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Endophytic fungi in the invasive spotted knapweed (Centaurea stoebe L.)
Alexey Shipunov, Anil Kumar Raghavendra, George Newcombe

Department of Forest Resources, University of Idaho


Spotted knapweed

Spotted knapweed (Centaurea stoebe L.) is a noxious, invasive plant which was introduced into North America from Eurasia. First reported in North America in 1893, knapweed now infests millions hectares of rangelands and pastures.


However, in native region (e.g., Russia and Ukraine), knapweed does not demonstrate invasion ability


"N o v e l w e a p o n s "
Many invasive North American plants have been reported to have antimicrobial, antiherbivore and allelopathic effects, which are most probably the consequences of unique (for American flora) secondary chemical compounds.

(From Cappucino & Arnason, 2006): invasive plants share their prominent secondary compounds with less native North American plants than non-invasive plants

Spotted knapweed is among plants which have significant phytotoxic (allelopathic) effect. Some secondary compounds were believed to have this effect: cnicin and catechins.


Catechin or not catechin

The m ost 1999 and c o n ta in e d s u p p re s s ( F e s t u c a,

a c c e p te d o p in io n ( C a lla w a y e t a l., m a n y o th e r s ) is th a t c a th e c h in r o o t e x u d a te s a r e c a p a b le to th e g r o w th o f n a tiv e g r a s s e s K o e le r ia e tc .) a n d o th e r p la n ts . H o w e v e r , r e c e n t e x p e r im e n ts ( B la ir e t a l., 2 0 0 5 , 2 0 0 6 ) s h o w th e a b s e n c e o f c a te c h in e ffe c t.


Fungal endophytes
· · Inhabit all plants Can produce secondary metabolites which are beneficial to the host plant, e.g., taxol from Taxus trees Specific endophytes may play specific roles, and many plants host very diverse arrays of endophytes We are hypothesizing that fungal endophytes are influencing plant competition with knapweed

·


Negative effects

Endophyte strain 124 (Fusarium sp.) suppresses the flowering of knapweed


Some endophytes have even pathogenic effect

T r a y s w ith in o c u la te d s e e d lin g s

T r a y s w ith c o n tr o l s e e d lin g s


Endophytes can benefit k n a p w e e d s e e d lin g s
Experiments with knapweed and Festuca idahoensis seeds


Endophytes are capable to suppress fescue seed growth
More than 2/3 endophyte strains have statistically significant termination effect on Festuca idahoensis seeds, whereas only 1/4 of them have similar effect on knapweed seeds. Moreover, some endophytes (Fusarium sp.) can kill fescue seeds.


Comparison of influence
As an example, Phoma herbarum (CID86) have selective effect: terminate fescue much more than knapweed


Competition experiment
Differences in biomass are significant

E+ E­ knapweed knapweed and fescue and fescue

Fescue alone: control


Liquid cultures and volatile com pounds


Gas chromatogram: highest pike corresponds with sesquiterpene

A t l east s om e endophytes c a n p ro d u c e sesquiterpenes


Insecticide effect

In a choice experiment, biocontrol weevils Larinus minutus demonstrated strong preference to non-inoculated flowers


Attraction of aphids

Some endophytes can attract other knapweed-eaters -aphids


Knapweed endophyte diversity


Isolation frequency varies from 0% to ~100%

Samples from Kamiah, ID

Samples from Grishneim (Germany)


Most endophytes belong to anamorphic fungi: it is nearly impossible to identify them without using molecular tools
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.


Recently (2005) founded singlecopy Alternaria allergene gene "Alt a 1" was used to identify Alternaria and Ulocladium species
Majority rule consensus tree from MP analysis of "Alt a 1" gene sequences


Some endophytes may represent n o v e l l in e a g e s
Putative new species

Botrytis (anamorph of Botryitinia, Sclerotiniaceae)


"Host-jumping" vs. co-introduction
Endophytes in introduced plants like knapweeds must either have been co-introduced in seeds of their host, or have `jumped' from other plants in the invaded range of their hosts This endophyte (CID250, from Germany) have 99% identity with GenBank sequences of Phoma tracheiphila, very dangerous pathogen of Citrus trees


Distribution am ong native and exotic ra n g e s

Present in both ranges

Data from 2004/2005 sampling


Are endophyte communities different?
exotic range

native range

Data from 2004/2005 sampling


Endophyte-free plants
W e c u ltiv a te d th e 2 n d g e n e r a tio n o f k n a p w e e d a n d in o c u la te th e m w ith liq u id fu n g a l c u ltu r e s o n th e flo w e r in g s ta g e . T h e n r e - is o la tio n w e r e d o n e . F r o m a ll p la n ts , w e o b ta in e d o n ly e n d o p h y te s w h ic h w e r e u s e d fo r in o c u la tio n . A lte r n a r ia s p e c ie s h a v e th e b e s t r e is o la tio n fr e q u e n c y . N o e n d o p h y te s w e r e is o la te d fr o m th e c o n tr o l. T h u s , w e h a v e fo u n d th e w a y to p r o d u c e e n d o p h y te - fr e e p la n s .


In progress
1) New samples: from California to Canada and from Germany to Russian Urals 2) Geographical distribution of endophytes, isolation frequency, germination rates and climate data 3) Repeated samples: succession in endophyte world 4) Hidden fungi: cultivation-free methods 5) Fungal coexistence and/or hyperparasitism 6) Plant morphology and fungi: age and flowering stalk development 7) Extended experiments with aphids, with fescue and with volatiles


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 for vegetation ecologists · Idaho State Government


Web-site of the project

http://uidaho.edu/~shipunov