network formation by rhizomorphs of armillaria lutea in natural soil: their description and...

R E S E A R C H A R T I C L E

Network formation by rhizomorphsofArmillaria lutea in naturalsoil: their descriptionand ecological signi¢canceAngelique Lamour1, Aad J. Termorshuizen1, Dine Volker1 & Michael J. Jeger2

1Biological Farming Systems, Wageningen University and Research Centre, Wageningen, The Netherlands; and 2Division of Biology, Imperial College

London, Wye Campus, Wye, Ashford, Kent UK

Correspondence: Michael J. Jeger, Division

of Biology, Imperial College London, Wye

Campus, Wye, Ashford, Kent TN25 5AH, UK.

Tel.: 144 207 5942719; fax: 144 207

5942601; e-mail: [email protected]

Received 10 January 2006; revised 8 May 2007;

accepted 8 May 2007.

First published online 20 July 2007.

DOI:10.1111/j.1574-6941.2007.00358.x

Editor: Jim Prosser

Keywords

Armillaria rhizomorphs; network structure;

graph theory; connectedness; ecological

persistence; robustness.

Abstract

Armillaria lutea rhizomorphs in soil were mapped over areas of 25 m2 at a Pinus

nigra (site I) and a Picea abies (site II) plantation. Rhizomorph density was 4.3 and

6.1 m m�2 soil surface with 84% and 48% of the total rhizomorph length in the

mapped area interconnected in a network at site I and site II, respectively. At site I

there were only two network attachments to Pinus stumps, but at site II many more

to Picea roots and stumps. Anastomoses of rhizomorphs resulted in cyclic paths,

parts of the network that start and end at the same point. Connections between

different rhizomorph segments were shown to allow gaseous exchange. The

network at site I consisted of 169 rhizomorphs (‘edges’), and 107 rhizomorph

nodes (‘vertices’). Disruption of two critical edges (‘bridges’) would lead to large

parts (13% and 11%) being disconnected from the remainder of the mapped

network. There was a low probability that amputation of a randomly chosen edge

would separate the network into two disconnected components. The high level of

connectedness may enhance redistribution of nutrients and provide a robust

rhizomorph structure, allowing Armillaria to respond opportunistically to spa-

tially and temporally changing environments.

Introduction

The clonal dispersal of plant pathogenic Armillaria species

occurs in temperate climatic zones by growth through soil of

specialized strands, called rhizomorphs. These shoestring-

like strands are 1–3 mm in diameter with a reddish brown to

black outer cortex layer (Cairney et al., 1988) usually in the

upper 30 cm soil layer (Redfern, 1973). Clones thus formed

may persist over centuries and may be of impressive size

(Smith et al., 1992; Ferguson et al., 2003) if there continue to

be sufficient sources of nutrition for absorption (Rizzo et al.,

1992) and translocation (Granlund et al., 1985; Cairney

et al., 1988; Gray et al., 1996) under turgor pressure (Eamus

& Jennings, 1984). Although rhizomorphs are in general

insulated from the environment the peripheral hyphae may

act as organs of nutrient uptake (Pareek et al., 2001) with

oxygen diffusing through a central gas-filled cavity (Pareek

et al., 2006). Contact of rhizomorphs with tree roots can

result in tree-to-tree spread of the fungus, even when direct

contact between diseased and healthy roots is not made. In

some species rhizomorphs grow epiphytically along roots

(Baumgartner & Rizzo, 2001). Rhizomorphs are produced

during the various stages of wood decay, but the extent of

growth is species-dependent and influenced by habitat and

environmental conditions and the presence of secondary

colonizers (e.g. Prospero et al., 2006).

Networks of fungal hyphae growing in pure culture

(Mihail & Bruhn, 2005) and soil microcosms (Bolton &

Boddy, 1993; Harris & Boddy, 2005) have been described in

terms of nutrient translocation (Watkinson et al., 2005) and

growth strategies in relation to grazing (Kampichler et al.,

2004), but fungal networks in undisturbed ecosystems have

been mapped only rarely (Thompson & Rayner, 1983).

Although the rhizomorph growth habits of 15 species of

Armillaria were described following the placement of inocu-

lum segments in small volumes of soil in plastic bags

(Morrison, 2004), the ecological relevance of Armillaria

rhizomorph networks has only partially been appreciated.

It is generally recognized that fungal networks occur com-

monly and their existence is of ecological relevance, e.g. for

translocation of nutrients and carbon by mycorrhizal fungi

(Leake et al., 2004), but quantitative tools to analyse fungal

networks have not been developed. In this study, we mapped

a rhizomorph network of Armillaria lutea in natural soil at

FEMS Microbiol Ecol 62 (2007) 222–232c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

two sites and analysed characteristics of the networks in

terms of foraging strategies (Dowson et al., 1989) and the

persistence of networks in time and space using graph-

theoretical concepts (Harary, 1969; Wilson, 1979).

In graph theory, the term ‘network’ is used in a technical

sense to mean a type of diagram, called a ‘graph’, to which

the numerical values of some quantity are attached. A graph

consists of a set of points or nodes, called ‘vertices’, and

connections between them called ‘edges’. A graph is ‘con-

nected’ if there is a ‘path’ (sequence of edges) that connects

every vertex in the graph. It is ‘disconnected’ if there is no

such path. A ‘cycle’ within a graph is a path that starts and

ends at the same point. We identify the branching or fusion

of rhizomorphs at a point in the network as a vertex, and

a rhizomorph connecting two vertices as an edge. Transport

of, for example, nutrients from one vertex to another is

determined by the presence or absence of edges, as nutrients

flow easily through the medulla of rhizomorphs (Granlund

et al., 1984). If a rhizomorph is removed, transport of

nutrients between two vertices is not prevented if these

vertices are connected by more than one rhizomorph. The

significance of vertices is that they bring flexibility to the

rhizomorph system, as multiple edges result in more ways to

transport nutrients.

Although the mathematical concepts of graph theory

have been widely applied, there have been few examples of

applications in population biology until relatively recently.

Network theory has been applied to networks at the gene

and protein level and increasingly existing techniques are

being applied to ecological systems (Proulx et al., 2005). A

particular theoretical question concerns the architecture of

biological networks. Recently, Southworth et al. (2005)

analysed Quercus garryana–mycorrhizal associations (20

trees/40 fungal morphotypes network) using graph-theore-

tic concepts. They concluded that all trees had about the

same linking to fungal morphotypes in the network, but that

certain morphotypes, e.g. Cenococcum geophilum, had more

links to trees than did other morphotypes. However, the

authors had no direct evidence of physical sharing of

resources through these links. In this study we apply graph

theory to an analysis of the rhizomorph network and discuss

an ecological interpretation.

Materials and methods

Mapping of rhizomorph networks

In two c. 40-year-old tree plantations (site I: Pinus nigra ssp.

maritima; site II: Picea abies) near Wageningen, the Nether-

lands, we prepared maps of rhizomorphs of A. lutea in soil

over a plot area of 25 m2. The plantation size at both sites

was c. 1 ha at an elevation of 30 m above sea level, with

horizontal aspect. The Pinus site had a dense shrub layer

consisting entirely of Prunus serotina. In the c. 10 years

before the study was undertaken both shrubs and pine trees

were heavily attacked by Armillaria, but in the sampling year

this was less so for Pr. serotina. Furthermore the Pinus site

had a moderate dense herb layer of Deschampsia flexuosa. In

the Picea plantation there was neither a shrub nor a herb

layer. Although both sites were situated on Pleistocene

moraine sand, the Picea plot was slightly podzolic, whereas

the Pinus plot was not. At each site the soil and surface litter

was hand-removed up to c. 25 cm depth and rhizomorphs

were located in 1 m2 grids and drawn on a two-dimensional

map at a scale of 1 : 10. The depth was not recorded because

this was small compared with the surface dimensions.

Isolates of the rhizomorphs from both sites were identified

by A. Perez-Sierra (Royal Horticultural Society, Wisley, UK)

as representing A. lutea Gillet [= Armillaria gallica Marxm.

and Romagn. = Armillaria bulbosa (Barla) Kile and Watl.]

with PCR–restriction fragment length polymorphism of the

IGS-region of the rRNA gene using species-specific primers

(Anderson & Stasovski, 1992; Chillali et al., 1997).

Observation of internal connectedness ofrhizomorph anastomoses

Anastomoses of rhizomorphs were frequently observed. To

investigate whether fused rhizomorph segments were intern-

ally connected or not, air was forced through water-immersed

rhizomorphs at one end and the occurrence of air bubbles

was observed distally beyond the point of fusion. For X-ray

microscopy (Skyscan-1072 desktop X-ray microtomograph),

two rhizomorph segments fused by anastomosis were cut

with a sharp blade to a length of c. 2 mm. The combination of

X-ray transmission technique with tomographical recon-

struction gave three-dimensional information about the

internal microstructure, constructed as a set of flat cross-

sections. Photographs were taken at 21 heights (steps of

0.091 mm), starting above the point where the rhizomorph

segments were fused, and ending below this point.

Decay time of dead rhizomorphs

The decay time of dead rhizomorphs was monitored to

determine whether dead remnants of Armillaria would be

present and mapped at the two sites. Rhizomorphs from site

I were killed by gamma irradiation (25 kGy), and 10 cm

pieces were incubated in pots containing forest soil of low

(–24.6 kPa) or high (� 3.9 kPa) water potential. Prior to

incubation, soil was air-dried for 1 week and sieved through

a 1.0 mm mesh. The fresh weight of the rhizomorph pieces

per pot was recorded after washing them with water and

drying between filter paper. To estimate their dry weight, the

water content of additional fresh rhizomorph pieces was

determined. The incubation temperatures were high (20 1C)

or low (10 1C), roughly encompassing the range of soil

FEMS Microbiol Ecol 62 (2007) 222–232 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

223Armillaria rhizomorph networks

temperatures over the spring to autumn period. A soil

temperature typical of winter was not used because of low

microbial activity and decay rates. At four harvest times (4,

10, 16 and 30 weeks), the soil was sieved through a 1.0 mm

mesh under tap water. The dry weight of the remaining

rhizomorph pieces was determined after 24 h at 105 1C, and

the percentage dry weight loss was used as a measure of the

state of decay (three replicates per treatment). Similarly, in

December 1997 two dead 10 cm rhizomorph pieces were put

in each of 24 nets (mesh size of 1.1 mm) containing sieved

forest soil. The nets were buried in the forest soil at a depth

of 5–10 cm and after 4, 10, 16 and 30 weeks the nets were

recovered and the dry weight of the remaining rhizomorph

pieces determined (six replicate nets on each occasion).

Results

Mapping of rhizomorph networks

Total rhizomorph length in the observed area was 109 (site I)

and 152 m (site II). At several places interconnected rhizo-

morphs (black lines in Fig. 1) crossed the boundary of the

mapped area, indicating that the rhizomorph system ex-

tended beyond the observed areas. Cyclic paths, parts of the

network that start and end at the same point, were observed

as the result of branching and subsequent anastomoses

between rhizomorph segments. In many cases, larger cycles

were embracing or closely connected to one or more smaller

cycles (e.g. the larger cycle A connected to the smaller cycle B

in Fig. 1a). Also, many small cycles were produced at this

finer scale (Fig. 2a), giving rise to a complex network

structure.

The largest connected component can readily be visua-

lized by reducing the mapped rhizomorph system to the

cyclic paths (Fig. 1, red lines). In a number of cases several

small cycles occurred closely together within the rhizo-

morph system (e.g. at C in Fig. 1a) but in other cases cycles

were larger and simpler in form (e.g. at D in Fig. 1a). At site

I, the largest connected component within the mapped area

was attached only twice to a dead stump of P. serotina,

although attachments may occur outside this area. At site II,

many more attachments to Picea stumps and roots were

observed � 73 in total. Also, at site II there were 253

rhizomorphs not being part of the largest connected com-

ponent, which were attached to tree roots and less than a few

centimetres in length.

Observation of internal connectedness ofrhizomorph anastomoses

Forcing air through one end of the rhizomorph segment

showed air bubbles at the distal end beyond the point of

fusion (Fig. 2b), indicating a continuity of air space between

the rhizomorphs. This was confirmed by X-ray cross-section

analysis of two fused rhizomorph segments (Fig. 2c). Of the

21 images taken at decreasing heights, the middle one

demonstrated clearly the presence of a continuum between

the two segments (Fig. 2d).

Decay time of dead rhizomorphs

Dead rhizomorph remnants that had decayed under con-

trolled conditions, or when buried in the forest soil, for 30

weeks were reduced to many small brittle pieces, which were

Fig. 1. Rhizomorph network at site I (a) and site II (b). Red lines: rhizomorphs contributing to the cyclic paths of the largest connected component. Black

lines: rhizomorphs connected to the cyclic paths. Blue lines: other rhizomorphs. Open squares: crossings of rhizomorphs without anastomosis. Green

dotted circles: Prunus serotina shrubs. Green hatched circles: tree plantation stems. Green lines: larger tree roots. Purple dots: points of attachment to

trees or roots. Letters refer to descriptions in the text.


224 A. Lamour et al.

hollow or reduced to spiral melanin sheaths. Such deterio-

rated pieces of rhizomorph were never encountered in the

network mapping at either site. Even by 16 weeks rhizo-

morph remnants occasionally had broken down into smaller

parts, but these retained some physical integrity and were

recorded. High temperature and high soil water potential

appeared to favour decay of dead rhizomorphs, but only soil

water potential affected weight loss (Fig. 3) significantly

(Po 0.01), and then only at 4 and 30 weeks.

Analysis of Rhizomorph networks

The mapping of the rhizomorph network in the observed

area at site I (Fig. 1a), here referred to as graph G1, is most

likely a ‘directed graph’, i.e. a graph in which arrows, single

or in both directions, can be assigned to the edges, if this

information is available. Flow of nutrients through rhizo-

morphs and mycelial cord systems can be simultaneously

bidirectional (Granlund et al., 1985; Cairney, 1992, 2005;

Gray et al., 1996; Olsson & Gray, 1998). However, for a given

nutrient at a given time and place in a rhizomorph network

the net flow may be one-directional, and indeed switch in

response to nutritional or other environmental cues. G1 is

not a connected graph but it can be expressed as the union

of connected graphs, each of which is defined to be a

‘component’ of G1. Thus, in G1 (Fig. 1a) the blue edges are

not connected to the red or black edges. The largest

connected component of G1 forms 84% of the total graph.

Focusing now only on this largest connected component

(red line in Fig. 1a), a topologically equivalent ‘planar’ graph

G2 (Fig. 4) can be constructed for ease of visualization in

which edges do not cross. G2 lies entirely within the

boundary of the mapped area.

Graph G2 (Fig. 4) consists of 107 vertices (n) and 169

edges (m). The degree of a vertex, d(v), is the number of

edges incident with this vertex, which mostly equals 3 (83%

of vertices) as a result of simple branching, but 17 vertices

(16%) have degree 4 (i.e. vertices 16, 20, 28, 30, 31, 41, 48,

59, 61–64, 74, 76, 77, 85 and 106). Although degree-4

Fig. 2. Rhizomorph connections. (a) Photograph of rhizomorphs of

Armillaria lutea showing various connections. (b) Demonstration of the

presence of a continuum between connected rhizomorphs. Air was

pressed through one end of the rhizomorph (above right) and air bubbles

were observed at the low left end of the rhizomorph (arrow). (c) Two

fused rhizomorph segments (2 mm each). (d) X-ray cross-section of the

rhizomorph connection depicted in (c). The outer black line is the

melanin sheath of the rhizomorphs. One rhizomorph is represented on

the left side by the vertical tube, and the other perpendicularly crossing

rhizomorph is represented by the right semicircle. For all photographs,

the diameter of the widest rhizomorphs is c. 3 mm.

Fig. 3. Decrease in dry weight of rhizomorph segments at four harvest

times (4, 10, 16 and 30 weeks) for different treatments incubated under

controlled conditions at a low (� 24.6 kPa) and high (� 3.9 kPa) soil water

potential and at 10 and 20 1C; and also incubated in forest soil.



vertices may be the result of multiple branching at one node,

our observations point to the possibility of anastomosis

of two crossing rhizomorphs. A ‘simple’ graph is a graph

without multiple edges between vertices and without loops,

where a ‘loop’ is an edge from a single vertex to itself. G2 is

not a simple graph, as it has 23 double edges, for example

between vertices 83 and 84, and five loops, for example at

vertex 107 (Fig. 4).

Fig. 4. Graph G2, which is a planar graph of G1 (red lines in Fig. 1a) in which the 107 vertices (bold) and 169 edges have been numbered. In a planar

graph, edges do not cross when drawn in the plane.



Connectedness is a basic characteristic of a network. The

degree to which the rhizomorph network is connected may

have ecological implications: a strongly connected network

will experience only small consequences from the amputa-

tion of rhizomorphs. The connectedness of a network can be

determined by calculating the number of edges that must be

removed in order to disconnect the graph. If removal of one

edge results in a disconnected graph then such an edge is

called a ‘bridge’ and, by inspection, occurs in G2 nine times,

namely edges 62, 65, 68, 73, 78, 81, 155, 163 and 168.

Disruption of bridges connecting two large parts of the

network has a major impact on the whole network. So,

amputation of rhizomorph 78 would disconnect 13% of the

rhizomorph system, based on number of vertices. For

rhizomorph 81 this percentage is similar (11%), but disrup-

tion of one of the other seven bridges would disconnect only

1–4% of the network. Thus, disruption of a bridge in the

original connected graph G2 gives rise to two connected

graphs (components), each with their own network proper-

ties. Depending on the location of resources, if a network is

separated by disruption (either of a bridge or a set of edges)

into two components of the same size, both components are

likely to persist, but if one is small there will only be minor

benefits of nutrient redistribution and the small component

may have little chance to persist. In G2 (n = 107) the removal

of edges 26, 150, 148 and 112 disconnects the graph into two

components of about the same size, containing 52 and 55

vertices.

In a planar graph the two-dimensional regions bounded

by the edges in the graph are called ‘faces’. Euler’s formula

(Wilson, 1979) states that for a connected graph

n�m1f = 2, where f is the number of faces. In G2 the

number of faces equals m� n12 = 169–10712 = 64. The

degree of a face, d(F), is defined as the number of edges on

the boundary of that face. A large face-degree may relate to a

large region where Armillaria has not foraged. A few faces

have a very large degree, but the median value is 3 (Fig. 5).

The frequency distribution of vertex-degrees has been used

to characterize different types of networks and has been

applied to a mycorrhizal association network (Southworth

et al., 2005). However, for the Armillaria rhizomorph net-

works the vertex degrees were either 3 or 4, which precludes

this form of analysis for vertices.

For a cycle in a connected graph, removal of any one edge

will still result in a connected graph. If this procedure is then

repeated with one of the remaining cycles, continuing until

there are no cycles left, then the graph that remains is still

one that connects all the vertices. This graph is called a

‘spanning tree’. The ‘matrix-tree theorem’ (Harary, 1969)

can be used to calculate the number of spanning trees in any

connected simple graph, obtained by removing any multiple

edges and loops. The number of spanning trees in G2 equals

5.6� 1021 and one spanning tree G3 is shown in Fig. 6. Due

to this high number of possible spanning trees, the prob-

ability that removal of a randomly chosen edge disconnects

the network into two components is infinitesimally small.

The number of spanning trees of a network may be inter-

preted as a measure of the robustness of the network, which

in turn could be related to ecological persistence, the ability

of the network to respond to spatially and temporally

changing environments.

In the rhizomorph mapping (Fig. 1a) a rhizomorph was

attached in only two instances to a root or stump, serving as

a nutrient source. The first source (S1) is attached to vertex

81 and the second source (S2) is attached to edge 44. Thus,

G2, the largest component of the rhizomorph network, is

attached to both nutrient sources, but disruption of these

two rhizomorphs would be sufficient to remove G2 from the

sources within the observed area. Disruption of rhizo-

morphs which cross the boundary of the observed area

would remove the mapped system from sources outside the

mapped area. The geographical distance to the furthest

vertex in G2 (vertex 62) measures 4.19 m from S1 and

4.56 m from S2; the distance between S1 and S2 is 2.2 m.

Assuming that all parts of the rhizomorph network need

access to nutrients, the distance over which the nutrients

and water have to be transported in relation to a source is

important and related to network structure. In a connected

graph, the ‘distance’ d(vi, vj) from vertex i to vertex j is the

length of a ‘shortest path’ from vertex i to vertex j. Here,

length is expressed in number of edges traversed rather than

the physical length (m) of edges. For example, vertex 81 is

connected to source S1 in graph G2 (Fig. 4); the number of

edges traversed in a shortest path from vertex 81 to vertex 62

is 15. There are 16 such paths, one of which, as an example,

follows the vertex sequence: 81 ! 89 ! 90 ! 91 ! 92

! 93 ! 94 ! 95 ! 66 ! 51 ! 52 ! 53 ! 54 ! 65

! 63 ! 62.

d(F)

0 8070605040302010

num

ber

of c

ases

0

5

10

15

20

25

Fig. 5. Frequency distribution of the degrees of faces, d(F), occurring in

graph G2 (Fig. 4). The degree of a face is defined as the number of edges

on the boundary of a face.



Fig. 6. The graph G3 is one of the spanning trees of G2 (Fig. 4). Application of Prim’s algorithm (Prim, 1957) shows that G3 is the minimal spanning tree,

namely the one with minimum weight across all edges.



Using the line intersection method developed for measur-

ing the length of root in a sample (Newman, 1966; Marsh,

1971), the length of each rhizomorph in G1 (the red lines in

Fig. 1a) was measured and assigned to graph G2 (Fig. 7).

Such a graph is then formally a ‘network’, and the number

assigned to each edge e is the ‘weight’ of e, in this case length

Fig. 7. The length of each rhizomorph segment, rounded to the nearest integer (cm), is assigned to each edge of the graph G2 (Fig. 4). The length of

each segment provides the weight assigned to each edge of G2.



in metres. The shortest path from vertex i to vertex j is then

the path with minimum total weight. A frequently used

algorithm is the one from Dijkstra (1959). The shortest path

from vertex 81 to vertex 62 measures 7.29 m and has edge

sequence: 126 (or 127) ! 125 ! 124 ! 120 ! 117 !115! 114! 112! 110! 109! 105! 103! 102!78! 80! 81! 101! 98! 96. This procedure can be

applied to each path in a spanning tree. The spanning tree that

minimizes the total length of the edges is termed a ‘minimal’

spanning tree. To construct a spanning tree with this property

the algorithm of Prim (1957) may be used. Using this algorithm

the spanning tree G3 (Fig. 6) is obtained as the minimal

spanning tree with a length of 24.98 m, which is 58% of the

total length of G2.

Discussion

Rhizomorphs of A. lutea at two locations in a natural forest

soil occurred in the form of networks. Outgrowth of

isolations made for identification of the Armillaria species

indicated that most rhizomorphs were viable. Accompany-

ing experiments indicated that the mapped rhizomorphs

were either alive or had died within a 16–30-week period

before the time of observations, and we are confident that

intact but dead rhizomorph remnants were not observed

extensively in the mapping of the networks.

In Armillaria, explorative growth is accomplished

through the formation of rhizomorphs, which are well

suited to this because of their insulation from the environ-

ment (Rayner et al., 1994). The ability of Armillaria to form

rhizomorph networks through anastomoses, as demon-

strated in our study, also reflects a strategy of persistence

(Reaves et al., 1993). Persistence is achieved through features

such as the number of spanning trees conferring robustness

to the network structure in relation to disturbance. Rhizo-

morph networks enable the search for nutrients in time as

well as exploration in space and their robustness would be of

particular value for Armillaria at the soil leaf–litter layer

interface, where rhizomorphs are frequently found and

which is often disturbed. The high frequency of cyclic

paths may limit the effects of unsuccessful exploration by

enabling the redistribution of nutrients within the network

and reducing the chance of a disconnected system when a

rhizomorph is amputated. Thus, the occurrence of cyclic

paths may explain in part the high age of some clones of

A. lutea (Smith et al., 1992). A robust network is one that is

not substantially weakened when one edge is disconnected

from part of the network. Graph G2 (Fig. 4) contains nine

bridges, of which amputation of any one of two of them

(edge 78 or 81) would disconnect a considerable part of the

rhizomorph system (based on the number of vertices).

Amputation of one of the other seven bridges would

disconnect only 1–4% of the original network (G2); if these

disconnected components were explorative parts of the

network they may not subsequently persist. The impact of

such disruption is lower on a densely connected network, for

which the number of possible spanning trees is high, as in

G2, than in a sparsely connected one.

The substrate of A. lutea, a weak pathogen and sapro-

troph (Rishbeth, 1982; Thompson & Boddy, 1983; Luisi

et al., 1996), probably consists of coarse and weakened

woody root material. At site I, both nutrient sources were

attached to the largest component of the rhizomorph net-

work, but disruption of the two rhizomorphs attached to

these nutrient sources would be sufficient to remove the

network from the sources, although there may be attach-

ments to other sources outside the mapped area. The

maintenance costs for the network are probably lower than

the costs for production of new rhizomorphs. We often

observed rhizomorphic growth against woody roots without

any sign of infection. This behaviour may be very similar to

the behaviour of Armillaria reported in tropical Africa

(Leach, 1939; Swift, 1972), where quiescent lesions on

woody host roots are common, ‘waiting’ until circumstances

are suitable for infection. Although Armillaria is able to

form basidiocarps, the success rate of basidiospores in

colonizing new substrate, e.g. freshly cut stumps, is extre-

mely low (Rishbeth, 1970; Termorshuizen, 2000), and there-

fore basidiospores contribute little to persistence.

Dense rhizomorphic networks may also occur at sites

where wood is or has been present, resulting in anastomosis

by rhizomorph encounters. For many Armillaria spp. there

is a good correlation between ectotrophic rhizomorph

abundance on root collars and wood and frequency in soil

(Marcais & Wargo, 2000; Lygis et al., 2005). Dense networks

of rhizomorphs of A. lutea, without differentiation into

individual hyphae, have often been observed on above-

ground parts of decomposing wood at these sites (A.J.

Termorshuizen, pers. commun.) and could occur below

ground for this saprotrophic species. In this type of network

formation, the length of the edges enclosing the faces would

be similar to the dimensions of host material, i.e. coarse

woody roots. Formation of anastomoses, and thus cyclic

paths, are more likely with growth of rhizomorphs over

living or dead roots, where there is a greater chance of

meeting other rhizomorphs, rather than by random encoun-

ters only. This would be true especially for very small cycles

(e.g. at C in Fig. 1a), which may arise from rhizomorphic

colonization of a single piece of root. However, these roots

were observed only in a few cases due possibly to differences

in rates of decay and persistence of roots and rhizomorphs.

Additionally, rhizomorphs that are not successful in attaining

food substrate and that are not part of cyclic paths would be

amputated relatively soon (Rayner, 1991).

The networks at site I and II showed some similarities,

including the total length of the cyclic paths, as well as some



differences (Table 1). The total rhizomorph system at site II

is 1.5 times longer than at site I, but the largest connected

component at site II is a twofold lower fraction of the total

rhizomorph system. We propose that the rhizomorph

system at site II is relatively young, showing many attach-

ments to Picea stumps and tree roots. The largest connected

component is characterized by many attachments to nutri-

ent sources. The rhizomorphs attaching the network to the

sources are assumed to be amputated when the food source

is exhausted. Also, young rhizomorphs with only a few

centimetres outgrowth from a nutritional source may have

little chance to persist if they fuse with a rhizomorph

network, by the time that the source is exhausted. Therefore,

an older network, as is assumed to occur at site I, is likely to

have fewer attachments to Pinus stumps and will have a

larger connected component and more cyclic paths.

In this study we have explored different possibilities for

graph-theoretic concepts to describe a rhizomorph network

and assist in the interpretation of growth strategies of

Armillaria. Our approach may be more generally valid for

those soil-borne mycelial fungi characterized by their ability

to form networks and provides a new tool to connect network

structure with function (Morris & Robertson, 2005). The

introduction of graph-theoretic properties to describe fungal

growth may lead to new insights in ecological understanding

of Armillaria rhizomorph networks in relatively undisturbed

environments. These networks provide a balance between

persistence and opportunism, a ‘wait-and-see’ strategy, in

relation to a changing biotic and abiotic environment.

Acknowledgements

We thank A. Perez-Sierra (Royal Horticultural Society, Wis-

ley, UK) for identification of the Armillaria isolates, Dr H.J.

Broersma (University of Twente, Enschede, The Netherlands)

for his helpful suggestions and comments in the development

of this paper, and anonymous reviewers for their helpful

comments on an earlier version of the manuscript.

References

Anderson JB & Stasovski E (1992) Molecular phylogeny of north-

ern hemisphere species of Armillaria. Mycologia 84: 505–516.

Baumgartner K & Rizzo DM (2001) Ecology of Armillaria spp. in

a mixed-hardwood forest of California. Plant Dis 85: 947–951.

Bolton RG & Boddy L (1993) Characterization of the spatial

aspects of foraging mycelial cord systems using fractal

geometry. Mycol Res 97: 762–768.

Cairney JWG (1992) Translocation of solutes in ectomycorrhizal

and saprotrophic rhizomorphs. Mycol Res 96: 135–141.

Cairney JWG (2005) Basidiomycete mycelia in forest soils:

dimensions, dynamics and roles in nutrient distribution.

Mycol Res 109: 7–20.

Cairney JWG, Jennings DH, Ratcliffe RG & Southon TE (1988)

The physiology of basidiomycete linear organs. II. Phosphate

uptake by rhizomorphs of Armillaria mellea. New Phytol 109:

327–333.

Chillali M, Idder-Ighill H, Guillaumin JJ, Mohammed C &

Botton B (1997) Species delimitation in the African Armillaria

complex by analysis of the ribosomal DNA spacers. J Gen Appl

Microbiol 43: 23–29.

Dijkstra EW (1959) A note on two problems in connection with

graphs. Numer Math 1: 269–271.

Dowson CG, Springham P, Rayner ADM & Boddy L (1989)

Resource relationships of foraging mycelial systems of

Phanerochaete velutina and Hypholoma fasciculare. New Phytol

111: 501–509.

Eamus D & Jennings DH (1984) Determination of water, solute

and turgor potentials of mycelium of various basidiomycete

fungi causing wood decay. J Exp Bot 35: 1782–1786.

Ferguson BA, Dreisbach TA, Parks CG, Filip GM & Schmitt CL

(2003) Coarse-scale population structure of pathogenic

Armillaria species in a mixed-conifer forest in the Blue

Mountains of northeast Oregon. Can J For Res 33: 612–623.

Granlund HI, Jennings DH & Veltkamp K (1984) Scanning

electron microscope studies of rhizomorphs of Armillaria

mellea. Nova Hed 39: 85–100.

Table 1. Characteristics of Rhizomorph Systems of Armillaria Lutea at two 25 m2 Plots within Plantations

Network characteristic Site I Site II

Total rhizomorph length 109 m (4.3� 0.4 m m�2) 152 m (6.1� 0.8 m m�2)

No. of short (o 5 cm) protrusions� 417 (16.7� 2.2 m�2) 491(19.6� 4.0 m�2)

No. of long (4 5 cm) protrusions 127 (5.1� 0.5 m�2) 268 (10.7� 3.5 m�2)

No. of cases that rhizomorphs crossed each other without anastomosis 90 (3.6� 0.7 m�2) 176 (7.0� 2.2 m�2)

Largest connected component 84% 48%

No. of cases that interconnected rhizomorphs left the mapped area 25 21

Total length of the cyclic paths 43 m 37 m

No. of cyclic paths 63 50

No. of connections of the largest connected component to stumps/rootsw 2 73

�Protrusions are dead-ending rhizomorph branches that terminate without further connection.wAs obtained from visual observations.

Site I, Pinus nigra ssp. maritima; site II, Picea abies.



Granlund HI, Jennings DH & Thompson W (1985) Translocation

of solutes along rhizomorphs of Armillaria mellea. Trans Br

Myc Soc 84: 111–119.

Gray SN, Dighton J & Jennings DH (1996) The physiology of

basidiomycete linear organs. III. Uptake and translocation of

radiocaesium within differentiated mycelia of Armillaria spp.

growing in microcosms and in the field. New Phytol 132: 471–482.

Harary F (1969) Graph Theory. Addison-Wesley, New York.

Harris MJ & Boddy L (2005) Nutrient movement and mycelial

reorganization in established systems of Phanerochaete

velutina, following arrival of colonized wood resources. Microb

Ecol 50: 141–151.

Kampichler C, Rolschewski J, Donnelly DP & Boddy L (2004)

Collembolan grazing affects the growth strategy of the cord-

forming fungus Hypholoma fasciculare. Soil Biol Biochem 36:

591–599.

Leach R (1939) Biological control and ecology of Armillaria

mellea (Vahl) Fr. Trans. Br Mycol Soc 23: 320–329.

Leake J, Johnson D, Donnelly D, Muckle G, Boddy L & Read D

(2004) Networks of power and influence: the role of

mycorrhizal mycelium in controlling plant communities and

agroecosystem functioning. Can J Bot 82: 1016–1045.

Luisi N, Sicoli G, Larario P, Dreyer E & Aussenac G (1996) Proc

Int Symp Ecology and physiology of oaks in a changing

environment. Ann Sci For 53: 389–394 [in French].

Lygis V, Vasiliauskas R, Larsson K-H & Stenlid J (2005) Wood-

inhabiting fungi in stems of Fraxinus excelsior in declining ash

stands of northern Lithuania, with particular reference to

Armillaria cepistipes. Scand J Forest Res 20: 337–346.

Marsh B a’B (1971) Measurement of length in random

arrangements of lines. J Appl Ecol 8: 265–267.

Marcais B & Wargo PM (2000) Impact of liming on the

abundance and vigor of Armillaria rhizomorphs in Allegheny

hardwoods stands. Can J For Res 30: 1847–1857.

Mihail JD & Bruhn JN (2005) Foraging behaviour of Armillaria

rhizomorph systems. Mycol Res 109: 1195–1207.

Morris SJ & Robertson GP (2005) Linking function between

scales of resolution. The Fungal Community (Dighton J,

White JF & Oudemans P, eds), pp. 13–26. Taylor and Francis,

London.

Morrison DJ (2004) Rhizomorph growth habit, saprophytic

ability and virulence of 15 Armillaria species. For Pathol 34:

15–26.

Newman EI (1966) A method of estimating the total length of

root in a sample. J Appl Ecol 3: 139–145.

Olsson S & Gray SN (1998) Patterns and dynamics of 32P-

phosphate and labelled 2-aminoisobutyric acid (14C-AIB)

translocation in intact basidiomycete mycelia. FEMS Microb

Ecol 26: 109–121.

Pareek M, Cole L & Ashford AE (2001) Variations in structure of

aerial and submerged rhizomorphs of Armillaria luteobubalina

indicate that they may be organs of absorption. Mycol Res 105:

1377–1387.

Pareek M, Allaway WG & Ashford AE (2006) Armillaria

luteobubalina mycelium develops air pores that conduct

oxygen to rhizomorph clusters. Mycol Res 110: 38–50.

Prim RC (1957) Shortest connection networks and some

generalizations. Bell System Tech J 36: 1389–1401.

Prospero S, Holdenrieder O & Rigling D (2006) Rhizomorph

production and stump colonization by co-occurring

Armillaria cepistipes and Armillaria ostoyae: an experimental

study. For Path 36: 21–31.

Proulx SR, Promislow DEL & Phillips PC (2005) Network think-

ing in ecology and evolution. Trends Ecol Evol 20: 345–353.

Rayner ADM (1991) The challenge of the individualistic

mycelium. Mycologia 83: 48–71.

Rayner ADM, Griffith GS & Ainsworth AM (1994) Mycelial

interconnectedness. The Growing Fungus (Gow AR & Gadd

GM, eds), pp. 21–40. Chapman & Hall, London.

Reaves JL, Shaw CG & Roth LF (1993) Infection of ponderosa

pine trees by Armillaria ostoyae: residual inoculum versus

contagion. Northw Sci 67: 156–162.

Redfern DB (1973) Growth and behaviour of Armillaria mellea

rhizomorphs in soil. Trans Br Mycol Soc 61: 569–581.

Rishbeth J (1970) The role of basidiospores in stump infection by

Armillaria mellea. Root Diseases and Soil Borne Pathogens

(Tousson TA, Bega R & Nelson P, eds), pp. 141–146. University

of California Press, Berkeley, CA.

Rishbeth J (1982) Species of Armillaria in Southern England.

Plant Path 31: 9–17.

Rizzo DM, Blanchette RA & Palmer MA (1992) Biosorption of

metal ions by Armillaria rhizomorphs. Can J Bot 70:

1515–1520.

Smith ML, Bruhn JN & Anderson JB (1992) The fungus

Armillaria bulbosa is among the largest and oldest living

organisms. Nature 356: 428–431.

Southworth D, He X-H, Swenson W, Bledsoe CS & Horwath WR

(2005) Application of network theory to potential mycorrhizal

networks. Mycorrhiza 15: 589–595.

Swift MJ (1972) The ecology of Armillaria mellea Vahl (ex Fries)

in the indigenous and exotic woodlands of Rhodesia. Forestry

45: 67–86.

Termorshuizen AJ (2000) Ecology and epidemiology of

Armillaria. Armillaria Root Rot: Biology and Control of Honey

Fungus (Fox RTV, ed), pp. 45–63. Intercept Ltd, Andover, UK.

Thompson W & Boddy L (1983) Decomposition of suppressed

oak trees in even-aged plantations. II. Colonization of tree

roots by cord- and rhizomorph-producing basidiomycetes.

New Phytologist 93: 277–291.

Thompson W & Rayner ADM (1983) Extent, development and

function of mycelial cord systems in soil. Trans Brit Mycol Soc

81: 333–345.

Watkinson SC, Boddy L, Burton K, Darrah PR, Eastwood D,

Fricker MD & Tlalka M (2005) New approaches to

investigating the function of mycelial networks. Mycologist 19:

11–17.

Wilson RJ (1979) Introduction to Graph Theory. Academic Press,

New York.



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