The endophyte community ofspotted knapweed (Centaureamaculosa Lam., Asteraceae)

Alexey Shipunov, Anil Kumar Raghavendra,George Newcombe

Department of Forest Resources, University ofIdaho, Moscow, Idaho 83844-1133

Spotted knapweed

Spotted knapweed (Centaureamaculosa Lam., but moreacceptable name is Centaureastoebe L.) is a noxious, invasiveplant which was introduced intoNorth America from Eurasia in the19th century. There are tworaces: diploids (biennials) andtetraploids (perennials), only thelast is present in North America.

“Novel weapons”

Many invasive North Americanplants have been reported tohave antimicrobial,antiherbivore and allelopathiceffects, which are mostprobably the consequences ofunique (for American flora)secondary chemicalcompounds.

(From Cappucino & Arnason, 2006):invasive plants share their prominentsecondary compounds with less nativeNorth American plants than non-invasiveplants

Catechin or not catechinSpotted knapweed is among plants which havesignificant phytotoxic (allelopathic) effect. Somesecondary compounds were believed to have this effect:cnicin and catechins.

Cnicin (sesquiterpene lactone) was extractedfrom aerial parts of knapweed in 1967 andhas been thought as main inhibitor ofneighbor plants growth (Kelsey & Locken,1987). However, some reporters told aboutlittle inhibitor effect of cnicin (Muir & Majak,1983)

CatechinsThe most accepted opinion(Callaway et al., 1999 andmany others) is that cathechin-contained root exudates arecapable to suppress the growthof native grasses (Festuca,Koeleria etc.) and other plants.

However,recentexperiments(Blair et al.,2005) show theabsence ofcatechin effect.

Fungal endophytes1) Inhabit every plant

2) Some endophytes areknown to producesecondary metaboliteswhich are beneficial tothe host plant

3) Have full spectrum fromparasitism tocommensalism

Therefore, the controversy could be explained ifinvestigated plants have different endophytecommunities and, as a consequence, differentsecondary compounds

Competition experiment

Fescuealone:control

Endophyte-free (E–)knapweed and fescue

E+knapweedand fescue

The differences in fescuebiomass are statistically

significantLeast Squares Means

1 2 CVAR00002

0

2

4

6

VAR00001

1 E– plants

2 E+ plants

C Control(Festucaidahoensisalone)

Liquid cultures and volatilecompounds

At least someendophytescan produce

sesquiterpenes

Gas chromatogram:highest pikecorresponds withsesquiterpene

We have also found thatthis particular endophytestrain has the insecticideeffect to weevils.

Endophytes and seedgermination

Experimentwith

knapweedseeds

(fungal cultureswere used )

Experimentwith

Festucaidahoensis

seeds(liquid culturefiltrates were

used, we tried toimitate Blair et al.,2005 experiment

conditions)

Some endophytes are capableto suppress seed growth

More than 2/3 endophytestrains have statisticallysignificant terminationeffect on Festucaidahoensis seeds,whereas only 1/4 of themhave similar effect onknapweed seeds.Moreover, someendophytes (Pleosporasp.) can kill fescueseeds.

Endophyteshave different

effects onknapweed

Endophyte strain 124(Fusarium sp.) suppresses theflowering of knapweed

Did fungi come with their hosts?

Two possibilities: “host-jumping” or co-introduction

Endophytes are usuallyisolated from the achenesof knapweed

endophyte-free achenes

Isolation

How diverseare

knapweedendophytes?

One of best MP trees from phylogenetic analysis of ITS1, 5.8S and ITS2 gene sequences.More then 65% of them have no exact matches in the NCBI GenBank nucleotide database.

Botrytisspp., 6 ITShaplotypes

Fusariumspp., 5 (allare newtoGenBank)Alternaria

spp., 5

Mostfrequent ITShaplotypes

Alternariaallergene gene

(alt a 1) wasused to identifyAlternaria and

Ulocladiumspecies

Majority rule consensus tree from MP analysis of “Alt a 1” gene sequences

Distributionamong

native andexoticranges

Present inbothranges

Are endophyte communitiesdifferent?

nativerange

exoticrange

Patterns of co-occurrence

Haplotypescommon tonativerange

ITShaplotypescommon toexoticrange

Toboth

At the time, we have support for both co-introduction and“host-jumping” hypotheses.

Endophyte-free plantsPlants from natural habitats are usuallyrich of endophytes (70%–90% of seeds).However, some of our samples contain noendophytes. We cultivated the 2ndgeneration of knapweed and inoculatethem with liquid fungal cultures on theflowering stage.

Re-isolation

Then re-isolation were done.From all plants, we obtainedonly endophytes which wereused for inoculation.Alternaria species have thebest re-isolation frequency.No endophytes wereisolated from the control.Thus, we have found theway to produce endophyte-free plans.

Sampling-2006

We are tryingnow to organize

much widersampling and

need your help!

Accumulation curve for 2004/2005(most of samples were collected inIdaho state or in southwesternEurope)

Places of interest

Acknowledgements• Cort Anderson

• Rebecca Ganley

• Sanford Eigenbrode

• Hongjian Ding

• Maryse Crawford

• The team of R project for statistical computing

• Jari Oksanen, author of “vegan” R package forvegetation ecologists

• Idaho State Government

Web-site of the project

http://uidaho.edu/~shipunov