred to as "sloughed root

 

90

cap cells") are deﬁned as those cells that are released into suspension by a brief
immersion into water (Hawes and Brigham, 1992). Different plant families vary
considerably in their capacity to produce BC, ranging from zero BC per root (e.g.
Brassicaceae and Chenopodiaceae) to thousands of BC per root (e. g. Fabaceae and
Cucurbitaceae) (Hawes and Pueppke, 1986). BC production is consistent between species

within a given family (Hawes and Pueppke, 1986), typically varying between 10-20%.

Materials and Methods
Reference list collection

Hawes and Pueppke (1986) determined the BC production levels of a number of
species, from a variety of families. Brigham et al. ,( 1995b) presented the average BC
production for a variety of families. In these studies, BC production was measured in
aseptic, newly-germinated seedlings. I surveyed mycorrhizal literature to collect AM
colonization data for over 40 plant species. I associated the published AM colonization
data for many of the species with the average BC production data of its family; this is
appropriate, given the comparatively low variability of BC production within families
(Hawes and Pueppke, 1986; Brigham et al.,1995a). Wherever possible, however, I
associated the AM colonization data with BC production values determined for that
particular species (Hawes and Pueppke, 1986). The AM colonization references used
were taken for consistency of data collection technique and for the breadth of species
tested, rather than for any other criteria. The growth conditions under which the AM
colonization data were collected varied; this broad based data set is speciﬁcally desirable

in order to fully test the suggestion of an AM-BC correlation.

91

Statistical analysis
The data from the six families best represented in the data set (Fabaceae, Poaceae,
Amaranthaceae, Solanaceae, Brassicaceae, Chenopodiaceae) were subjected to cluster

analysis using the statistical program NCSS.

Results
AM colonization, BC production

The collection of data is presented in Table A. 1. The AM percent colonization
in this collection of references refers to the percent of AM colonized root fragments as
determined by the line intersect method, a widely used measure of AM association
(Brundrett and McGonigle, 1994).
Cluster analysis

Cluster analysis of the plot of AM colonization % vs. the log transform of the
number of BC produced per root places the data into distinct clusters (Figure A. 1).
These clusters correspond to families which are minimally- or non-mycorrhizal,

moderately mycorrhizal and highly mycorrhizal.

Discussion

The data from the literature suggests a connection between a family’s AM
propensity and its capacity to produce BC. The production of BC appears to be strongly
correlated with mycorrhizal colonization. In general, families that were BC capable had
a mycorrhizal propensity, while families that were not BC capable were minimally- or

non-mycorrhizal (e. g. Brassicaceae and the Chenopodiaceae). The data used to formulate

92

Table A. 1. Arbuscular mycorrhizal (AM) colonization and border cell (BC)
production.

3 AM% is percent root colonization.

b BC: Border cell production is in number of cells released per root. Border cell
production data is species speciﬁc (*, from Hawes and Pueppke, 1986) or the
average for the family (**, from Brigham et al. , 1995a).

93

 

Family Species AM BCb Ref.
% a
Fabaceae Glycine max 77 3260* Skipper and
Struble (b)
Fabaceae Glycine max 63 3260* Pacovsky, et al.
Fabaceae Glycine max 63 3260* Skipper and
Struble (a)
Fabaceae Glycine max 47 3260* Zambolim and
Schenck
Cucurbitaceae Cucumis sativus 69 3070* Pearson and
J akobsen
Fabaceae Phaseolus vulgaris 70 3070* Kucey, et al.
Fabaceae Phaseolus vulgaris 58 3070* Daft and El-
Giahami
Fabaceae Phaseolus vulgaris 20 3070* Kruckleman
Fabaceae Coronilla varia 85 3000** Daft, et al.
Fabaceae Robinia hispida 44 3000** Daft, et al.
Fabaceae Robinia 66 3000** Daft, et al.
pseudacacia
Fabaceae T rifolium repens 88 3000** Kruckleman
Fabaceae Vicia sativa 98 3000** Kruckleman
Malvaceae Gossypium hirsutim 69 3000* Smith and
Roncadori
Malvaceae Gossypium hirsutim 55 3000* Price, et al.
Fabaceae Pisum sativum 70 2680* Jakobsen and
Nielsen
Poaceae Zea mays 73 2350* Kruckleman
Poaceae Zea mays 20 2350* Daniels-
Hetrick, et al.
Poaceae Agrostis tennuis 41 2150** Daft, et al.
Poaceae Dactylis glomerata 38 2150** Daft, et al.

Poaceae

Poaceae
Poaceae
Poaceae
Poaceae

Poaceae

Poaceae
Poaceae
Poaceae
Poaceae

Poaceae

Poaceae

Poaceae

Poaceae

Poaceae

Poaceae
Agavaceae
Amaranthaceae
Amaranthaceae
Amaranthaceae
Amaranthaceae

Amaranthaceae

Amaranthaceae

Amaranthaceae

94

F estuca
arudinaceae

F estuca ovina
Holcus lanatas
Lolium perenne
Poa annum
Poa compressa
Avena sativa
Avena sativa
Avena sativa
Secale cereale

Secale cereale

T riticum aestivum

Triticum aestivum

T riticum aestivum

Triti cum aestivum

T riticum aestivum
Yucca baccata
Achyranthes aspera
Achyranthes aspera
Aerva javanica

Altemanthera
sessilis

Amaranthus
caudatus

Amaranthus
caudatus

Amaranthus
crucutus

62

41
84
46
30
13
78
48
28
41
35

76
70

68
50

40
45
34
15

2150**

2150**
2150**
2150**
2150**
2150**
1645*
1645*
1645*
1485*
1485*

1195*
1195*

1195*
1195*

1195*

1050**

140**
140**
140**
140**

140**

140**

140**

Daft, et al.

Daft, et al.
Daft, et al.
Daft, et a1.
Kruckleman
Daft, et al.
Strzemska
Kruckleman
Wang, et al.
Strzemska

Jakobsen and
Nielsen

Strzemska

Dodd and
Jefferies

Dodd, et al.

Jakobsen and
Nielsen

Kruckleman
Daft, et al.
Neeraj, et al.
Neeraj, et al.
Neeraj, et al.

Neeraj, et al.

Neeraj, et al.

Neeraj, et al.

Neeraj, et al.

Amaranthaceae
Amaranthaceae

Amaranthaceae

Amaranthaceae
Amaranthaceae
Amaranthaceae
Amaranthaceae

Solanaceae

Solanaceae
Solanaceae
Solanaceae

Solanaceae

Solanaceae

Brassicaceae

Brassicaceae

Brassicaceae

Brassicaceae
Chenopodiaceae

Chenopodiaceae

Chenopodiaceae

95

Amaranthus gracilis
Amaranthus persica

Amaranthus
spinosus

Celosia argentia
Celosia argentia
Celosia cristata
Celosia cristata

Capsicum annum

Solanum nigrum
Solanum tuberosum
Solanum tuberosum

Lycopersicon
esculentum

Lycopersicon
esculentum

Arabidopsis
thaliana

Brassica napus

Capsella
bursa-pastoris

Raphanus vulgaris
Beta vulgaris

Chenopodium
album

Spinacia oleracea

47
12
35

60
26
46
4 1
38

57
28
25
48

42

15

140**
140**
140**

140**
140**
133*
133*
63*

55**
55**

55**
16*

16*

O**

O**
0*III

0*!!!
O**
0**

0101!

Neeraj, et al.
Neeraj, et al.

Neeraj, et al.

Neeraj, et al.
Neeraj, et al.
Neeraj, et al.
Neeraj, et al.

Haas and
Krikun

Kruckleman
Wang, et al.
Kruckleman
Kruckleman

Caron, et al.
Kruckleman

Dodd, et al.
Kruckleman

Kruckleman
Kruckleman
Kruckleman

Kruckleman

 

96

Figure A. 1. Cluster analysis of the major families represented in the data set
(Fabaceae, Poaceae, Amaranthaceae, Solanaceae, Brassicaceae,
Chenopodiaceae), log(BC) vs. AM colonization. Patterns represent distinct
clusters.

AM colonization (%)

110

97

 

100 —

90-

80—

70—

60—

50—

40—.

30—

 

 

 

 

 

 

 

C) Fabaceae
Cl Poaceae
A Amaranthaceae
V Solanaceae
O Brassicaceae
<> Chenopodiaceae
A
V A
V A
l I l I l l T
0.5 1.0 1.5 2.0 2.5 3.0 3.5

log(BC)

 

4.0

98

this hypothesis also seemed to suggest that the range of AM propensity may follow the
range of BC capability. This is supported by cluster analysis, which shows that distinct
clusters corresponding to intersections of AM propensity/BC capability. Thus, plants that
produce larger numbers of BC appeared to have a greater mycorrhizal propensity, while
plants that produce fewer BC appeared to have a lesser mycorrhizal propensity.

Species in the family Amaranthaceae were shown to produce BC on the order of
hundreds of BC per root, comparable with the established mycorrhizal family Solanaceae
(Brigham et al. ,19953). On the basis of increasing evidence, AM colonization of species
in this family is more widespread than was previously believed (Saif and Iffat, 1976; Saif
et al.,1977; Neeraj et al.,1991). An early review of the mycorrhizal status of plants
(Gerdemann, 1968) included the Amaranthaceae in a list of families that were considered
to be "possibly non-mycorrhizal or rarely mycorrhizal". Gerdemann’s review (1968)
incorrectly cited Koch (1961) as the basis for this assertion, because Koch (1961) did not
mention the Amaranthaceae. In light of the most recent data regarding the mycorrhizal
status of the Amaranthaceae, this example of BC capability in a mycorrhizal family,
previously thought to be non-mycorrhizal, tends to support this hypothesis.

The family Pinaceae also is of particular interest among the families surveyed for
BC production (Hawes and Pueppke, 1986; Brigham et al. ,1995a). Species in the family
Pinaceae were determined to produce 3000 to 5500 BC per root (Hawes and Pueppke,
1986). The species in this family are known to exhibit a characteristically obligate
dependence on ectomycorrhizal fungi in nature (Harley and Smith, 1983). This example
of BC capability in a family with a strong ectomycorrhizal propensity suggests that our

hypothesis may possibly be broadened to include ectomycorrhizal, as well as AM

99

interactions. However, the larger data set of BC production by ectomycorrhizal plant
species needed to reliably expand this hypothesis is lacking.

There are other interesting physiological points about a possible AM-BC
connection. Many plant-produced compounds are known to have a profound impact on
soil pathogens, even at very low concentrations (Morris and Ward, 1992; Vedenyapina
et al. , 1996). Upon dissociation from the root, BC generate a novel complement of gene
products that are rapidly released into the external medium (Brigham et al. ,1995b). The
area of the rhizosphere distal to the root cap where the majority of BC are distributed is
also the primary site of root penetration by AM and by a variety of important fungal root
pathogens (Agrios, 1988; Harley and Smith, 1983; Hawes, 1990; Hawes and Brigham,
1992). BC have been shown to be speciﬁcally chemoattractive to pythiaceaous fungi
(Goldberg et al.,1989). Thus, I speculate that BC-produced bioactive compounds may
inﬂuence the behavior of the mycorrhizal population in the rhizosphere. Conventional
root studies probably exclude the BC, and therefore exclude from consideration gene
products generated exclusively in the BC (Hawes and Pueppke, 1986). The hypothesis
put forward by Brigham et al. , (Brigham et al. , 1995b) that BC constitute a uniquely
specialized tissue of the root system, may have important implications for the role of BC-
produced compounds in the establishment of mycorrhizal symbioses, as well as in the
course of development of root pathogens.

This is, to my knowledge, the ﬁrst discussion of a positive association of AM
colonization with BC production. This hypothesis, that mycorrhizal colonization is
correlated with BC production, should be evaluated by further experimentation. These

experiments could include i) a more complete determination of the mycorrhizal status of

100

species in the family Amaranthaceae; ii) measurement of BC production by species in
veriﬁably non-mycorrhizal families other than Brassicaceae and Chenopodiaceae; iii)
measurement of BC production by species in Ericaceae, a family with a strong ericoid
endomycorrhizal propensity; iv) measurement of BC production by other species in
Pinaceae, a family with a strong ectomycorrhizal propensity; and v) measurement of the
AM propensity of mutants of characteristically mycorrhizal species which have been
rendered incapable or less capable of producing BC. Preliminary results of ongoing
experiments to determine the AM propensity of species in Amaranthaceae (Celosia
cristata, Gomphrena globosa, Amaranthus tricolor, Amaranthus caudatus) suggest that
these species are fully capable of sustaining an AM association (unpublished results).
The overall physiological signiﬁcance of BC has not been fully elucidated, nor
has the role of BC-produced compounds. Hopefully, an evaluation of this hypothesis will
lead to a greater understanding of the role that BC and BC-produced compounds may

play in the establishment of mycorrhizal associations.

101

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