Identification of durum wheat (Triticum turgidum ssp. durum L.) germplasm from Iran, Italy, Argentina, and Bulgaria with resistance at the seedling stage to race Ug99 of Puccinia graminis f. sp. tritici

Abstract

We evaluated seedling responses of a diverse set of durum wheat accessions originating from Serbia, Afghanistan, Portugal, Bulgaria, Argentina, Iraq, Italy, and Iran to Puccinia gramini f. sp. tritici. Morphologically superior durum accessions (118 entries) were screened against two isolates of the pathogen from Lorestan and Khuzestan provinces of Iran previously typed as TTKSK (Ug99) at the seedling stage. The putatively resistant entries were tested in replicated experiments along with germplasm known to possess resistance genes, such as ‘Amigo’, ‘Fleming’, ‘Eagle’, ‘Agatha’, ‘Sage’, and ‘RL5711’. We identified putatively novel sources of resistance to Ug99, characterized by infection types 0, 0;, ;1, ;2, ;1+, and 22+. We also observed a previously unreported highly susceptible seedling response in germplasm containing the gene Sr13 to Ug99. The resistant durum accessions include ‘Wc-47208’ from Bulgaria (IT = 0;), ‘Wc-47191’ from Argentina (IT = 0;), ‘Tn-12716’ from Iran (IT = ;1), and ‘P.S.No18’ and ‘P.S.No19’ from Italy (IT = ;2 or ;1+). This study emphasized that local landraces in national gene banks are rich in disease resistance genes and may be useful in breeding efforts to combat Ug99 stem rust.

Résumé

Nous avons évalué la réaction de diverses séries d'obtentions de blé dur provenant de la Serbie, d'Afghanistan, du Portugal, de Bulgarie, d'Argentine, d'Iraq, d'Italie et d'Iran à Puccinia gramini f. sp. tritici. Des obtentions (118) de blé dur supérieur sur le plan morphologique ont été criblées, au stade de semis, avec deux isolats de l'agent pathogène appartenant à la race classifiée comme TTKSK (Ug99), provenant des provinces du Lorestan et du Khuzesta, en Iran. Ces obtentions présumées résistantes ont été testées au cours d'expériences répétées avec des germoplasmes possédant des gènes de résistance, comme ceux contenus dans les cultivars tels que ‘Amigo’, ‘Fleming’, ‘Eagle’, ‘Agatha’, ‘Sage’ et ‘RL5711’. Nous avons identifié de présumées sources de résistance à Ug99 caractérisées par les types d'infection 0, 0;, ;1, ;2, ;1+ et 22+. Nous avons également observé une très forte réaction des semis à Ug99 chez le germoplasme contenant le gène Sr13, réaction qui, jusqu’à maintenant, n'avait pas été rapportée. Les obtentions de blé dur résistantes incluent ‘Wc-47208’ de Bulgarie (IT = 0;), ‘Wc-47191’ d'Argentine (IT = 0;), ‘Tn-12716’ d'Iran (IT = ;1) ainsi que ‘P.S.No18’ et ‘P.S.No19’ d'Italie (IT = ;2 ou ;1+). Cette étude a mis l'accent sur le fait que les variétés locales contenues dans les banques de gènes nationales sont riches en gènes de résistance et peuvent être utiles lors des efforts de sélection déployés pour combattre la race Ug99 causant la rouille noire des céréales.

Keywords:

• durum wheat
• stem rust
• breeding
• disease resistance
• seedling infection

Mots clés:

• Blé dur
• rouille noire des céréales
• sélection
• résistance à la maladie
• infection des semis

Introduction

Over 200 million hectares of agricultural lands are under wheat cultivation worldwide (Rosegrant et al., 1995). Wheat contributes to one-fifth of the food calories and the protein consumed by billions of people in around 100 developing countries (Braun et al., 2010). Wheat productivity is limited by many biotic and abiotic stresses. Rust diseases (e.g., stripe, leaf, and stem rusts) are of prime importance, and severe wheat yield losses due to stem rust epidemics have been reported in Europe, Asia, Australia, and the United States before the middle of the 20th century (Roelfs, 1978; Saari & Prescott, 1985). A new virulent race of Puccinia graminis f. sp. tritici, capable of infecting wheat lines carrying Sr31, was identified in Uganda wheat nurseries. This new isolate, typed as TTKSK, became popularly known as Ug99 after the country and the year of discovery (Pretorius et al., 2000). Similarly virulent isolates were observed in 2001 in Kenya, 2003 in Ethiopia, and 2007 in Yemen and Iran (Nazari et al., 2009). All of the stem rust resistance (Sr) genes identified to date (McIntosh et al., 2008) are race specific except for the gene Sr2 (McIntosh et al., 1995; Spielmeyer et al., 2003; Singh et al., 2006). Disease response phenotyping tests have revealed that only a few of the characterized genes are effective in bread wheat against Ug99 (Jin et al., 2007).

In bread wheat germplasm, the genes Sr24, Sr25, Sr26, and Sr39 are amongst the resistant genes effective against TTKSK race of stem rust. Currently, the only known effective resistance gene against Ug99 in durum wheat is Sr13 (Simons et al., 2011), which is present within US durum wheat cultivars. This gene originates from the Ethiopian landrace ST464 and the domesticated emmer wheat (T. turgidum ssp. dicoccon L.) ‘Khapli’ (McIntosh, 1972; Klindworth, 2007). Sr13 was located to the distal region of the long arm of chromosome 6A by McIntosh (1972). To combat the threat posed by Ug99, breeders require effective sources of resistance and knowledge about existing sources of resistance to stem rust disease. Here, we report on the seedling stage response to Ug99 of a diverse set of durum wheat accessions from Serbia/Montenegro (formerly known as Yugoslavia), Afghanistan, Portugal, Bulgaria, Argentina, Iraq, Italy, and a relatively larger number of durum landraces from Iran.

Materials and methods

Plant materials and screening strategy

A total 116 durum wheat accessions from Iran (84 accessions), Serbia (formerly Yugoslavia, 2 accessions), Afghanistan (6 accessions), Portugal (4 accessions), Bulgaria (1 accession), Argentina (2 accessions), Iraq (3 accessions), Italy (14 accessions), along with two durum wheat cultivars (‘Dena’ and ‘Zardak’), and two breed wheat cultivars ( ‘Vee/Nac’ and ‘Soissons’) were tested (see Supplemental Information). All accessions were initially evaluated using race TTKSK at the seedling stage; germplasm showing resistant phenotypes were subsequently tested using two independent isolates typed as TTKSK in replicated experiments (r = 6) (see Supplemental Information). We also included control lines known to carry specific stem rust resistance genes, plus the susceptible check line ‘McNair701’ (Table 1).

Table 1.  Infection type (IT) data for control germplasm and resistant durum accessions are shown

Control entries	Sr genes	CPU01	CPU02
Fleming	Sr24, SrAmigo	;	;1
Amigo	SrAmigo	;1	;1
Triumph	SrTmp	2	2
RL6087	Sr40	;2	;1
RL5711	Sr39	;1	;1
Eagle	Sr26	;1	;1
Agatha	Sr25	;1	;1
SWSr22TB	Sr22	;1	;1
Tam200	Sr24	;1	;1
Sage	Sr24	;1	;1
Kota	Sr28	;2	;2
Chinese Spring*5/Thatcher 3B	Sr12	4	4
Prelude*4/2/Marquis*6/Khapstein	Sr13	4	33+
W2691Sr13	Sr13	3	3
W2691*2/Khapstein	Sr14	33+	4
McNair-701	susceptible	3+	33+
Durum selections		CPU01	CPU02
Wc-47208-Bulgaria	unknown	0;	0;
Wc-47191-Argentina.1	unknown	0;	0;
Wc-47191-Argentina.2	unknown	0;	0;
Tn-12716-Dezful, Iran .1	unknown	;1	;1
Tn-12716-Dezful, Iran.2	unknown	;2	;1
P.S.No18-Italy.1	unknown	;2	;1+
P.S.No18-Italy.2	unknown	;2	;1+
P.S.No19-Italy.1	unknown	;1+	;2
P.S.No19-Italy.2	unknown	;1+	;2
P.S.No19-Italy.3	unknown	;1+	;2
Dena	unknown	;1+	;1+
IT equals to 0;, ;, ;1, ;2, 22+, and 2 indicate resistance and IT equals to 3, 33+, or 4 demonstrate susceptible. Lines with the same identifiers but differing in the last digit are different pure lines head-rowed from each landrace. Most of these lines seem to be identical but the durum wheat breeding program at CRD, SPII kept them separate by different identifiers.

Disease phenotyping

Two isolates previously typed as TTKSK (CPU01 and CPU02) were used to inoculate fully expanded primary leaves of 7-day-old (2-leaf stage) seedlings of wheat grown under a controlled environment (18 °C temperature day and night and 16 / 8 h photoperiod). For inoculum preparation, urediniospores of P. graminis f. sp. tritici stored in a –80°C freezer were heat-shocked at 40°C for 10 min. After incubation in a rehydration chamber for 2 h at approximately 80% relative humidity, urediniospores were mixed with talc powder in a 1 to 3 ratio and dusted homogenously onto seedling leaves by using a small paint brush. Plants were immediately placed in a dew cabinet (dark chamber) maintained at 18°C overnight (approximately 14 to 16 h) and then kept in a greenhouse set at 18 ± 2°C with a photoperiod of 16 h. We assessed infection types (IT) as described by Stakman et al. (1962) 14 days post-inoculation. After the first round of screening, 120 entries with TTKSK resistant lines were evaluated with isolates CPU01 and CPU02 in replicated experiments (6 reps) along with control entries (3 reps).

Agronomic performance

The agronomic performance of the resistant durum accessions is included in this report. Lines were planted in the fall in three rows of one meter long each. We assessed variations in plant height (PLNTHT), spike length (SPKLNG), days to heading (DAYSHD), days to maturity (DAYSMT), spikelets per spike (SPLSPK), grain numbers per spike (GRNSPK), 1000 kernel weight (THOUWT), and grain yield (GRNYLD). Description of traits and abbreviated names are in accordance with trait dictionary for phenotyping recommended by integrated breeding platform (available online at: https://integratedbreeding.net/crop-information/wheat). PLNTHT was measured on five randomly selected culms. SPKLNG, SPLSPK, GRNSPK, and THOUWT were measured on five randomly selected spikes. DAYSHD and DAYSMT were recorded when 50% of tillers within each plot headed or matured, respectively.

Results and discussion

The best strategy to mitigate the threat of Ug99 is to identify resistant sources among existing breeding materials well adapted to the prevalent environments. In this study, we characterized resistance to Ug99 and agronomic performance of a diverse collection of 118 durum wheat accessions. The standard check ‘McNair-701’ demonstrated a susceptible phenotype (IT = 3+), indicating successful establishment of stem rust disease in our experiments (Table 1). Lines known to possess Ug99 resistance (‘Fleming’, ‘Amigo’, ‘RL6087’, ‘RL5711’, ‘Eagle’, ‘Agatha’, ‘SWSr22TB’, ‘Tam200’, ‘Sage’, and ‘Kota’) displayed low phenotypes (IT = ;, ;1, or ;2) to both isolates. Out of 118 durum accessions tested, 10 durum accessions along with a national check cultivar ‘Dena’ were found resistant to Ug99, demonstrating infection types of 0, 0;, ;1, ;2, ;1+, 22+, and 2+3 as shown in Table 1. These accessions are from Italy, Argentina, Bulgaria, and Iran. Their Genbank accession numbers are as follow: ‘Wc-47208’ from Bulgaria (IT = 0;), Wc-47191 from Argentina (IT = 0;), ‘Tn-12716’ from Iran (IT = ;1), and ‘P.S.No18’ and ‘P.S.No19’ from Italy (IT = ;2 or ;1+).

Simons et al. (2011) reported that Sr13 is one of the effective resistance genes against TTKSK in US durum wheat germplasm. We examined the infection type of Chinese Spring*5/Thatcher 3B (Sr12), Prelude*4/2/Marquis*6/Khapstein (Sr13), ‘W2691Sr13’ (Sr13), and W2691*2/Khapstein (Sr14) when inoculated with TTKSK and all were susceptible (Table 1). While Sr12 and Sr14 were previously reported to be susceptible to TTKSK (Jin et al., 2007), Sr13 should be effective.

Low incubation temperature can cause a higher IT response for gene Sr13 (McIntosh et al., 1995); this may explain the susceptible response. Another reason may be that the race used in this study is virulent on Sr13. While the resistant durum lines identified in this study may have Sr13, they must have another gene(s) since they exhibited ITs that are lower than what is normally conditioned by Sr13 (IT = 22⁺).This requires further genetic investigation to determine the mode of action and genetics of resistance for these putative novel Ug99-resistant tetraploid wheat accessions.

The identified durum germplasm in this study may be considered amongst the best available sources of resistance for breeding to Ug99. Accessions from Italy and Iran yielded at least one standard deviation greater than the average of germplasm tested (Table 2). Accession from Bulgaria yielded below average of the entire 118 accessions, whereas two accessions from Argentina yielded grain yields around average (Table 2). Nevertheless, this study was successful in identifying new sources of durum wheat with resistance to Ug99 and good agronomic background which should be useful to durum breeders worldwide. Inquires for germplasm may be directed to durum wheat program leader Dr. Mostafa Aghaee-Sarbarzeh at [email protected].

Table 2.  Traits collected from the field for resistant germplasm and descriptive statistics for the entire population are presented

Germplasm	PLNTHT	SPKLNG	DAYSHD	DAYSMT	SPLSPK	GRNSPK	THOUWT	GRNYLD
Ug99 resistant durum accessions
Wc-47208-Bulgaria	110	8.6	187	216	102	244	36.48	72
Wc-47191-Argentina.1	131	9.5	188	216	109	264	46.21	100
Wc-47191-Argentina.2	125	9	187	216	111	261	41.38	90
Tn-12716-Dezful, Iran .1	93	8.5	185	223	106	276	48.19	144
Tn-12716-Dezful, Iran.2	92	9	184	223	105	306	41.18	158
P.S.No18-Italy.1	90	9.3	183	222	100	305	47.54	226
P.S.No18-Italy.2	95	10	184	223	98	247	46.56	128
P.S.No19-Italy.1	90	8.6	183	219	95	278	36.69	160
P.S.No19-Italy.2	87	8	184	218	98	278	35.61	120
P.S.No19-Italy.3	92	9	183	217	90	285	35.44	144
Statistics of 118 durum accessions
Average	116.7	8.2	186	219	102	236	49	105
SD	17.3	1.16	3.48	2.74	21.9	49.8	7.1	34.8
Min	71.7	5.5	179	216	73	113	27	24
Max	180	12.2	198	225	313	359	64	226
PLNTHT is height of plant at maturity in cm, SPKLNG is measurement of spike length in cm, DAYSHD is number of days to heading, DAYSMT is number of days to physiological maturity, SPLSPK is number of spikelets per spike based on the average of five spikes, GRNSPK is count of total number of grains on an average of five spikes, THOUWT is measurement of 1000 well-developed grains in grams, and GRNYLD is measurement of total grains harvested from three rows of one meter long each in grams.

Acknowledgements

This work was conducted at the Seed and Plant Improvement Institute (SPII) and financially supported under SPII grant numbers 02-03-03-89044 to MAS (durum wheat program leader) and 2-03-03-90011 to MM. The authors are thankful to crew members of the Cereals Disease Facilities at SPII for technical help and to Mr. Mehran Patpour for granting the use of isolates. Special thanks are also to Dr. William Yajima for providing critical review of the text.

Supplemental Electronic Information (XLS format). The one replicate Ug99 test for all 120 entries along with replicated Ug99 test for resistant genotypes are presented. Agronomic traits are also presented for all entries for traits including PLNTHT, SPKLNG, DAYSHD, DAYSMT, SPLSPK, GRNSPK, THOUWT, and GRNYLD. Traits definitions are given in footnote of Table 2.