Wheat is one the most important cereals for human consumption worldwide, and it occupies second place in production [1]. Wheat production in Mexico is concentrated in the states of Sonora (54.16%), Guanajuato (13.96%), Baja California (7.99%), Sinaloa (8.44%), Michoacan (6.60%), Jalisco (3.65%), and Chihuahua (2.25%) [2]. This crop provides more nourishment than any other source of food, and it is most important source of carbohydrates in most countries in temperate countries [3]. This cereal is used primarily for flour and whole flour production, semolina, beer, and a wide variety of food products [4]. The crop may be affected by pests and diseases caused by bacteria, mycoplasma, fungi, nematodes, viruses and viroids, which may affect the foliage, roots, stems, spikes, and grains [5], and consequently will affect the grain yield and quality. Therefore, production costs might increase due to the use of agrochemicals for the control of pests and diseases. Karnal bunt (KB) caused by the fungus Tilletia indica Mitra (syn. Neovossia indica (Mitra) Mundkur) occurs in nature in bread wheat [6], durum wheat (T. durum Desf.), and triticale [7]. Infected grains generally are partially affected [8-10] (Figure 1). This disease was first reported in India [6], then, in Mexico [11], Pakistan [12], Nepal [13], Brasil [14], the United States [15], Iran [16], South Africa [17] and Afghanistan [18].

KB is a quarantine disease in Mexico which was detected in the Yaqui Valley during the wheat season 1969-1970 [11]; the importance of KB is directly related to the negative effect on the organoleptic characteristics of the wheat flour [19-21]. This in turn, has caused economic losses to wheat producers due to rejection of wheat lots by the industry, loss of wheat seed exports, and phytosanitary regulations [22]. Control of Tilletia indica is difficult since teliospores are resistant to physical and chemical factors [23-26]. Chemical control can be accomplished by applying fungicides during flowering [27-37]; however, this measure is not feasible when quarantines do not allow tolerance levels for seed production [38]. The use of resistant wheat cultivars is the best control method for this disease, and it also would reduce the possibilities of its introduction into karnal bunt-free areas. Since the 1940’s several species of Triticum have been evaluated for resistance to karnal bunt [9,39-41]. Bread wheat is the species most affected by the disease; under artificial inoculation some lines may show more than 50% infected grain [42,43]; therefore, it is important to keep evaluating the new advanced lines and wheat cultivars. The objective of this work was to evaluate experimental bread wheat germplasm for the reaction to the fungus Tilletia indica under artificial inoculation in the field.

Figure 1 (a,b): Symptoms of Karnal bunt (Tilletia indica) in the grain of bread wheat (Triticum aestivum)

One hundred and fifty-seven elite advanced bread wheat lines were evaluated for resistance to Karnal bunt (Tilletia indica) by artificial field inoculation during the season 2020-2021 at the Norman E. Borlaug Experimental Station which belongs to the Mexican National Institute for Livestock, Agriculture and Forestry Research (INIFAP). The station is located in block 910 in the Yaqui Valley, Sonora, Mexico at 27º22´3.01” N, 109º55´40.22” W, and 37 masl. Sowing dates were November 28, December 8 and 17, 2020, using 8 g of seed for a row 0.7 m long on a bed with two rows in a clay soil with pH 7.8. For the agronomic management, INIFAP´s technical recommendations were followed [44]. For field inoculations, the inoculum was prepared by isolation of teliospores from one year old infected grains through shaking in a test tube with a water-tween solution, sieving (60 μm) (Figure 2), incubation of teliospores in the water-tween solution at 22-24°C for 24 h, followed by centrifugation at 3,000 rpm in a 0.5% sodium hypochlorite solution, decanting, centrifugation in a sterile water, and decanting. Plating of teliospores was done on 2% water-agar Petri plates. After teliospore germination, fungal colonies were transferred and multiplied on potato-dextrose-agar [45].

Inoculations were carried out by injecting 1 mL of an allantoid sporidial suspension (10,000/mL) during the boot stage (stage 49 according to Zadoks et al. scale [46]) in five spikes per line following the method described by Chona et al. [10], modified by Fuentes-Dávila et al. [47] (Figure 3). The susceptible wheat KBSUS was also used during the evaluation as a reference check. High relative humidity in the experimental area was provided by an automatic mist spray-irrigation-system five times a day for 20 min each time (Figure 4). To avoid bird damage, an anti-bird net system was installed in the area used for evaluation of the wheat lines. The period of inoculation of the wheat nursery started on February 1 and finished on March 1, 2021, making a total of 20 days with inoculation in this particular nursery. Harvest was carried out manually, and the counting of healthy and infected grains was done visually to determine the percentage of infection. The wheat lines originated from the collaborative project between the Global Wheat Program of the International Maize and Wheat Improvement Center (CIMMYT) and INIFAP.

Figure 2 A,B,C,D: A) Sieving teliospores through a 60 μm mesh; B-D) teliospore germination; E-G) identification of areas where teliospores are germinating; G) transfer of fungal growth structures on the lid of petri plates with potato-dextrose-agar; H) multiplication of the fungus on PDA

Figure 3: Inoculation with Tilletia indica by injection of 157 elite advanced bread wheat lines during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 4: Antibird net and mist irrigation system in the experimental area

The range of the percentage of infection of the elite advanced lines in the first date was 1.56 to 69.2%, with an average of 29.18%; there were not lines without any infected grains (Figure 5). Within the arbitrary infection categories, three lines were within the range 0.1-5.0%, seven within 5.1-10.0%, 81 within 10.1-30%, and 66 with more than 30% infection (Figure 6). Lines with the lowest percentage of infection in the first sowing date were: ESTOC/7/2*KISKADEE#1/5/KAUZ*2/ MNV//KAUZ/3/MILAN/4/BAV92/6/WHEAR//2*PRL/2*PASTOR with 1.56, SUP152/BAJ#1//KIDEA with 2.87, and WBLL1*2/BRAMBLING*2//BAVIS*2/3/KACHU#1/KIRITATI//KACHU with 4.96, while those lines with the highest percentage of infection in the same date were: PRL/2*PASTOR/3/WBLL1*2/SHAMA//KACHU/4/KACHU//WBLL1*2/BRAMBLING with 69.02%, SOKOLL/3/PASTOR/ /HXL7573/2*BAU/4/PASTOR//MILAN/KAUZ/3/BAV92/5/2* MUCUY with 68.52%, and WBLL1*2/BRAMBLING/ /TAM200/TUI/3/VILLAJUAREZF2009/4/KACHU*2/3/ND643//2*PRL/2* PASTOR with 63.64%.

Figure 5: Percentage of infection with Karnal bunt, in 157 elite advanced bread wheat lines artificially inoculated in the first sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 6: Karnal bunt infection categories (%) of 157 elite advanced bread wheat lines artificially inoculated in the first sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 7: Percentage of infection with Karnal bunt, in 157 elite advanced bread wheat lines artificially inoculated in the second sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 8: Karnal bunt infection categories (%) of 157 elite advanced bread wheat lines artificially inoculated in the second sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

The range of the percentage of infection of the elite advanced lines in the second date was 0.61 to 71.84% with an average of 29.63%; similarly to the first sowing date, there were not lines without any infected grains (Figure 7). Within the infection categories, two lines were withing the range 0.1-5.0%, six within 5.1-10.0%, 79 within 10.1-30%, and 70 with more than 30% infection (Figure 8). Lines with the lowest percentage of infection in the second sowing date were: ESTOC/7/2*KISKADEE #1/5/KAUZ*2/MNV//KAUZ/3/ MILAN/4/BAV92 /6/ WHEAR//2*PRL/2*PASTOR with 0.61, and ND643// 2*PRL/2* PASTOR*2/3/HEILO/4/KAKURU with 3.38, while those lines with the highest percentage of infection in the same date were: TUKURU//BAV92/RAYON/ 3/ND643/2*WBLL1/4/BORL14 con 71.84%, BORL14*3/ /LALBMONO1*4/PVN with 70.49%, and PRL/2*PASTOR/ /KACHU/3/SEHER06/4/KACHU/3/WHEAR//2*PRL/2* PASTOR con 64.47%. The range of the percentage of infection of the elite advanced lines in the third date was 1.12 to 68.97% with an average of 27.96%; as in the first and second sowing dates, there were not lines without any infected grains (Figure 9). Within the infection catagories, three lines were within the range 0.1-5.0%, five within 5.1-10%, 89 within 10.1-30%, and 60 with more than 30% infection (Figure 10). Lines with the lowest percentage of infection in the third sowing date were: ND643//2*PRL/ 2*PASTOR*2/3/HEILO/4/ KAKURU with 1.12, WBLL1*2/ BRAMBLING*2//BAVIS*2/3/KACHU#1/KIRITATI//KACHU with 1.67, and SUP152/BAJ#1//KIDEA with 4.33, while those lines with the highest percentage of infection in the same date were: KASUKO/4/CIRO16*2/3/MUU #1/SAUAL// MUU with 68.97%, PFAU/WEAVER*2/TRANSFER#12,P88.272.2/3/WHEAR//2*PRL/2*PASTOR/4/SWSR22T.B./2*BLOUK#1//WBLL1*2/KURUKU/5/SWSR22T.B./2*BLOUK#1//WBLL1*2/KURUKU with 68.86%, and CROC_1/AE.SQUARROSA(205)// BORL95/ 3/PRL/SARA//TSI/VEE#5/4/FRET2/5/CIRO16/6/ONIX/KBIRD/7/KACHU/3/WHEAR//2*PRL/2*PASTOR with 65.97%.

Figure 9: Percentage of infection with Karnal bunt, in 157 elite advanced bread wheat lines artificially inoculated in the third sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 10: Karnal bunt infection categories (%) of 157 elite advanced bread wheat lines artificially inoculated in the third sowing date during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 11: Percentage of infection with Karnal bunt, in 157 elite advanced bread wheat lines artificially inoculated in three sowing dates during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

The overall range of infection of this nursery of elite advance bread wheat lines was 2.89 to 62.68% with an average of 28.92% (Figure 11). Within the infection categories, three lines were within the range 0.1-5.0%, two within 5.1-10.0%, 83 within 10.1-30%, and 69 with more than 30% infection (Figure 12). The range of infection of the susceptible check was 65.9% on February 24, 2021, to 100% on February 12, with an average of 85.6%. the average of the three highest percentages of infection of the susceptible check was 99.08%. Lines that showed an overall percentage of infection lower than 5% in the three sowing dates are shown in Table 1. The lines that showed the overall highest percentage of infection were: TUKURU//BAV92/RAYON/3/ND643/2*WBLL1/ 4/BORL14 with 62.68%, BORL14*3//LALBMONO1*4/ PVN with 57.01%, and W15.92/4/PASTOR//HXL7573/2*BAU/3/ WBLL1/6/VEE/MJI//2*TUI/3/2*PASTOR/4/ BERKUT/5/PFAU/ MILAN/7/ROBINK with 56.18%.

Table 1: Elite advanced bread wheat lines that showed an overall percentage of Karnal bunt infection lower than 5.0%, after artificial inoculation in three sowing dates during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Figure 12: Karnal bunt infection categories (%) of 157 elite advanced bread wheat lines artificially inoculated in three sowing dates during the crop season 2020-2021, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico

Althoug there were only three lines with an overall percentage of infection lower than 5%, two other lines had an overall percentage of infection lower than 10%; the first three qualify as sources of resistance and also, similar to the last two lines, as candidates for commercial release. The group of lines evaluated in this work had 1.81% of resistant germplasm to Karnal bunt and 43.9% susceptible. Since the identification of resistant Aegilops spp. to Tilletia indica, and the generation of synthetic wheats by interspecific hybridization between Triticum turgidum x Triticum tauschii (Coss.) Schmalh. (syn. Aegilops squarrosa L.) followed by chromosome doubling of F₁ hybrids, synthetic hexaploid derivatives have been produced with the objective to incorporate resistance into bread wheat from the best synthetic hexaploids with good agronomic characteristics and cytogenetic stability.

Out of the 157 elite advanced bread wheat lines of the present work, 7.64% carry in their pedigree a synthetic with the potential to be a contributor for the resistance to Karnal bunt of a particular bread wheat line. In the case of lines having in their pedigree CROC_1/AE. SQUARROSA (205), their average percentage of infection was 25.14 which place them in the category of moderately susceptible, with the exception of one line that had 48.49%. In the case of two lines with T. DICOCCONPI94624/AE.SQUARROSA (40), the average percentage of infection was 32.43 which is classified as susceptible to Karnal bunt, and both lines were susceptible; in the case of other two lines with CROC_1/AE:SQUARROSA(213), the average percentage of infection was 25.86, but one lines was moderately susceptible and the other one susceptible; in the case of one line with CROC_1/AE.SQUARROSA(224), it showed a susceptible reaction with 32.88%; and in the case of the line with CROC_1/AE.SQUARROSA(210), the reaction was as moderately susceptible with 20.92%.

In the case of lines carrying in their pedigree the resistant line to Karnal bunt MUNAL#1 (Fuentes-Dávila et al., 2014) which is used as check as well as in combination with other lines (Fuentes-Dávila et al., 2018b), out of 9 lines, one line was moderately resistant, six were moderately susceptible, and two were susceptible; the average percentage of infection of this group was 21.98%. Medina-Uriarte et al. (2013) evaluated 180 elite bread wheat lines, from which 27.2% showed resistance to Karnal bunt and 9.4% were susceptible; those lines with the lowest percentage of infection were: BAJ#1/3/KIRITATI//ATTILA*2/PASTOR with 0.28%, SAUAL/KIRITATI//SAUAL with 0.33%, TAM200/ PASTOR//TOBA97/3/HEILO with 0.40%, another sister lines of SAUAL/KIRITATI//SAUAL with 0.47%, SAUAL/4/ CROC_1/AE.SQUARROSA(205)//KAUZ/3/ATTILA/5/SAUAL with 0.71%, and two sister lines of MUNAL#1 with 0.75 and 0.76%. Fuentes-Dávila et al. (2014) evaluated 46 elite bread wheat lines out of which 41.3% were resistant to KB and 4.3% susceptible; in this evaluation, lines that showed the lowest percentage of infection were: SWSR22T.B./5/KAUZ//ALTAR84/AOS/3/KAUZ/4/SW94.15464/6/2*PRL/2*PASTOR with 0.35%, MUNAL#1 with 0.82%, WHEAR/VIVITSI//WHEAR/3/PANDORA with 0.99%, NINGMAI96035/FINSI//HEILO/3/NAVJ07 with 1.13%, MUNAL/3/HUW234+LR34/PRINIA//PFAU/ WEAVER/4/ MUNAL#1 with 1.36%, BAVIS/VORB/5/ CROC_1/AE.SQUARROSA(205)//BORL95/3/PRL/SARA//TSI/VEE#5/4/FRET2 with 1.48%, PRL/2*PASTOR/3/ PFAU/WEAVER*2//CHAPIO with 1.89%, and BABAX/ LR42//BABAX/3/ER2000/4/NAVJ07 with 1.94%. Fuentes-Dávila et al. reported that 27.3% out of 168 elite bread wheat lines were resistant to KB. They also reported that out of 85 elite bread wheat lines evaluated for resistance to Kb, 23.5% were resistant while 1.17% were susceptible (2018b). For the nursery evaluated in the present work, 43.9% of the group of lines was susceptible to KB, while only 1.9% was resistant. Therefore, evaluation of the new bread wheat germplasm generated must be a continuous effort in order to assure aceptable resistance levels to KB.

Out of 157 elite advanced bread wheat lines evaluated for their reaction to the fungus Tilletia indica, causal agent of Karnal bunt, only three lines showed a percentage of infection lower than 5% after field artificial inoculation in three sowing dates: ESTOC/7/2*KISKADEE#1/5/ KAUZ* 2/MNV//KAUZ/3/MILAN/4/BAV92/6/WHEAR//2*PRL/2*PASTOR, ND643//2*PRL/2*PASTOR*2/3/HEILO/4/ KAKURU, and WBLL1*2/BRAMBLING*2//BAVIS*2/3/ KACHU#1/KIRITATI//KACHU. These lines may be used as sources of resistance to Karnal bunt in wheat breeding programs, and if other agronomic and quality traits are expressed by these lines, they may be also considered as candidates for commercial release as cultivars.

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