Zhensheng Kang, Northwest A&F University, China

1. New evidence supporting the occurrence of
sexual reproduction in the wheat stripe
rust fungus on barberry in China
Zhensheng Kang
State Key Laboratory of Crop Stress Biology for Arid Areas,
Northwest A & F University,Yangling, Shaanxi, China

2. Wheat stripe rust is a destructive disease
throughout all winter wheat regions in China
and is considered the most important disease
of wheat.
Ø Diseased area: between 3-6 million ha.
Ø Yield losses: 10-50%.
Years Losses
(million tonnes)
1950 6.0
1964 3.2
1990 2.6
2002 1.4

3. Western over-summering areas
Over-wintering areas
Eastern epidemic areas
Based on historical epidemiological data for stripe rust,
the wheat-growing regions in China can be divided into
three areas:
Epidemics of Pst in China

4. In the western mountainous areas, stripe rust can over-
summer on volunteer wheat and late-maturing spring wheat.
Terrace lands for growing wheat in
Over-summering areas in south Guansu
Diseased volunteer wheat
in south Guansu
Pst survival in over-summering areas in China

5. Migration pathway of Pst in China
Shandong
Jiansu
Anhui
Hubei
Henan
Shaanxi
Gansu
Shanxi
Sichuan
Qinghai
Hebei
Ningxia Eastern epidemic areas
Infecting autumn
sown wheat

6. Ø The most new races (≈90%) were first detected in
these regions in recent history of surveillance;
Ø A high genetic diversity within the regional Pst
populations has been reported by different research
groups (Lu et al., 2011; Duan et al., 2010; Mboup et al., 2009);
Ø The genetic recombination for Pst was found in these
regions (Lu et al.,2011; Duan et al., 2010; Mboup et al., 2009).
The western over-summering areas has been caught
much national and international attentions, and are
considered as a “hot-spot” for Pst.

7. Virulence variation for rusts maybe due to
Ø sexual hybridization
Ø mutations
Ø somatic hybridization
However, the mechanism of sexual hybridization
for Pst has been neglected since the sexual
stage was presumed to be absent.
Why does the western over-summering areas
become the “hot-spot” for Pst in China?

8. In 2010, some Berberis spp. were shown to serve as
alternate hosts for the wheat stripe rust pathogen.
Ø B. chinensis,
Ø B. holstii,
Ø B. koreana
Ø B. vulgaris.

9. Question: Does the sexual stage of
wheat stripe rust occur under natural
conditions, particularly in China?
Ø Is there any susceptible barberry species in
China?
Ø Can wheat stripe rust be isolated from
infected Berberis spp.?
Ø Does the sexual stage of wheat stripe rust
contribute to variation in virulence?

10. Surveys for Berberis spp.in China
Ø 215 of the ~500 described Berberis spp. in the world
are endemic to China;
Ø Many of China's Berberis spp. distribute in the
western over-summering areas (hot-spot).
Western China:
Sichuan：81; Chongqin： 30; Yunnan： 78; Tibet: 55; Guansu： 26;
Shaanxi: 20; Guizhou： 19; Qinghai：13; Xinjiang： 5; Ningxia： 3;
Central China:
Hubei： 24; Henan： 7; Shanxi： 10; Hunan： 9; Anhui： 2;
Eastern China:
Hebei： 6; Jiangxi： 5; Guandong：4; Guanxi：4; Hujian： 5;
Distribution of Berberis species in different regions of China

11. Surveys for Berberis spp.in China

12. No barberry plant
Barberry plants distributed
Barberry plants with rusts
30 barberry species were surveyed and
identified.
Over-summering area

13. B. brachypodaB. shensiana
Berberis soulieana B. potaninii

14. Are these Berberis spp.
susceptible to wheat
stripe rust ?

15. Identification of Berberis spp. as alternate
hosts of wheat stripe rust
Dew chamber
We collected seeds and seedlings of Berberis spp. from the
fields and inoculated using telia of Pst in the greenhouse.

16. Identification of Berberis spp.
as alternate hosts of wheat stripe rust
Normally, we see pycnia on the leaves of susceptible
Berberis spp. 11-14 days after inoculation.
Infection of basidiospore and development of pycnia
Pycnia on Berberis

17. Identification of Berberis spp.
as alternate hosts of wheat stripe rust
About 20 days after inoculation, we see aecia develop on the leaves.
Aeciospores can infect wheat through the stoma and produce typical
rust symptoms (uredinia).

18. 23 Berberis species: alternate host for Pst
Successful production of pycnia, and aecia on
upper and back sides of barberry leaf after inoculation
Zhao, J., et al. Phytopathology, 2013, 103(9):927-934

19. A B C D E F
G H I J K L
M N O P Q R
Zhao, J., et al. Phytopathology, 2013, 103(9):927-934

20. No. Berberis spp. Orgin Distribution
1 B. aggregata Gansu, China Gansu, Sichuan, Hubei, Qinghai, Shanxi
2 B. brachypoda Gansu, China Gansu, Sichuan, Hubei, Qinghai, Shanxi,Henan, Shanxi
3 B. potaninii Gansu, China Gansu, Shaanxi, Sichuan
4 B. soulieana Gansu, China Gansu, Shaanxi, Sichuan,Hubei
5 B. dasystachya Shaanxi, China Gansu, Shaanxi,Hubei,Shanxi
6 B. shensiana Shaanxi, China Shaanxi,Gansu
7 B. atrocarpa Sichuan, China Sichuan, Yunnan, Hunan
8 B．ferdinandi-coburgii Yunnan, China Yunnan
9 B. phanera Yunnan, China Yunna, Sichuan
10 B. aggregate var. integrifolia Yunnan, China Gansu, Sichuan,Qinghai,Hubei,Shanxi
11 B. davidii Yunnan, China Yunnan
12 B. stenostachya Gansu, China Gansu, Shaanxi, Shanxi
13 B. wangii Yunnan, China Yunnan
14 B. circumserrata Shaanxi, China Shaanxi, Hubei, Gansu, Qinghai,Henan
15 B. poiretii Beijing, China Shaanxi, Qinghai, shanxi, Hebei, Jilin, Liaoning,
16 B. guizhouensis Guizhou, China Guizhou
17 B. wilsonae Yunnan, China Yunnan, Tibet, Shaanxi, Qinghai, Hubei, Guizhou
18 B. sp. (unidentified) Tibet, China Tibet
19 B. jamesiana Yunnan, China Yunnan，Qinghai, Chongqing, Tibet, Sichuan
20 B. coryi Yunnan, China Yunnan
21 B. wilsonae Yunnan, China Yunnan, Sichuan, Tibet, Gansu
22 B. sp. Tibet, China Tibet
23 B. sp. Tibet, China Tibet
Species of barberry identified as alternate hosts for Pst by
artificial inoculation using germinated teliospores in China

21. Ø Some species are evergreen, distributed in southwest regions.
Ø Others are deciduous, distributed in northwest regions.
Ø Some susceptible species (e.g., Berberis soulieana, B. brachypoda,
and B. shensiana) are widely distributed in the western over-
summering areas.
Berberis soulieana
B. shensiana

22. Can barberry species be infected by the
stripe rust fungus (Pst) in nature?
It is very common to see pycnia and aecia on
infected Berberis leaves in fields in early May.

23. Obtaining of Pst isolates from rust-
infected barberry growing in nature
20 Pst isolates recovered from the infected barberry
Ø 4 Pst isolated from 3 barberry species in 2011
Ø 16 Pst isolates from 3 barberry species in 2013
collected the infected
Berberis leaves
Single aecium (SA) was
used for inoculation
Recovered SA isolate
on wheat leaf

24. Berberis spp. No. of aecia
No. of isolate Rate (%)
IT0 IT1-2 Pst Pgt Pst Pgt
B. brachypoda 1519 479 1032 2 6 0.13 0.39
B. shensiana 410 35 374 1 0 0.24 0.00
B. soulieana 384 367 16 1 0 0.26 0.00
B. potaninii 742 711 28 0 3 0.00 0.40
B. aggregata 648 633 13 0 2 0.00 0.31
Total 3703 2225 1463 4 11 0.11 0.30
Pst isolates recovered from rust-infected
Berberis spp. in Gansu, and Shaanxi in 2011
*IT0 indicates no any necrosis, or/and chlorosis on wheat after inoculation,
IT1-2 indicates necrosis, or/and chlorosis;
Pst=Puccinia striiformis f. sp. tritici; Pgt=Puccinia graminis f. sp. tritici

25. Comparison of infection types on Chinese differential
hosts of eight major races of Pst and the four Pst
isolates recovered from three barberry species in 2011
Virulence tests demonstrated that the infection types of
the four barberry-recovered cultures are different
compared to the major Chinese races.
Cultures
and
races
Origin of cultures
Differential hosts
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
B2011-1 Berberis brachypoda A V A V V V A V A VA V A A V A A A A A
B2011-2 B. brachypoda A V A V V A A V A A V A A A A A A A A
B2011-3 B. shensiana A V A V V A V V A AV V A A AV A A A A A
B2011-4 B. soulieana A V A A V V A V A A A A A V A V A A A
CYA33 Triticum aestivum V V V V V V V V V V V V V V A V A A A
CYA32 T. aestivum V V V V V V V V V V V V V V A V V A A
CYA31 T. aestivum V V V V V V V V V A V V A V A V V A A
CYA30 T. aestivum V V V V V V V V V A V V A A A V V A A
CYA29 T. aestivum V V V V V V V V V A V V A A A V A A A
CYA28 T. aestivum V V V V V V V V V A V A A A A V A A A
CYA23 T. aestivum V V V V A V V V V A V A A A A A A A A
CYA17 T. aestivum VA V A VA A VA V A A A AV A A A A A A A A

26. Berberis spp. No. of aecia
Number of isolate* Rate (%)
IT 0 IT 1-2 Pst Pgt Pst Pgt
B. shensiana 3677 3630 40 4 3 0.11 0.08
B. brachypoda 4477 4406 59 8 4 0.17 0.15
B. sp. (Tibet) 1163 1150 6 4 3 0.34 0.26
Total 9317 9174 105 16 12 0.17 0.13
*IT 0 indicates no any necrosis, or/and chlorosis on wheat after inoculation,
IT1-2 indicates necrosis, or/and chlorosis;
Pst=Puccinia striiformis f. sp. tritici; Pgt=Puccinia graminis f. sp. tritici
Pst isolates obtained from the infected Berberis spp.
in Gansu, Shaanxi and Tibet in 2013

27. Berberis
(origin)
Isolate
No.
Chinese differential hosts for Pst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
20
(ck)
Race /
race group
U1 AV V V V V V V V A V V A V VA A AV A A A V SuG
U2 A V V V V V V V A V AV A A V A A A A A V SuG
B.aggregata
(Gansu)
U3 A V A AV V V V V A V AV A A V A A A A A V SuG
U4 AV A V V AV V V V A V V V A V A V A A A V SuG
U5 V V V V V V V V V V V V V V A V V A A V CYR32
U6 A V AV V V V V V A V V V A V A A A A A V SuG
U7 V V V V V V V V A V V AV A V A A A A A V SuG
U8 V V V V V V V V V V V V V V A V V A A V CYR32
B.shensiana
(Shaanxi)
U 9 V V V V V V V V V V V V V V A V V A A V CYR32
U10 V V V V V V V V V V V V V V V V A A A V SuG
U11 A V A V V V V V A AV AV A A V A A A A A V SuG
U12 A V A AV V V V V A V AV A A V A A A A A V SuG
B.sp.
（Tibet)
U13 AV V V V A V V V A V V A A V A A A A A V SuG
U14 A V V V V V VA V A V V A A V A A A A A V SuG
U15 AV V V AV A V V A A V A A V V A A A A A V SuG
U16 A V A AV A V V V A A AV A A V A A A A A V SuG
• 3 recovered isolates were identified as race CYR 32.
• Other 13 isolates have different virulence patterns as compared
to the major Chinese races , and can be considered as new races.
Chinese differential hosts for Pst
A=avirulent ; V=virulent; AV/VA =segeration of resistant and susceptible; ck=Mingxian 169 susceptible to Pst.
SuG=race group virulence for Suwon 11(YrSu); HyG=race group virulence for Hyrbrid 46 (Yr3b,Yr4b)
CYR= chinese yellow rust
Virulence patterns of 16 recovered isolates on
Chinese differential host sets

28. Single Aecia
from barberry
Recovered isolate
from single aecium
Single uredium
(Su) isolates
Virulence test on
NILs
Does the sexual reproduction under natural
conditions contribute to virulence variation for
Pst ?
• 120 single-uredium (SU) isolates were make from 16 recovered
isolates from infected barberry.
• Virulence test for each SU isolates on near-isogenic lines.
• Remarkable variation in virulence patterns among the SU isolates.

29. Virulence patterns of single-uredium isolates
from recovered Pst isolate on near-isogenic lines
SU isolate
Yr NILs
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Yr2 Yr5 Yr6 Yr7 Yr8 Yr9 Yr10 Yr15 Yr17 Yr24 Yr26 Yr27 Yr44 YrSp YrTr1 YrExp2 Yr25 Yr28 CK
U10*
VA A V A A A A A A A AV A V A A A V V V
U10-1 A A V A A V A A A A A A V A A A V V V
U10-2 A A V A A A A A A A A A V A A A V V V
U10-3 A A V A A A A A A A A A V A A A V V V
U10-4 A A V A A V A A A A A A V V A A V V V
U10-5 A A V A A V A A V A A A V V A A V V V
U10-6 V A V AV A A A A V A A A V V A A V V V
U10-7 A A V A A V A A V A A V V V A A V V V
U10-8 A A V A A V A A A A A A V A A A V V V
U10-9 A A V AV A V A A A A A A V A A A V V V
• SU isolates U10-2 and U10-3 share the same virulence patterns,
• Other 7 SU isolates have different virulence patterns.
*U10 isolate recovered from Berberis shensiana in Shaanxi

30. SU
isolate
Yr NILs
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Yr2 Yr5 Yr6 Yr7 Yr8 Yr9 Yr10 Yr15 Yr17 Yr24 Yr26 Yr27 Yr44 YrSp YrTr1 YrExp2 Yr25 Yr28 CK
U2* A A A AV V A A A A A V A A A A V A A V
U2-1 A A A AV V A A A A A V A A A A V A A V
U2-2 A A A A V A A A AV A V A V A A V A A V
U2-3 A A VA AV V A A A A A V A A A A A A A V
U2-4 A A A A V A A A V A V A V A A V A A V
U2-5 A A AV A V A A A A A V A A A A V A V V
U2-6 A/V A V V V A A A A A V A V A A V A A V
U2-7 A A A A V A A A A A V A A A A V A A V
U2-8 VA A V A/VA V A A A V A V A VA A A V A A V
U2-9 A A AV A V A A A A A V A V A A V A A V
U2-10 A A A A V A A A A A V A A A A V A A V
U2-11 A A AV VA V A A A A A V A A A A V A A V
U2-12 A A AV A V A A A A A V A A A A V A A V
U2-13 A/VA A A/V A V A A A V A V A A A A V A A V
U2-14 A/V A A/V A/VA V A A A A A V A A/V A A V V VA V
“/” indicates various infection type on first and second leaf of genotype wheat cultivar. AV, VA= segregation of
resistant and susceptible.
*U2 isolate recovered form Berberis aggregata in Gansu.
• U2-7 and U2-10, share the same virulence patterns,
• Other 12 SU isolates have different virulence patterns.
Virulence patterns of single-uredium isolates
from recovered Pst isolate on near-isogenic lines

31. Single Aecia
from barberry
Recovered isolate
from single aecium
Single uredium
(Su) isolates
Virulence test on
NILs
Does the sexual reproduction under natural
conditions contribute to virulence variation for Pst?
• The single-uredium isolates originally from aecia on naturally
infected barberry plants consisted of diverse of virulence
patterns, which may be due to sexual reproduction on the
barberry plants.

32. Ø 23 of the 30 tested barberry species show susceptibility to Pst,
indicating that there is a great diversity of potential alternate
hosts for Pst in China.
Ø 20 Pst isolates obtained from different barberry species in the
fields indicated that the sexual stage of Pst occurs under
natural conditions.
Ø Virulence tests for single-uredium isolates recovered from a
single aecium demonstrate that the sexual reproduction
contributes to virulence variation for Pst.
Ø 23 isolates of wheat stem rust were obtained from 4 barberry
species in the fields, indicating that the sexual stage of wheat
stem rust also occurs under natural conditions in China.
Summary

33. Ø The earmarked fund for
Modern Agro-industry
Technology Research System in
China
Ø National Basic Research
Program of China (973)
Ø Nature Science Foundation of
China
Ø The 111 Project from the
Chinese Ministry of Education
Acknowledgement
Dr. Jie Zhao Prof. Lili Huang
Dr. Hongchang Zhang Dr. Gangming Zhan
Dr. Qingmei Han Dr. Yonghong Zhang
Dr. Dejun Han Dr. Xiaojie Wang
Dr. Jun Guo Dr. Xueling Huang
Dr. Wenming zheng Mrs. Guorong Wei
All PhD and Master students in My Lab.
Dr. X M. Chen, Dr. H. Buchenauer,
Dr. Robert McIntosh , Dr. Colin
Wellings, Dr. Scot H. Hulbert,
Dr. Jin-Rong Xu, Dr. Shiping Wang,
Dr. Hei Leung, Dr. J. Chong
Dr. Yue Jin, Dr. Ravi Singh,
Dr. Zacharias Pretorius

34. Thanks for your attention!