New evidence supporting the occurrence of sexual reproduction in the wheat stripe rust fungus on barberry in China

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

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

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

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

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

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.

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?

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.

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?

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

Surveys for Berberis spp.in China

No barberry plant Barberry plants distributed

Barberry plants with rusts

30 barberry species were surveyed and identified.

Over-summering area

B. brachypoda B. shensiana

Berberis soulieana B. potaninii

Are these Berberis spp. susceptible to wheat stripe rust ?

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.


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


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).

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

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

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

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

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.

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

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

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

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

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

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.

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

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

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.

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

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

Thanks for your attention!