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

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 Plant 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, Kenya. 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.

Berberis holstii, native to the highlands of East Africa, is susceptible to Puccinia graminis and P. striiformis through artificial inoculations (Jin et al. 2010; Jin 2011). 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 wheat stem rust 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.

This research is funded by USDA-ARS and the Durable Rust Resistance of Wheat (Cornell). The authors thank Sam Gale, Lucy Wanschura, Kim P. Nguyen, and Melissa Carter for their technical assistance.

Field surveys of B. holstii distributions and collections of aecial infections.

Field surveys conducted since 2008 in Kenya and 2009 in Ethiopia, focused on areas where small grain cereals, primarily wheat and barley, are grown. B. holstii herbarium collections were used to locate plants in Ethiopia. Aecial infections were collected, air-dried, shipped to USA, and stored at 4C until use in inoculation experiments. Inoculation on cereals and grasses.

A series of cereal species, termed identification series, and consisted of wheat (Triticum aestivum cv. Morocco and Line E), barley (Hordeum vulgaris cv. Hiproly), rye (Secale cereale cv. Prolific), and oat (Avena sativa cv. Marvelous), was used to isolate different 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-planting) and incubated in a dew chamber for 48 hrs. When a sample was limited, aeciospores were collected, suspended in mineral oil, and spray-inoculated to seedlings. Two incubation temperature regimes, 18-20C and 12-15C, were used in the infection period. 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 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. graminis (Pg) and two sets were used to test for P. striiformis (PsFAM2 and PsFAM4).

Barnes, C., and L. Szabo. 2007. Phytopathology 97:717-727. Jin, Y., L.J. Szabo, and M. Carson. 2010. Phytopathology 100:432-435. Jin, Y. 2011. Euphytica 179:105-108.

Materials and Methods

Acknowledgements

References

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 grown. Aecial infections were observed in August in the Mt Kenya area. In Ethiopia, B. 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 infection intensity in August to October. For the majority of samples, aeciospore viability was lost. Using relatively fresh samples collected in North Shewa in 2012 and 2014, inoculations resulted in stem rust infections on Line E, Prolific, Hiproly, and Marvelous (Table 1). DNA assays confirmed the presence of P. graminis in these samples (Table 2). Inoculations and DNA assays did not detect the presence of P. striiformis. While it is likely that the rust pathogen infecting Line E, Hiproly, and Prolific is P. graminis f. sp. secalis (Pgs), inoculation and DNA assays did not provide sufficient resolution to 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 its sexual cycle in Ethiopia. The contribution of the sexual cycle to the observed variation within the Pgt population in the region remains unclear.

Line E wheat

Prolific rye

Hiproly barley

Marvelous oats

# Samples 13 13 13 13

# Infections 11 8 4 1

Introduction Results and Discussion

Table 2. Real-Time PCR results of 13 aecial samples from Ethiopia.

A B

A B C

A B

Fig 2. Aecial infection of barberry leaves (A and B), and release of aeciospores during mist incubation (C).

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

# Samples # Subsamples DNA assay for Puccinia graminis

Positive Weak positive* Negative Failed

13 130 69 26 31 4

Fig 3. Aeciospore deposits on identification series (A), and stem rust infection on Line E (B).

Fig. 1. Barberry survey and collections in Kenya (A) and Ethiopia (B).

* Difference between the Cp of the specific assay and the internal control is >5.0.