Ecological Archives E088-076-A2

Ingrid M. Parker and Gregory S. Gilbert. 2007. When there is no escape: the effects of natural enemies on native, invasive, and noninvasive plants. Ecology 88:1210–1224.

Appendix B. Isolation, identification, and geographical origin of fungi from clovers.

This appendix includes: (1) Details of isolation procedures and culturing of fungi, (2) How fungi were identified, (3) Table B1: Fungi isolated and identified from common-garden clovers in each year, (4) Host and geographic ranges of clover pathogens, and (5) Isolations done from greenhouse seedlings before outplanting.

Details of isolation procedures

Close to the time of harvest, we collected leaf material (generally a single leaflet from the third leaf from the top) in the field using sterilized dissecting scissors. Leaflets were transferred to 1.5-mL microfuge tubes and processed within 1–3 h. Each leaf was surface sterilized by placing it in 70% ethanol for 1 min followed by 10% commercial bleach (0.525% sodium hypochlorite) for 1 min. Leaves were placed on either potato dextrose agar (200 g potato infusion, 20 g dextrose, 15 g agar per L) or malt extract agar with chloramphenicol to reduce bacterial contamination (20 g malt extract, 15 g agar, 0.2 g chloramphenicol per L). We checked plates regularly under a stereomicroscope and recorded growth of fungi from the leaflets. When multiple fungal types were found in single leaves, they were transferred onto separate plates. When possible, cultures were grown to sporulation and identified.

Identities of fungi associated with clovers in the common gardens.

In each of the four years we identified the fungi from a subset of all the leaves from which we made isolations (Table B1). In Years 2, 3, and 4 the identifications were made from leaves isolated on the harvest date. In Year 1, identifications were made from isolations one week prior to harvest. Of 1005 leaves, 85.9% produced at least one fungus, with a total of 1178 fungi identified to genus, species, or morphotype. Five fungal genera – Stemphylium, Cladosporium, Alternaria, Colletotrichum, and Leptosphaerulina – account for 95.6% of all examined fungi. A dozen or more other fungal morphotypes were encountered one to a few times each (e.g., Fusarium, Ulocladium, Cylindrocarpon, Uromyces, sterile mycelia); the frequencies of these fungi were too low for meaningful analysis.

TABLE B1. Fungi isolated and identified from common-garden clovers in each year. Total percentages of fungi within a year can sum to more than 100% because some leaves had multiple infections.


Year 1

Year 2

Year 3

Year 4


Number of leaves






Number of isolates






Percent leaves infected






Percent of leaves with:


Stemphylium spp.






Cladosporium cladosporioides






Alternaria spp.






Colletotrichum trifolii






Leptosphaerulina trifolii






Other (# morphotypes)

7.0 (8)

6.8 (3)

5.7 (9)




Host and Geographic Ranges of Clover Pathogens

The most common fungi found on clovers at the Bodega Marine Reserve (BMR) comprise both cosmopolitan species that are common on both Europe and California clovers (Alternaria alternata, Alternaria infectoria, Cladosporium cladosporioides, Stemphylium Group E) and species that are rare or absent on clovers in Europe (Stemphylium solani, Colletotrichum trifolii, Leptosphaerulina trifolii). Most species are broadly polyphagous, although Colletotrichum trifolii has been reported primarily on Trifolium and Medicago and some other legume species, and Stemphylium solani, previously not reported on clovers, has most often been reported from Solanaceae.

We used morphology and nuclear rDNA sequences (ITS 1 and 2) of representative fungal strains from our site at BMR to identify isolated fungi. Because simple Blast searches of Genbank sequence data using “best match criteria” often produce ambiguous or misleading identifications, we drew on published genus-level phylogenies and congeneric sequences from Genbank to confirm species names for our isolates. We used CLUSTAL-W, PHYLIP, and Tree View X to create trees that integrated the ITS sequences of our fungi and other strains from GenBank with published phylogenies. We combined that information with records in the USDA plant-fungus database (Farr et al.) to evaluate whether the fungi are most likely California natives, European natives brought to California, or cosmopolitan species. GenBank and USDA databases were accessed in May 2006. For each of the five most common fungal genera from clovers at BMR, we present the current understanding of the host range and geographic distribution.

In addition, in two collecting trips across Europe (France, Spain, and Denmark), we isolated 353 fungal cultures from more than 30 Trifolium and Medicago species. We identified 240 European fungi using ITS sequences and then used these identifications as an independent source of information regarding the distribution of BMR clover pathogens in the native range of the introduced clovers.

The following are GenBank accession numbers for ITS sequences of fungi used in this study. See GenBank records for detailed information on hosts and collection locations and dates.


From California:  EF104177, EF104178, EF104179, EF104180

From Europe:  EF104181, EF104182, EF104183, EF104184, EF104185, EF104186, EF104187, EF104188, EF104189, EF104190, EF104191, EF104192, EF104193, EF104194, EF104195, EF104196, EF104197, EF104198, EF104199, EF104200, EF104201, EF104202, EF104203, EF104204, EF104205, EF104206, EF104207, EF104208, EF104209, EF104210, EF104211, EF104212, EF104213, EF104214, EF104215, EF104216, EF104217, EF104218, EF104219, EF104220, EF104221, EF104222, EF104223, EF104224, EF104225, EF104226, EF104227, EF104228, EF104229, EF104230, EF104231


From California:  EF104246, EF104247, EF104248, EF104249, EF104250

From Europe:  EF104235, EF104236, EF104237, EF104238, EF104239, EF104240, EF104241, EF104242, EF104243, EF104244, EF104245


From California:  EF104233, EF104234


From California:  EF104152, EF104153, EF104154, EF104156, EF104157

From Europe:  EF104155, EF104158, EF104159, EF104160, EF104161, EF104162, EF104163, EF104164, EF104165, EF104166, EF104167, EF104168, EF104169, EF104170, EF104171, EF104172, EF104173, EF104174, EF104175, EF104176

Stemphylium spp.

Phylogenetic position and relation to European collections: We found two species of Stemphylium at BMR; one is S. solani and the other falls within the Câmara et al. (2002) complex Group E (including S. trifolii, S. sarciniforme, S. loti, S. triglochinicola). Of 30 strains of Stemphylium in GenBank allied with Stemphylium solani, none are from Europe (18 from N or S America; the remainder from Australia, New Zealand, or Asia), and none of the 21 strains of Stemphylium we isolated from clovers in Europe are similar to S. solani. Of the 31 GenBank strains most similar to our Stemphylium Group E, 20 are from N America, 2 from the UK, and the remainder from Australia, New Zealand, or Iran; 3 of our European collections from clovers belong to Group E. The remaining 18 European sequences of Stemphylium are most closely allied with Câmara Groups B and C (S. herbarum, S. vesicarum, S. botryosum, S. alfalfae), which are commonly reported on clovers.

USDA fungus-host distributions database: S. solani: 65 USDA records from 12 genera, mostly from Solanaceae; widespread throughout the US, Brazil, and China, but not reported from Europe and never reported from Trifolium or Medicago. S. trifolii: 6 USDA records all from Trifolium in the USA. S. sarciniforme: 38 USDA records on 10 genera, all legumes. On Trifolium and Medicago widespread in North and South America, Asia; reports from Portugal, Scotland, and Uzbekistan. S. loti: 22 USDA records, limited Lotus and Trifolium, reported only from Eastern USA. S. triglochinicola: 2 USDA records, only reported from Triglochin (Juncaginaceae) in the UK.

Summary : Of the two Stemphylium species found at BMR, one (S. solani) is very likely native to North America and is apparently absent from Europe. The second (Stemphylium Group E) has many close associates known from North America but may be cosmopolitan.

Cladosporium cladosporioides

Phylogenetic position and relation to European collections: Cladosporium cladosporioides was common among BMR clover isolates. Of 11 European collections of Cladosporium from clovers, 6 were this species. The remaining European isolates were C. herbarum (teleomorph Davidiella tassiana), which we did not find in California. Genbank submissions show that C. cladosporioides is cosmopolitan and known from a wide diversity of environmental and plant samples in both North American and Europe.

USDA plant-fungus database: C. cladosporioides: 317 USDA records from 176 genera; cosmopolitan; from Trifolium or Medicago in Oregon and China, and one record from Poland.

Summary. C. cladosporioides is cosmopolitan, cryptogenic, present on clovers in Europe, and has a broad host range.

Alternaria spp.

Phylogenetic position and relation to European collections: We found two species of Alternaria at BMR; one is clearly placed within Alternaria infectoria (teleomorph Lewia infectoria), and the second within A. alternata. Genbank submissions show that both species are cosmopolitan. Alternaria spp. made up 25% of all fungi we collected from clovers in Europe. From our European collections, 35 of 51 Alternaria collections were of A. alternata, 9 from A. infectoria, and 6 of A. longipes. A. longipes was not found among our California collections. ITS sequences place the California A. alternata close to the strains we isolated from European clovers, and closely allied with A. alternata from published phylogenies (Pryor and Gilbertson 2000).

USDA plant-fungus database: A. alternata: 694 USDA records from 256 genera; cosmopolitan; known from Trifolium or Medicago in California, Florida, Africa, Asia, but not reported from clovers in Europe except Russia. A. infectoria: 48 USDA records from 35 genera, mostly grasses; cosmopolitan. It has not been reported previously from Trifolium and Medicago.

Summary. Both A. alternata and A. infectoria are cosmopolitan and present on clovers in Europe, and have broad host ranges.

Colletotrichum trifolii

Phylogenetic position and relation to European collections: Colletotrichum trifolii was commonly found on clovers at BMR. Of 6 Colletotrichum collections from European clovers, three aligned most closely with C. trifolii in Blast searches.

USDA plant-fungus database: Colletotrichum trifolii: 142 USDA records from 10 genera (mostly Trifolium, Medicago and other Fabaceae); widespread in US, China, Australia; rare in Europe; Widespread in North America on Trifolium or Medicago, mostly absent from Europe except one report each in Poland and Greece.

Summary. C. trifolii is widespread in the US but rare in Europe. Host range is mostly restricted to clovers and some other legumes. This fungus appears to be a more important pathogen on California clovers than European clovers, but its origin is cryptogenic.

Leptosphaerulina trifolii

Phylogenetic position and relation to European collections: Leptosphaerulina trifolii was regularly encountered on clovers at BMR, but was not found in our collections from European clovers, nor has it been reported previously on European clovers.

USDA plant-fungus database: Leptosphaerulina trifolii: 211 USDA records from 64 genera; North America, Australia, New Zealand, and Southeast Asia, only 2 European reports). From Trifolium or Medicago in North and South America, Africa, Australia, New Zealand, Asia, one report each England and Portugal. This fungus appears to be absent or rare in Europe, but widespread on numerous host species in North America, the South Pacific, and southeast Asia.

Summary: L. trifolii is widespread in the U.S. but rare or absent in Europe. It has a broad host range. This fungus is likely to have a much more intimate history with California clovers than European clovers.

Isolations done from greenhouse seedlings before outplanting

In preliminary testing of seedlings grown in the BMR greenhouse for common garden experiments, we found that 12% of initial leaves were infected by fungi at the time of outplanting (N = 3 plants per species, 17 species). The 6 infected individuals were distributed across 5 different clover species, and the fungi comprised three sterile morphotypes that could not be identified. These infections could have come from the field-collected soil, from natural sources of windborne spores (the greenhouse was on-site and open to ambient sources of spores), or from “weedy” greenhouse taxa. However, they did not appear to persist in or spread among outplanted individuals, because they were not species we commonly saw in isolations from field plants.


Câmara, M. P. S., N. R. O’Neill, and P. van Berkum. 2002. Phylogeny of Stemphylium spp. based on ITS and glyceraldehydes-3-phosphate dehydrogenase gene sequences. Mylocogia 94:660–672.

Pryor, B. M., and R. L. Gilbertson. 2000. Molecular phylogenetic relationships amongst Alternaria species and related fungi based upon analysis of nuclear ITS and mt SSU rDNA sequences. Mycological Research 104:1312–1321.

Farr, D. F., A. Y. Rossman, M. E. Palm, and E. B. McCray. (n.d.) Fungal Databases, Systematic Botany & Mycology Laboratory, ARS, USDA. Retrieved May 31, 2006, from

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