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Roles of 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., Eastern Europe, include 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

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

Cnicin (sesquiterpene lactone) was extracted from aerial parts of knapweed in 1967 and has been thought as main inhibitor of neighbor plants growth (Kelsey & Locken, 1987). However, some reporters told about little inhibitor effect of cnicin (Muir & Majak, 1983)

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 every plant Some endophytes are known to produce secondary metabolites which are beneficial to the host plant, e.g., taxol from Taxus trees Have full spectrum from parasitism to commensalism

·

Therefore, the controversy could be explained if investigated plants have different endophyte communities and, as a consequence, different secondary compounds


Endophytes have different effects on knapw eed

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


Different effects


Some endophytes have strong negative effect on knapweed (i.e., they are close to pathogens)
Even close species can be different
E ffe c t o f C ID 1 0 7 ( F u s a riu m s p . 1 )

E ffe c t o f C ID 4 4 ( F u s a riu m s p . 2 ) 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 and seed germination

Experiment with knapweed seeds
(fungal cultures were used )

Experiment with Festuca idahoensis seeds
(liquid culture filtrates were used, we tried to imitate Blair et al., 2005 experiment conditions)


Some endophytes are capable to suppress 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.


Competition experiment
E+ knapweed and fescue

Fescue alone: control

Endophyte-free (E­) knapweed and fescue


The differences in fescue biomass are statistically significant
Least Squares Means
6 1 0 04 0 0 R A V2

1 E­ plants 2 E+ plants C Control (Festuca idahoensis alone)
1 2 C VAR00002

0


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


Did fungi come with their hosts?

Two possibilities: "host-jumping" or co-introduction


K n a p w e e d c a n b rin g " m a l s e c c o " d is e a s e ?
This endophyte (CID250, from Germany) have 99% identity with GenBank sequences of Phoma tracheiphila, very dangerous pathogen of Citrus trees


Isolation
Endophytes are usually isolated from the achenes of knapweed

endophytefree achenes


Isolation frequency varies from 0% to ~100%

Samples from Kamiah, ID

Samples from Grishneim (Germany)


H o w d iv e rs e a re knapw eed endophytes?

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.


Alternaria a lle rg e n e g e n e (a lt a 1 ) w a s used to identify Alternaria and U lo c la d iu m s p e c ie s
Majority rule consensus tree from MP analysis of "Alt a 1" gene sequences


M ost frequent endophytes

Botrytis spp.

Fusarium spp.

Alternaria spp.


Distribution am ong native and exotic ra n g e s

Present in both ranges


Are endophyte communities different?
exotic range

native range


Endophyte-free plants
P la n ts fr o m n a tu r a l h a b ita ts a r e u s u a lly r ic h o f e n d o p h y te s ( 7 0 % ­ 9 0 % o f s e e d s ) . H o w e v e r , s o m e o f o u r s a m p le s c o n ta in n o e n d o p h y te s . 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 i n 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 .


Re-isolation
Then re-isolation were done. From all plants, we obtained only endophytes which were used for inoculation. Alternaria species have the best re-isolation frequency. No endophytes were isolated from the control. Thus, we have found the way to produce endophytefree plans.


S a m p lin g -2 0 0 6
Accumulation curve for 2004/2005 (most of samples were collected in Idaho state or in southwestern Europe)

T h is y e a r w e h a v e m u c h w id e r s a m p lin g


· Cort Anderson

Acknowledgements

· 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