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Endophytic fungi associated with Fallopia japonica(Polygonaceae) in Japan and their interactions with Pucciniapolygoni-amphibii var. tovariae, a candidate for classicalbiological control

Daisuke KUROSEa,*, Naruto FURUYAb, Kenichi TSUCHIYAb, Seiya TSUSHIMAa,Harry C. EVANSc

aNatural Resources Inventory Center, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604,

JapanbLaboratory of Plant Pathology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, JapancCABI Europe-UK, Bakeham Lane, Egham, Surrey, TW20 9TY, UK

a r t i c l e i n f o

Article history:

Received 22 August 2011

Received in revised form

16 April 2012

Accepted 18 April 2012

Available online 28 April 2012

Corresponding Editor:

Barbara Schulz

Keywords:

Endophyte diversity

ITS sequences

Japanese knotweed (Fallopia japonica)

Phomopsis

Puccinia polygoni-amphibii var. tovariae

Synergistic interaction

* Corresponding author. Tel.: þ81 29 838 8257E-mail addresses: daikuro@affrc.go.jp,

cabi.org1878-6146/$ e see front matter ª 2012 The Bdoi:10.1016/j.funbio.2012.04.011

a b s t r a c t

Fallopia japonica (Polygonaceae), or Japanese knotweed, is now spreading globally, causing se-

rious problems in Europe and North America in both natural and urban habitats. There is an

urgent need for alternativemanagement solutions, and classical biological control, using co-

evolved natural enemies found in the native range, is currently being investigated. Here, we

isolated fungal endophytes from F. japonica in Japan, its natural habitat, to find endophytes

that might increase the virulence of a coevolved rust pathogen, Puccinia polygoni-amphibii var.

tovariae. A total of 1581 fungal endophytes were recovered from F. japonica and classified into

15 taxa. Five genera (Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, and Alternaria) were

dominant as endophytes in F. japonica. A greenhouse study of the dominant endophy-

teepathogen interactions revealed three types of reactions: suppressive, synergistic, and

neutral. In particular, one Phomopsis isolate e closely related to Diaporthe medusaea, based

on ITS sequences e promoted the pathogenic aggressiveness of P. polygoni-amphibii var.

tovariae and, therefore, this interaction is potentially useful to increase the effectiveness

of the rust fungus as a biological control agent of F. japonica in its invasive range.

ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction 19th Century (Synge 1956). This invasiveness has imposed

Fallopia japonica (Houtt.) Ronse Decr., or Japanese knotweed,

belongs to the family Polygonaceae and is a problematic inva-

sive weed in both North America and Europe, especially in the

UK, following its introduction into Europe from Japan in the

; fax: þ81 29 838 8604.nafuruya@agr.kyushu-u.a

ritish Mycological Societ

a significant cost for urban development, as well as posing

a threat to biodiversity (Bailey & Conolly 2000). Control is dif-

ficult to achieve as the plant has an extensive rhizome system

that survives in spite of apparently successful above-ground

chemical control, necessitating the issue of official knotweed

c.jp, kentsuch@agr.kyushu-u.ac.jp, seya@affrc.go.jp, h.evans@

y. Published by Elsevier Ltd. All rights reserved.

786 D. Kurose et al.

management guidance documents in the UK (Welsh

Development Agency 1998; Environment Agency 2007). Thus,

there is an urgent need for sustainable control measures for

F. japonica in its invasive range. Classical biological control e

involving the introduction of coevolved natural enemies

from the plant’s centre of origine is being investigated as a po-

tential approach for management of this weed in the UK.

Surveys in the Japanese endemic range revealed a guild of

specialised natural enemies which can severely reduce the

vigour and change the population dynamics of F. japonica

(Kurose et al. 2006). In particular, two fungal agents, the rust

fungus, Puccinia polygoni-amphibii var. tovariae (Kurose et al.

2009b) and a leaf-spot fungus,Mycosphaerella polygoni-cuspidati

(Kurose et al. 2009a), caused severe damage to F. japonica in the

field. During attempts to isolate Mycosphaerella and other

necrotrophic fungal pathogens from the surface-sterilised,

infected leaves of F. japonica, pathogen colonies were fre-

quently overgrown by faster-growing fungal ‘contaminants’.

Because samples were taken from plants in their natural

range, and isolations were made only from the advancing

edges of fresh legions, we assumed that a proportion of these

may represent specialist or coevolved endophytic fungi rather

than generalist or opportunistic saprophytes.

Fungal endophytes occur within the living tissues of

plants (Petrini 1991; Clay 1993) without producing any appar-

ent symptoms, and their presence may confer certain advan-

tages to the host plant (Carroll 1991). Mutualistic endophytic

fungi offer a variety of potential benefits to their host plants:

growth enhancement; tolerance to abiotic factors, including

drought, heat, and heavy metals; resistance to pests and dis-

eases (Redman et al. 2001; Rudgers et al. 2004; Schulz & Boyle

2005). Evans (2008) reported that the endophytic mycobiota

of F. japonica is relatively depauperate in its weedy invasive

range in the UK, especially in urban situations; whilst

Huang et al. (2008) reported the common occurrence of

Fig 1 e Map of collection sites of Fallopia japonica for isol

fungal endophytes in Polygonum cuspidatum (syn. F. japonica)

in part of its native range in China. Our preliminary results

show that there is a rich and diverse endophytic mycobiota

associated with leaves of F. japonica in Japan (Daisuke Kurose,

unpublished) compared to the UK (Evans 2008).

From the viewpoint of the efficacy of classical biological

control, it is important to understand the interaction not

only between endophytes and hosts, but also between endo-

phytes and pathogens. Synergistic effects, following co-

inoculation with two pathogens or more, have been observed

during studies to enhance the impact of biological control

agents (Crawley et al. 1985; Morin et al. 1993a, b; Guske et al.

2004). However, interactions between fungal endophytes and

the pathogens have not been reported previously. If endo-

phytes have a potential role of promoting aggressiveness of

pathogens in their hosts, this interaction could possibly be

exploited for the biological control of weeds.

In this study, we investigated the fungal endophytes asso-

ciated with F. japonica in part of its native range in southern

Japan. Then, we monitored the interactions of dominant

endophytes with the rust pathogen, P. polygoni-amphibii var.

tovariae to find ones that synergistically interact with the

rust in F. japonica.

Materials and methods

Collection of samples

Samples were collected from Fallopia japonica, located at: Kusu

(33� 16. 6660N, 131� 09. 6210E; 350 m a.s.l.) in Oita Prefecture; Mt.

Hiko (33� 29. 1020N, 130� 53. 8170E; 488 m a.s.l.) in Fukuoka Pre-

fecture; and Unzen (32� 43. 8530N, 130� 16. 0600E; 693 m a.s.l.) in

Nagasaki Prefecture; all situated on Kyushu Island in Japan

(Fig 1) from where the European introductions originated

ation of fungal endophytes on Kyushu Island, Japan.

Endophyte-rust interactions in Japanese knotweed 787

(Bailey & Conolly 2000). Data from these three sites were used

for the analysis of the composition of fungal endophytes. Dur-

ing the study period, the annual rainfall at three sites was

1410, 2142, 2365 mm, and the median temperature was

14.1 �C, 15.3 �C, 12.7 �C, at Kusu, Mt. Hiko, and Unzen,

respectively.

Plants with more than five expanded leaves were sampled

at the beginning of this study, because preliminary experi-

ments using unopened leaves showed no fungal colonisation.

Fifty apparently healthy leaves were collected from ten indi-

vidual plants from each site in May, September, and Decem-

ber 2005. Samples were brought to the laboratory in paper

bags and stored in a refrigerator at 4 �C for no longer than

48 h prior to use.

Isolation of endophytes

Leaf pieces, approximately 0.5� 0.5 cm, were excised from ap-

parently healthy leaves of Fallopia japonica. The number of leaf

pieces depended on the size of the leaf. Surface sterilisation

was carried out by immersion in 70 % ethanol solution for

3e5 s and 1.5 % sodium hypochlorite solution for 10 min.

The leaf pieces were thoroughly rinsed three times in sterile

distilled water. After surface sterilisation, four leaf pieces

were placed on potato carrot agar (PCA; decoction of 20 g po-

tato and 20 g carrot, and 20 g agar l�1) in 9-cm diam Petri

dishes and incubated at 25 �C for 2 weeks. The absence of

strictly epiphytic bacteria, yeasts and zygomycetes from the

cultures indicated that the sterilisation protocol was effective

(Arnold et al. 2000). All fungal colonies growing from the leaf

pieces were subcultured onto PCA.

Morphological characterisation

The morphological identification of endophytic fungal iso-

lates to genus level was based on the macro-morphology

of the fungal culture as well as micro-morphology using

standard mycological manuals (e.g. Ellis 1971; Sutton

1980). To induce sporulation, each of the fungal isolates

was subcultured on potato dextrose agar (PDA; decoction

of 200 g potato, 15 g dextrose, 20 g agar l�1), PCA, and water

agar (WA; 20 g agar l�1), prior to placing an autoclaved leaf

piece of Fallopia japonica on each medium. After incubation

for 2 m at 25 �C, the isolates that failed to sporulate were

classified in the group Mycelia sterilia. Cultures of the rep-

resentative isolates in each taxon were stored under min-

eral oil or sterile distilled water (Smith 2002) at room

temperature.

Genotypic characterisation of dominant fungal endophytesand an unidentified Ascomycete

DNA isolation and PCR amplificationFive dominant taxa (Colletotrichum, Pestalotiopsis, Phoma, Pho-

mopsis, and Alternaria) and a member of an unidentified asco-

mycete genus were subjected to identification at the species

level using sequencing of the internal transcribed spacer

(ITS) region, including the 5.8S rDNA. Based on morphological

classification, different morphospecies in each taxon were

selected for molecular analysis. Mycelia grown on PCA at

20 �C were harvested after 1 m. Genomic DNA was extracted

with the DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA)

according to the supplier’s protocols. ITS region including

the 5.8S rDNA was amplified as a single fragment using the

polymerase chain reaction (PCR) with Taq DNA polymerase

and with the primer pairs ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and ITS4 (50-TCCTCCGCTTATTGATATGC-30)(White et al. 1990). Optimal conditions for PCR were deter-

mined with a 50-ml reaction volume containing 5.0 ml of 10�Taq buffer (100 mMTriseHCl, pH 9.0, 500 mMKCl and 20% Tri-

ton X-100), 1.25 U of Taq polymerase, 1.5 mM MgCl2, 200 mM

dNTPs, 1.0 ml of DNA template solution (10 ng) and 0.2 mM

each primer. Amplification of the desired fragment was per-

formed with a MyCycler Thermal Cycler (Bio-Rad, Hercules,

CA, USA) under the following conditions: 5 min initial dena-

turation at 96 �C, 36 cycles of denaturation for 45 s at 94 �C,annealing for 30 s at 50 �C, and extension for 1.5 min at

72 �C; the reaction was terminated after a final extension at

72 �C for 8 s.

DNA sequencingPCR products were purified using the QIAquick PCR purification

kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s

protocol and prepared for sequencing using a BigDye Termina-

tor v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosys-

tems, Foster City, CA, USA) with the same primers used for

PCR amplification under the following conditions: 1 cycle of

96 �C for 1 min, 25 cycles of 96 �C for 10 s, 53 �C for 5 s, 60 �Cfor 4 min. Cycle sequencing reaction products were finally puri-

fied using a BigDye XTerminator Purification Kit (Applied Bio-

systems), and then sequenced using an ABI 3730xl DNA

Analyzer (Applied Biosystems). The DNA sequences obtained

in this study were deposited in the DDBJ/EMBL/GenBank data-

base systems under accession numbers AB640840-AB640842,

AB640844-AB640845, AB640847-AB640848, AB697732-AB697738,

and AB698854.

Inoculation and re-isolation of endophytes

Leaf inoculations were conducted in order to determine if the

isolated fungi could colonize healthy plants of Fallopia japonica

asymptomatically. Rhizomes collected from natural popula-

tions were grown in commercial proprietary compost, and

maintained in a greenhouse with supplementary lighting

and temperature of 20e25 �C. By using this methodology, the

shoots emerging from these rhizomes were confirmed free

of endophytes based on the fact that no microorganisms

were isolated from the leaves in pre-trials.

Freely-sporulating strains of five dominant taxa, Colletotri-

chum TH-SZ2d, Pestalotiopsis TH-SZ1c, PhomaHND-Bc, Phomop-

sis HS-SZ1j, and Alternaria W2374i, isolated during this study

and subsequently identified bymolecular phylogeny, were se-

lected for further inoculation experiments. Control plants

were inoculated with 0.05 % Tween-80 only. Plants of F. japon-

ica at the five-leaf stage were inoculated with spore suspen-

sions containing approximately 1.0� 106 spores ml�1 in

0.05 % Tween-80, using a fine brush to paint both adaxial

and abaxial sides of leaves. After treatment, all plants were

transferred to a dew chamber at 20 �C and 100 % relative

Table 1 e Isolation prevalence and number taxa of fungalendophytes isolated from Fallopia japonica at each site.

Characteristic Kusu Mt. Hiko Unzen

No. of samples 513 513 513

No. of isolates recovered 469 571 541

Total taxa identified 14 11 11

Isolate prevalence (%)a 79.7 88.1 84.2

a Isolate prevalence (%) ¼ (total number of leaf pieces yielding

more than one isolate)/(total number of leaf pieces in that

trial) � 100.

788 D. Kurose et al.

humidity, without light, for 48 h. Subsequently, all inoculated

plants were maintained in the greenhouse together with the

control plants. Four weeks after inoculation, re-isolation

from all the inoculated leaves, and non-inoculated upper

leaves which grew after treatment, as well as the control

plants, was undertaken using the same procedure described

above in order to investigate colonisation of and systemic in-

fection by the selected strains. Three replicate plants, with

five leaves per plant, were used.

Endophytes/Puccinia interaction

The interaction of fungal endophyteswith the rust fungus, Pucci-

nia polygoni-amphibii var. tovariae, was examined in glasshouse

experiments to determine if they have a significant synergism

on the pathogenic activity of the rust in Fallopia japonica. The

five dominant endophytes selected previously, Colletotrichum

TH-SZ2d, Pestalotiopsis TH-SZ1c, Phoma HND-Bc, Phomopsis

HS-SZ1j, and Alternaria W2374i, were inoculated 1 week prior to

inoculation with P. polygoni-amphibii var. tovariae. The same

inoculationmethod of fungal endophyteswas used as described

previously. For Puccinia, urediniospores weremixedwith talcum

powder (spores: talc ¼ 1:10) (w/w) and then applied to both leaf

surfaces using a fine brush. In the control treatment, only

Tween-80 was applied 1 week before the rust fungus. An addi-

tional three replicates in each experiment were inoculated

with eachendophyte only, inorder to confirmendophyte coloni-

sation in case the Puccinia or the talcum powder impeded endo-

phyte re-isolation. Inoculated plants were placed in a dew

chamber at 20 �C, without light, for 48 h. Subsequently, all inoc-

ulated plants weremaintained in the greenhouse, together with

thecontrolplants inoculatedwith thePucciniaorendophyteonly.

Eachexperiment contained tenplants,withfive leavesper plant,

per treatment, and thisexperimentwas repeated four times.The

Puccinia infection was assessed in terms of the number of rust

pustules produced on the abaxial surface of each leaf per leaf

area (cm2) at every 3 days post-inoculation (dpi).

Statistical analysis

The percentage of isolate prevalence was counted as the ratio

of the number of leaf pieces infected with endophytes to the

total number (Petrini et al. 1982). Relative isolation frequency

was expressed as the proportion of the number of isolates

within one endophyte taxon to the total number. The result-

ing data from each experiment were performed by analysis

of variance (ANOVA) before using TukeyeKramer’s Honestly

Significant Difference (HSD) test to compare means. The sta-

tistical software package JMP� 7 for Windows (SAS Institute

Inc., Cary, NC, USA) was used for the analysis.

Results

Isolate prevalence

A total of 1539 leaf pieces were processed from three sites; 1581

fungal isolates were recovered. The overall isolate prevalence

(%) ateachsite isgiven inTable1. Inall cases, ahigh isolateprev-

alence (79.7e88.1 %) of fungal endophytes was shown.

Composition of endophytic assemblages

The 1581 fungal endophytes of fungal endophytes resulting

from the study were categorized into 15 taxa based on mor-

phological characteristics. These comprised ten genera of

Hyphomycetes, three genera of Ascomycetes, and an uniden-

tified Ascomycete genus, as well as the Mycelia sterilia group

(Fig 2). Out of these 14 genera plus Mycelia sterilia, Colletotri-

chum, Phomopsis, Pestalotiopsis, and Mycelia sterilia were sepa-

rated into two ormoremorphospecies, on the basis of conidial

size or colony characteristics. The unidentified Ascomycete is

considered to represent a new genus close to Pezicula (Derma-

teaceae, Helotiales), based on sequence data, which also pro-

duces a highly distinctive anamorph with arthrosporic

conidiogenesis, similar to that of Neozythia (Sutton 1980).

As shown in Fig 2, of all the taxa isolated from Fallopia ja-

ponica leaves, Colletotrichum spp. had the highest average in

relative isolation frequency. The second most frequent endo-

phytic taxa were Phoma sp., Phomopsis spp., Pestalotiopsis spp.,

and Alternaria sp. with mean relative isolation frequencies of

13.9 %, 12.7 %, 10.4 %, and 9.3 %, respectively (Fig 2).

Isolates in the five dominant genera, Colletotrichum, Phoma,

Phomopsis, Pestalotiopsis, and Alternaria, were identified to spe-

cies level based on ITS sequences. The Colletotrichum isolates

could be separated into Colletotrichum acutatum and Colletotri-

chum gloeosporioides; and Pestalotiopsis spp. comprised three

species, Pestalotiopsis microspora, Pestalotiopsis sydowiana, and

Pestalotiopsis vismiae. The Phomopsis isolates were grouped

into seven different species, Phomopsis eucommicola, Phomopsis

liquidambari, Phomopsis longicolla, Phomopsis loropetali, Phomop-

sis phoenicicola, Diaporthe medusaea, and Diaporthe phaseolorum.

Phoma and Alternariawere identified as Phoma macrostoma and

Alternaria alternata, respectively.

Re-isolation of endophytes

The five selected dominant endophytes, Colletotrichum TH-

SZ2d, Phoma HND-Bc, Phomopsis HS-SZ1j, Pestalotiopsis TH-SZ1c,

and Alternaria W2374i, which are closely related to Colletotri-

chum gloeosporioides, Phoma macrostoma, Diaporthe medusaea,

Pestalotiopsis vismiae, and Alternaria alternata, respectively,

were successfully re-isolated from the inoculated leaves 28

dpi, but not from the non-inoculated upper (youngest) leaves

(data not shown). No fungal endophyte was obtained from

the control plants. All the inoculated leaves remained

asymptomatic.

0

10

20

30

40

Mean

relative iso

latio

n f

req

uen

cy (%

)

Taxon

b

a

bb

b

cccd

cdcdcd

cdcd

cd d

Fig 2 e Mean relative isolation frequencies of different endophytic fungal taxa isolated from Fallopia japonica. Vertical bars

indicate standard errors (n [ 9). Columns with different letters are significantly different according to TukeyeKramer’s HSD

test (P < 0.05).

Endophyte-rust interactions in Japanese knotweed 789

Endophyte/Puccinia interaction

The results show three types of reactions in effectiveness on

rust disease development; ‘suppressive fungi’, ‘promoting

30

2)

Alternaria W2374i

20

25

f a

re

a (c

m2

Colletotrichum TH-SZ2d

Pestalotiopsis TH-SZ1c

Phoma HND-Bc

15

ed

in

ia / leaf

Phomopsis HS-SZ1j

Control (the rust only)

10

mb

er o

f u

re

0

5

Nu

m

0 3 6 9Days post-inocula

Fig 3 e The impact of endophyte inoculation on the performanc

Alternaria W2374i, Colletotrichum TH-SZ2d, Pestalotiopsis TH-SZ1

week before P. polygoni-amphibii var. tovariae. In control treatm

The Puccinia infection was assessed in terms of the number of ru

leaf area (cm2) at 3-d intervals post-inoculation. Photographs we

amphibii var. tovariae on Fallopia japonica. The photographs are

trichum TH-SZ2d, (C) Phoma HND-Bc. Vertical bars indicate stan

each of the dpi are not significantly different according to Tukeye

four times.

fungi’, and ‘non-effective or neutral fungi’ (Fig 3). Colonisation

by Alternaria W2374i and Phoma HND-Bc inhibited rust coloni-

sation; whilst that of Phomopsis HS-SZ1j significantly pro-

moted disease development 9, 12 and 15 dpi compared with

A

a aa

a a

ab ab

Bab

bb

b

bc

bc

C

cc

c

c

c c

bc

c

12 15tion

e of Puccinia polygoni-amphibii var. tovariae. Five endophytes,

c, Phoma HND-Bc, and Phomopsis HS-SZ1j, were inoculated 1

ent, only Tween-80 was applied 1 week before the Puccinia.

st pustules produced on the abaxial surface of each leaf per

re taken at 15 dpi of disease symptoms caused by P. polygoni-

of leaves inoculated with (A) Phomopsis HS-SZ1j, (B) Colleto-

dard errors (n [ 40). Values followed by the same letter for

Kramer’s HSD test (P < 0.05). This experiment was repeated

790 D. Kurose et al.

the control plants. No significant effects of Colletotrichum TH-

SZ2d, and Pestalotiopsis TH-SZ1c, were observed. Endophytes

were re-isolated from all the inoculated leaves, and none of

the fungal endophytes were obtained from the control leaves

treated with Tween-80 only.

In the plants pre-inoculated with the Phomopsis

straindalthough rust uredinia did not appear on the lower

leaf surface until 6 dpidafter 9 dpi the number of uredinia

per leaf area (cm2) was significantly increased as compared

with the plants inoculated with other endophytes, or with

Puccinia only.

Discussion

Our data show that Fallopia japonica has a rich and diverse en-

dophyticmycobiota in Japan. In the field, the unopened leaves

of F. japonicawere not colonized by endophytic fungi and older

leaves tended to have more endophytes than younger opened

leaves. This is in general agreement with investigations un-

dertaken with other host plants (e.g. Petrini 1991; Rodrigues

1994). Additionally, in the greenhouse, no endophytes were

isolated from the non-inoculated plants grown from the rhi-

zomes collected in the field, and none of the endophytes

that we inoculated colonised the test plants systemically.

These results suggest that the plants in the field are infected

by air-borne spores and are not systemically infected.

Five fungal genera, Colletotrichum, Pestalotiopsis, Phoma, Pho-

mopsis, and Alternaria, have been found as endophytes in nu-

merous other herbaceous host plants (e.g. Schulz et al. 1993;

Kumaresan & Suryanarayanan 2001; Photita et al. 2001, 2005;

Gange et al. 2007). In the present study, these were the domi-

nant endophyte representatives in F. japonica in Japan. Al-

though Phomopsis was also one of the dominant genera

isolated from this host in China, Colletotrichum, Pestalotiopsis,

Phoma, and Alternaria were not recorded (Huang et al. 2008).

The differences in Japan and China suggest that geography in-

fluences the distribution patterns of fungal endophytes, as

reported by Fisher et al. (1994).

An understanding of the fungal endophytes colonising the

target weed might be useful for understanding the success or

failure of classical biological control (Evans 2008). Endophytes

had already increased in September (data not shown) when

rust disease causedby Puccinia polygoni-amphibii var. tovariaebe-

gan to appear at the Kusu site. This pathogen caused severe

damage to F. japonica by December, and isolate prevalence of

endophytes was also high in December (data not shown).

Therefore, endophytic and pathogenic fungi may interact

with each other in plant tissues. In the present study, an inves-

tigation of the interaction between a rust fungus and the dom-

inant endophytic genera e such as Colletotrichum, Pestalotiopsis,

Phoma, Phomopsis, and Alternaria e was conducted. It has been

shown that endophytic fungi can induce resistance to patho-

gens (Arnold et al. 2003; Lee et al. 2009). Cheung & Barber

(1972) and Dingle & McGee (2003) reported that inoculation of

non-pathogenic fungal endophytes significantly reduced

stem/leaf rust disease in wheat, which is in agreement with

the results attained following inoculation with Alternaria and

Phoma in this study. However, in our study, we also found that

Phomopsis HS-SZ1j increased the number of uredinial pustules

of P. polygoni-amphibii var. tovariae. Thus, this Phomopsis strain

may be a synergist of the rust, thereby increasing its potential

as a biological control agent of F. japonica. To our knowledge,

this is the first report that an endophyte can act in synergy

withaplantpathogen, followingpre-inoculationwith theendo-

phyte ahead of the pathogen. The other four endophytes either

do not interact or have a different form of interaction.

Phomopsis spp. have been recorded as plant pathogens of

economically important crops, as well as inducers of latent in-

fection, and as asymptomatic endophytes in weeds and other

plant hosts (Cerkauskas et al. 1983; Kulik 1984; Okane et al.

1998; Bussaban et al. 2001; Kumaresan & Suryanarayanan

2001). Molecular analysis of the ITS region, including the 5.8S

rDNA sequences, indicates that PhomopsisHS-SZ1j is closely re-

lated toDiaporthe medusaeawhich is the teleomorph of Phomop-

sis rudis, previously recorded as a pathogen of Japanese pear in

Japan (Watanabe 1991). In the present study, no symptoms

were observed in F. japonica inoculated with the endophytic

Phomopsis strain. A fungus that occupies plant tissues asymp-

tomatically may also be a weak pathogen, or a virulent strain

detected during latency, or possibly a passive inhabitant of

a niche just waiting for an opportunity to propagate (Schulz

& Boyle 2005). Accordingly, this does not exclude the possibility

that PhomopsisHS-SZ1j may become pathogenic when the host

is stressed. Furthermore, the Phomopsis endophyte assemblage

associatedwith F. japonica in Japanwould appear to bemore di-

verse than that of other genera since seven distinct species

have been identified using morphology and molecular phylog-

eny. This result may imply that strains other than HS-SZ1j also

have the ability to increase the rust disease development.

In contrast,Alternaria and Phoma isolates inhibited rust col-

onisation; whilst Colletotrichum and Pestalotiopsis isolates were

non-effective or neutral on rust development. These results

offer evidence that there are three types of endophytic fungal

interactions, which could have an impact on plant pathogens;

‘suppressive fungi or bodyguards’, ‘promoting or synergistic

fungi’, and ‘non-effective or neutral fungi’, implying that un-

derstanding of the endophytic mycobiota and their potential

interactions is necessary when analysing the success or fail-

ure of classical biological control agents.

Overall, the endophyte assemblages isolated from F. japonica

in Japanwere diverse anddominated by five fungal genera:Colle-

totrichum, Pestalotiopsis, Phoma, Phomopsis, and Alternaria. In con-

trast, Evans (2008) reported that this plant in urban areas in the

UK lacks endophytes. More in-depth comparisons of the myco-

biota between Japan and theUKappear to bewarranted. In addi-

tion, the mechanisms underlying these competitive or

synergistic fungal interactions are not clear. Further study on

the interaction between these dominant endophytes and P. poly-

goni-amphibiivar. tovariae, couldenable thedevelopmentofanew

strategy of classical biological control using both coevolvedpath-

ogens and endophytes to increase disease impact.

Acknowledgements

This study was partly supported by a Grant-in-Aid (22380181,

227223) from the Japan Society for the Promotion of Science

for NF and DK.

Endophyte-rust interactions in Japanese knotweed 791

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