Berberis holstii is Functional as an Alternate Host of Puccinia graminis in

M. Lim1, G. Wolderufael2, E. Hailu2, R. Wanyera3, M. Newcomb1, P. Olivera1, D. Hodson4, I. Hale5, L. Szabo6, D. Luster7, A. Berlin8, J. Yuen8, and Y. Jin6

1Department of Pathology, University of Minnesota, St. Paul, MN, USA. 2Ethiopian Institute of Agricultural Research, Ambo Agricultural Research Center, Ethiopia. 3Kenya Agricultural and Livestock Research Organization, Njoro, . 4CIMMYT-Ethiopia, Addis Ababa, Ethiopia. 5Department of Biological Sciences, University of New Hampshire, Durham, NH, USA. 6USDA-ARS Cereal Disease Laboratory, University of Minnesota, St. Paul, MN, USA. 7USDA-ARS Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD, USA. 8Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden.

Introduction Results and Discussion

Berberis holstii, native to the highlands of East , is susceptible to Puccinia graminis and P. striiformis through artificial inoculations (Jin et al. 2010; Jin 2011). A B However, it is not known whether these pathogens complete their sexual cycles in the region. In an attempt to understand the roles of B. holstii in pathogen variation and epidemiology of and stripe rust in East Africa, we investigated the functionality of B. holstii as an alternate host by surveying the presence of P. graminis and P. striiformis on this barberry species through inoculation experiments and DNA assays.

Fig 3. Aeciospore deposits on identification series (A), and stem rust

infection on Line E (B). Materials and Methods

In Kenya, B. holstii was found in the highlands of the Rift Valley and northern slope of Mt Kenya in an elevation range of 2000 - 3000m (Fig. 1A), where wheat is normally Field surveys of B. holstii distributions and collections of aecial grown. Aecial infections were observed in August in the Mt Kenya area. In Ethiopia, B. infections. holstii was found primarily in North Shewa Zone, with a similar range of elevations (Fig. 1B). Aecial infections (Fig. 2) were observed from June to December with highest Field surveys conducted since 2008 in Kenya and 2009 in Ethiopia, focused on areas infection intensity in August to October. where small grain cereals, primarily wheat and , are grown. B. holstii herbarium collections were used to locate in Ethiopia. Aecial infections were collected, air- For the majority of samples, aeciospore viability was lost. Using relatively fresh samples dried, shipped to USA, and stored at 4C until use in inoculation experiments. collected in North Shewa in 2012 and 2014, inoculations resulted in stem rust infections on Line E, Prolific, Hiproly, and Marvelous (Table 1).

Table 1. Ethiopian aecial inoculations on identification series and infection A B observed in wheat, , barley, and .

Line E Prolific Hiproly Marvelous wheat rye barley oats

# Samples 13 13 13 13

# Infections 11 8 4 1

Table 2. Real-Time PCR results of 13 aecial samples from Ethiopia. Fig. 1. Barberry survey and collections in Kenya (A) and Ethiopia (B). DNA assay for Puccinia graminis

Inoculation on cereals and grasses. # Samples # Subsamples Positive Weak positive* Negative Failed A series of cereal species, termed identification series, and consisted of wheat (Triticum 13 130 69 26 31 4 aestivum cv. Morocco and Line E), barley (Hordeum vulgaris cv. Hiproly), rye (Secale cereale cv. Prolific), and (Avena sativa cv. Marvelous), was used to isolate different * Difference between the Cp of the specific assay and the internal control is >5.0. formae speciales (f. sp.) of P. graminis on cereals from infected barberry leaves.

Infected barberry leaves were suspended over seedling plants (7 to 10 days post- DNA assays confirmed the presence of P. graminis in these samples (Table 2). planting) and incubated in a dew chamber for 48 hrs. When a sample was limited, Inoculations and DNA assays did not detect the presence of P. striiformis. While it is aeciospores were collected, suspended in mineral oil, and spray-inoculated to likely that the rust pathogen infecting Line E, Hiproly, and Prolific is P. graminis f. sp. seedlings. Two incubation temperature regimes, 18-20C and 12-15C, were used in the secalis (Pgs), inoculation and DNA assays did not provide sufficient resolution to infection period. distinguish Pgs from P. graminis f. sp. tritici (Pgt). Stem rust infections observed on Marvelous were assumed to be P. graminis f. sp. avenae. Experiments are in progress to characterize isolates derived from these samples, and to determine if other rust fungi are present. Based on these preliminary data, we conclude that P. graminis completes A B C its sexual cycle in Ethiopia. The contribution of the sexual cycle to the observed variation within the Pgt population in the region remains unclear.

Acknowledgements

Fig 2. Aecial infection of barberry leaves (A and B), and release of This research is funded by USDA-ARS and the Durable Rust Resistance of Wheat aeciospores during mist incubation (C). (Cornell). The authors thank Sam Gale, Lucy Wanschura, Kim P. Nguyen, and Melissa Carter for their technical assistance.

DNA Assay.

Ten individual aecia from each sample were used for DNA extractions. DNA was extracted following a modified protocol of the CTAB method. A SmartCycler was used to References perform real-time PCR reactions (Barnes & Szabo 2007). Three specific probes and primers were used for the real-time PCR reactions. One set was used to test for P. Barnes, C., and L. Szabo. 2007. Phytopathology 97:717-727. graminis (Pg) and two sets were used to test for P. striiformis (PsFAM2 and PsFAM4). Jin, Y., L.J. Szabo, and M. Carson. 2010. Phytopathology 100:432-435. Jin, Y. 2011. Euphytica 179:105-108.