1. Endophytes: biocontrol killers?
Sarah Dodd, Daniel Than,
Stanley Bellgard and Chris Winks (LCR)
Rebecca Ganley (Scion)
Trevor James (AgR)

2. Endophytes All plants are infested with microbes Symptomless Disease
Epiphytes/Endophytes
Balanced status of symbiosis
= Majority
Disease
Pathogens
Unbalanced status of symbiosis

3. Endophyte/Plant relationship
Endophyte gains
shelter and nutrients
Plant cost
nutrients and resources
(e.g. more susceptible to pests and diseases: BCAs)
Plant gains
Growth promotion
(enhanced nutrient uptake)
Increased tolerance to harsh environments
(e.g. drought tolerance)
Induced Resistance to pests and diseases (BCAs)

4. Endophytes – weeds and weed biocontrol
Harry Evan’s (2007) proposed:
Weediness: Endophytes influence the fitness of a plant and therefore its invasiveness
Biocontrol: Endophytes interact with plants and/or biocontrol agents either enhancing or reducing their activity

5. Microbial interactions
Pathogens
Endophytes/epiphytes
Antibiosis
Parasitism
Competition for nutrients/niche
Cell lysis
Antibiosis
Parasitism
Competition
Cell lysis

6. Microbe interactions – under the microscope
Speculated activity of endophytes:
Harry Evans Hyopthesis:
Enhanced plant growth
Protect plant against pathogens
Drought resistance
In absence, more vulnerable to these.
Drain on plant resources making more vulnerable to pathogens
Mutualist hypothesis: Recent work has indicated that mutualists may be critical for successful plant invasion. Richardson et al. (2000) extensively reviewed the literature on the importance of pollinators, nitrogen-fixing bacteria, soil mycorrhizae and vertebrate seed-dispersers in plant invasions and also noted that there has been growing interest in the role of leaf fungal endophytes in conferring an advantage to plants in the presence of herbivores (e.g. Clay 1996). As with arbuscular mycorrhizal fungi, these associations appear to be widespread in the plant kingdom. Yet the specificity and nature of the associations (mutualistic or pathogenic) are poorly known, as is their role in invasion.
We hypothesise that non-invasive exotic species may lack key mutualist species. Indeed, the probability of an exotic species being imported without its natural enemies, while retaining or acquiring new key mutualist species is likely to be very low. Furthermore, while mutualist species may afford plants some protection from natural enemies, if their presence comes at a metabolic cost, they may actually be a disadvantage in the introduced range where natural enemies may be absent.
Earlier this year, a new hypothesis was presented by Dr Harry Evans, the ‘endophyte-enemy release hypothesis (E-ERH)’, which we believe warrants further investigation. Dr Evans proposes, with the support of circumstantial evidence, that it is the presence or absence of mutualistic endophytes that plays a key role in determining why some alien plants become invasive.
Endophytes are micro-organisms that colonize the tissues of healthy plants. There is compelling evidence of plant/endophyte mutualism, with mutualistic endophytes offering a variety of potential benefits to their host plants: 1) growth enhancement (Varma et al. 1999); 2) tolerance to abiotic factors (such as drought, heat and heavy metals; Rodriguez & Redman 2005; Rodriguez et al. 2004; Marquez et al. 2007)); and 3) resistance to pests and disease (Latch 1993; Christensen 1996; Clay 1997; Schardl & Phillips 1997; Stone et al. 2000). See also Redman et al. (2001); Rudgers et al. (2004); and Schulz & Boyle (2005). In return, endophytes are thought to benefit from the comparatively nutrient rich, buffered environment found inside plant tissues.
A. The endophyte-enemy release hypothesis
How mutualistic endophytes could enhance plant invasiveness: Plants arriving in a new location without co-evolved natural enemies, but with mutalistic coevolved endophytes, or forming mutualistic associations with indigenous endophytes, would have a greater resistance to natural enemies and abiotic factors, giving them an advantage over local competitors.
How a lack of mutualistic endophytes could enhance plant invasiveness: There is a cost to the plant in harbouring endophytes, and consequently those alien plants arriving and remaining endophyte-free, without coevolved natural enemies, would have a distinct competitive advantage since they would have more resources to allocate to growth and reproduction.The endophyte-enemy release hypothesis therefore explains why classical biological control can be unpredictable as a management strategy. In the absence of endophytes, exotic plants with weedy traits would be vulnerable to co-evolved natural enemies. Hence, subsequent introductions of a single biological control agent can successfully, and often unexpectedly, bring about the complete control of a rampant invasive weed. In contrast, weedy exotic plants complete with their cohort of co-evolved endophytes would be protected from introduced natural enemies, making such enemies unsuitable for biological control.

7. Weed / Endophyte systems under investigation in NZ
Sclerotinia on Californian thistle
Phoma on Old Man’s Beard
1. Californian thistle
Sclerotinia BCA vs endophyte population
2. Phoma on Old Man’s Beard
3. Endophytes of wilding ginger
Invasiveness
introduced vs place of origin (India)
4. Privet endophytes
5. Pampas endophytes
Wilding Ginger
Privet
Pampas

8. Sclerotinia on Californian thistle - a mycoherbicide
1. Kills entire plant
2. Partially kills plant
Plant recovers
3. No effect on plant

9. Aim
To identify microbes that influence the success/failure of a pathogen biocontrol agent
To ultimately manipulate interactions to improve consistency of weed biocontrol activity

10. Progress: step 1 determine population variation
When looking for microbe populations associated with a particular activity, it is important to know the level of natural variation in such populations as this will impact on the number of samples required to expose them.
As there is currently no data available on this, initial experiments have been designed and implemented to determine the natural variation and diversity of such populations.
Different plant tissue samples (roots, shoot, flowers and seeds) have been collected from Californian Thistle plants from different sites, and from within a site, for comparison.
Roots, shoots,
flowers & seeds

11. Methods - to identify endophytes
Culturing
isolate & identify key endophyte fungi
● DGGE
- DNA profile comparisons
- endophyte fungi and bacteria
- DNA sequence to identify bands (microbes)

12. Culturing – isolation and identification
An array of fungal strains have been isolated from plant material collected
In process of identifying these using physical characters and DNA sequence data.
To date, cultures have been isolated from half the plant material and these are currently being identified using morphological and molecular techniques. DNA has been extracted from the remaining samples in preparation for molecular analysis of the resident microbial populations using a technique called DGGE. Results from culturing and molecular analysis will highlight those microbes likely to play a role in the success/failure of a pathogen. Their activity will then be verified in glasshouse trials

13. Molecular -DGGE
Samples are all assessed and bands(microbes) identified
Variability between tissues/plants/fields has been assessed
PCR amplification and run on gels for analysis. Bands have been sequenced.

14. Lots of things detected L=leaf, s=seed, r=root and p=papus on seed.
Endophytic microbes from thistle
(closest GenBank match)
Culturing
DGGE
Known biology
Fungi - Ascomycota
Alternaria sp.
Aureobasidium pullulans
Bionectria sp.
Botryosphaeria laricina
Cladosporium cladosporioides
Cladosporium sp.
Codinaeopsis sp.
Colletotrichum acutatum
Cryptococcus rajasthanensis
Curvularia sp.
Cylindrocarpon sp.
Davidiella tassiana
Epicoccum nigrum
Eudarluca caricis
Exophiala sp.
Fusarium cortaderiae
Fusarium oxysporum
Fusarium solani
Fusarium sp. 1.
Hypocrea/Trichoderma
Leptodondium orchidicola
Lewia infectoria
Neonectria radicola
Nigrospora oryzae
Phaeococcomyces chersonesos
Phoma exigua
Phoma sp.
Phomopsis theicola
Phomopsis sp.
Pichia fermentans
Plectosphaerella cucumerina
Plectosphaerella sp.
Preussia isomera
Preussia sp.
Pyrenochaeta terrestris
Sclerotinia sp.
Stachybotrys echinata
Stemphylium sp.
Verticillium dahliae
Xylariaceae sp.
L,S S L -
L,S,P R
L,R
S,R LR PS SR
L,S,R,P
L,R,P
L,P
saprobe
possible plant pathogen or saprobe
possible plant pathogen
possible secondary pathogen
yeast-like saprobe
grass pathogen
rust mycoparasite
Possible mycopathogen
plant pathogen
possible secondary pathogen or saprobe
Possible secondary pathogen or saprobe
yeast
coprophilous saprobe
onion path/soil saprobe
Note:
Lots of things detected
L=leaf, s=seed, r=root and p=papus on seed.
Some detected by both methods, but many by one or other. So traditional culturing and molecular methods complement each other.
Note array of known biology's. From saprobe to known pathogens of thistle and other hosts
Rust mycoparasite – could explain inconsistent disease of Californian thistle rust.

15. Endophytic microbes from thistle (closest GenBank match) Culturing
DGGE
Known biology
Fungi - Basidiomycota
Bovista plumbea
Ceratobasidium sp.
Cyathus stercoreus
Exidiopsis sp.
Flammulina velutipes
Gloeoporus dichrous
Kuehneromyces rostratus
Langermannia gigantean
Limonomyces roseipellis
Melampsora laricis-populina
Mycena sp. / Nolanea sp.
Panaeolus sphinctrinus
Peniophora pini / aurantiaca
Pleurotopsis longinqua
Pleurotus pulmonarius
Puccinia chrysanthemi / carduorum
Puccinia cnici-oleracei
Rogersella griseliniae / Hyphodontia crustosa
Schizophyllum commune
Tomentellopsis submollis / bresadoliana - R S L P
L,P
S,R
saprobe
possible plant pathogen
plant pathogen (rust)
Bacteria
Pantoea sp.
L,R
unknown
Note, here only molecular detected this important group of fungi, including rust fungi.
Note Cali thistle rust was not detected, but rust pathogens of other hosts were .
Remember from Boneseed talk that rusts are highly host specific, could explain why get some invasion in host plant testing. Maybe can exist as endophytes in non-hosts.
Yet to do molecular work on bacteria.
Melampsora laricis-populina (host = Poplus trees)
Puccinia chrysanthemi / carduorum
P. chrysanthemi = Black rust of cultivated chrysanthemum).
Puccinia carduorum, a rust fungus from Italian thistle in Tunisia, was most aggressive on young growth stages of the weed in greenhouse tests. Repeated inoculations with the fungus significantly reduced weed biomass. Host-range tests suggest the fungus may be a safe biological control agent of Italian thistle in Tunisia.
Puccinia cnici-oleracei causes these lovely pale bulges on the veins and stems of Aster tripolium. When the galls mature they become covered with brown black spores.

16. Testing influence of endophytes
Glasshouse trials – assess disease development
a) BCA + key endophyte
b) BCA - key endophyte
Will also have implication for biocontrol of plant diseases on commercial crops…ie grasses.

17. Preliminary glasshouse trials
Can endophytes influence Sclerotinia disease?
No influence
Enhanced Sclerotinia disease
Reduced Sclerotinia disease (e.g. Colletotrichum sp.)
Colletotrichum – induced resistance response. A bit like immunising the plant against pathogens.

18. Summary
We now have evidence to support the theory that endophytes DO influence the activity of pathogen biocontrol agents
We now have evidence that some endophytes do influence the activity of pathogen biocontrol agents.
Probably will influence insect BCAs too. Need to test this as well.

19. The Future Sclerotinia on Cali thistle Other plant systems
Identify key bacterial endophytes
Continue glasshouse testing
Identify key endophyte modes of action
Improve pathogen biocontrol agent activity
Other plant systems
Identify key endophytes and test in glasshouse
Endophytes and insect BCAs

20. Californian thistle rust
Puccinia punctiformis
Californian thistle Phoma