Biological properties of isolates of Triticum mosaic virus from the Great Plains states of the USA

Abstract

Triticum mosaic virus (TriMV) is a recently discovered virus infecting wheat. A total of 170 isolates of TriMV, collected in 2010 and 2011 from wheat (Triticum aestivum L) or jointed goatgrass (Aegilops cylindrica Host) from Colorado, Kansas, Nebraska and South Dakota, were compared with the 06-123 Kansas isolate. These isolates were compared for the percentage of infected plants in N28Ht maize, ‘Gallatin’ barley and ‘Mace’ wheat (with temperature-sensitive resistance to Wheat streak mosaic virus (WSMV). The isolates were also compared for the effect of inoculum virus titre on the percentage of infected plants in WSMV-susceptible ‘Tomahawk’ wheat by mechanically inoculating the cultivar using 1:10 w/v, 1:300 v/v or 1:600 v/v dilutions of extracts. None of the isolates infected N28Ht maize but all isolates infected ‘Gallatin’ barley, ‘Mace’ and ‘Tomahawk’ wheat. Some isolates from Colorado, Kansas and Nebraska had low relative titre in wheat compared with the 06-123 isolate. This information is important in critical selection of TriMV isolates for use in greenhouse and field studies and in resistance screening protocols.

Résumé

Le virus de la mosaïque du Triticum (TriMV) est un virus récemment découvert qui infecte le blé. En tout, 170 isolats de TriVM, collectés en 2010 et 2011 sur du blé (Triticum aestivum L.) ou de l’égilope cylindrique (Aegilops cylindrica Host.) au Colorado, au Kansas, au Nebraska et au Dakota su Sud, ont été comparés à l’isolat 06-123 du Kansas. Ils ont été comparés relativement au pourcentage de plants infectés chez le maïs N28Ht, l’orge ‘Gallatin’ et le blé ‘Mace’ (avec résistance thermosensible au virus de la mosaïque-bigarrure [WSMV]). Les isolats ont également été comparés quant à l’effet du titre en virus de l’inoculum ou au pourcentage de plants infectés chez le cultivar de blé ‘Tomahawk’ réceptif au WSMV, en l’inoculant mécaniquement avec les dilutions d’extrait suivantes : 1:10 w/v, 1:300 v/v ou 1:600 v/v. Aucun des isolats n’a infecté le maïs N28Ht, mais tous les isolats ont infecté l’orge ‘Gallatin’ et les cultivars de blé ‘Mace’ et ‘Tomahawk’. Certains des isolats provenant du Colorado, du Kansas et du Nebraska affichaient de faibles titres relatifs chez le blé, comparativement à l’isolat 06-123. Cette information est importante sur le plan de la sélection critique des isolats de TriMV quant à leur utilisation dans les études menées en serre et en champ ainsi que sur celui des protocoles de criblage à l’égard de la résistance.

Keywords:

• host range
• titre
• Triticum mosaic virus
• Wheat streak mosaic virus

Mots clés:

• gamme d’hôtes
• titre
• virus de la mosaïque-bigarrure
• virus de la mosaïque du Triticum

Introduction

Triticum mosaic virus  (TriMV) is a member of the family Potyviridae and is the type member in the new genus Poacevirus (Tatineni et al. 2009; http://www.dpvweb.net/notes/showgenus.php?genus=Poacevirus). It was first identified infecting wheat (Triticum aestivum L.) in Kansas in 2006 (Seifers et al. 2008). The virus is mechanically transmissible and is associated with a coat protein (CP) of approximately 35 kDa. Antiserum raised to the TriMV CP preferentially reacted only to TriMV in enzyme-linked immunosorbent assay (ELISA) and Western blot assay (Seifers et al. 2008). Symptomatic plants have been shown to be associated with flexuous rods when analysed by electron microscopy (Seifers et al. 2008).

TriMV has been identified from wheat in the Great Plains in Colorado, Nebraska, Oklahoma, South Dakota, Texas and Wyoming (Burrows et al. 2009). Field surveys were conducted in Colorado, Kansas, Nebraska and South Dakota in the spring and autumn of 2010 and 2011 to determine TriMV prevalence and incidence and the frequency of TriMV co-infection with Wheat streak mosaic virus (WSMV) or High Plains virus (HPV) in winter wheat (Byamukama et al. 2013). In that study, TriMV was detected in all four states, and WSMV was the most prevalent virus followed by TriMV and HPV. Ninety-one per cent of TriMV-positive samples were co-infected with WSMV, whereas WSMV and HPV were mainly detected as single infections. In another study, two isolates of TriMV from Colorado demonstrated biological diversity manifested as a weak reaction with antiserum prepared to the 06-123 isolate of TriMV and low virus titre in wheat when compared with 06-123 (Seifers et al. 2013). Initially, only naturally infected wheat and barley (Hordeum vulgare L.) were confirmed as hosts of TriMV (Seifers et al. 2008). The reaction of row crop species to infection by TriMV and WSMV has been documented and the differential hosts for these viruses have since been identified (Seifers & Martin 2009; Tatineni et al. 2009; Seifers et al. 2010).

Wheat curl mites (Aceria tosichella Keifer) (WCM) are the vector of TriMV (Seifers et al. 2009). The WCM transmits TriMV both singly and in combination with WSMV (Seifers et al. 2009). Thus, in addition to TriMV, WCM also transmits WSMV (Slykhuis 1955) and the HPV (Seifers et al. 1997).

Four isolates of TriMV – 06-123 (Seifers et al. 2008) and RW (Tatineni et al. 2009), as well as C10-492 and C11-775 from Colorado (Seifers et al. 2013) – have been used for biological studies. Except for the 06-123, C10-492 and C11-775 isolates, no side-by-side comparisons of biological characteristics have been made for isolates of TriMV. In this study, we tested isolates of TriMV (collected in 2010 and 2011 from Colorado, Kansas, Nebraska and South Dakota) for infection of N28Ht maize (Zea mays L.), ‘Gallatin’ barley, ‘Mace’ wheat (with temperature-sensitive resistance to WSMV (Graybosch et al. 2009) and ‘Tomahawk’ wheat (WSMV and TriMV susceptible cultivar) and determined relative virus concentration (titre) in ‘Tomahawk’ wheat.

Materials and methods

Virus source and maintenance

Isolate 06-123 of TriMV was collected from wheat KS06HW79 at the Kansas Agricultural Research Centre-Hays (KSU-ARCH), at Hays, Kansas in 2006 (Seifers et al. 2008). Information on the source of TriMV isolates collected in 2010 and 2011 are presented in Table 1. When the original plant sample reacted in ELISA against antibodies of WSMV and TriMV, the isolate of TriMV was freed of WSMV by passaging through ‘Arica’ triticale (× Triticosecale rimpani Wittmack) because it is susceptible to TriMV but not WSMV (Seifers et al. 2010). Leaves of the symptomatic triticale plants were then used to inoculate ‘Tomahawk’ wheat. Symptomatic ‘Tomahawk’ wheat plants were analysed by ELISA to verify presence of TriMV and not WSMV. The tissues were then frozen at −80 °C and later used to increase the respective isolates in different sets (Supplementary Tables 1–15).

Table 1. Triticum mosaic virus isolates used to inoculate maize, barley, and wheat.

Isolate^A	County
C10-43 to C10-57	Baca
C10-386	Prowers
C10-403 to C10-409	Unknown
C10-492 to C10-524	Baca
C10-1290	Bent
C10-1371 to C10-1447	Cheyenne
S10-2889	Hugh
N10-3323 to N10-3324	Perkins
N10-3374	Keith
N10-4437 to N10-4712	Cheyenne
N10-4930	Scottsbluff
N10-5071	Cherry
N10-5249 to N10-5263	Kimball
K10-7407 to K10-7410	Edwards
K11-80 to K11-85	Stevens
K11-94 to K11-118	Ness
K11-135 to K11-138	McPherson
K11-142 to K11-143	Osborne
K11-246 to K11-248	Clark
K11-256 to K11-261	Cheyenne
K11-262 to K11-266	Thomas
K11-276 to K11-279	Mitchell
K11-292	Pawnee
K11-636 to K11-643	Lane
K11-646	Mitchell
K11-654 to K11-658	Rawlins
C11-773 to C11-784	Unknown
C11-874 to C11-929	Sedgwick
N11-1504 to N11-1558	Box Butte
N11-1583 to N11-1615	Sioux
N11-1657	Box Butte
N11-1661 to N11-1678	Cheyenne
N11-2595 to N11-2625	Chase

^A The total number of isolates was 170. C, K, N and S indicate Colorado, Kansas, Nebraska and South Dakota, respectively. The numbers 10 and 11 indicate the years 2010 and 2011, respectively and the numbers following the dashes are the field sample number. All samples were wheat except N11-2595 to N11-2625 which are jointed goatgrass (Aegilops cylindrica Host).

Propagation of TriMV isolates

Seeds of ‘Tomahawk’ wheat were planted in 7 rows in each of 3 soil-filled (Harney clay loam soil, fine montmorillonitic, mesic type Argiustoll) flats (21 × 35 cm), with 8–10 seeds per row. The flats were held in a growth chamber (Percival Model PGC-15WC) at 22°C with 12 h/day fluorescent lighting (250 µEs⁻¹m⁻²) until the plants were at the single-leaf stage. The plants were mechanically (finger-rub) inoculated with a 1:10 (w/v) dilution of extract prepared from single wheat plants that tested positive in ELISA for TriMV only (see above). The inoculated plants were returned to the growth chamber. At 14 days post-inoculation, the plants were separately harvested for each isolate and several 1-gram portions (comprising 4–5 plants) were frozen at −80 °C. TriMV isolates were propagated in 15 sets with each set having 14 or fewer isolates. Each set was compared independently of the other sets. Isolate 06-123 was used as the positive control and was propagated in ‘Tomahawk’ wheat along with the other isolates of TriMV for a given set.

Planting and inoculation procedures

Seeds of N28Ht maize (provided by S. Wegulo, Univ. Nebraska, Lincoln, NE), ‘Gallatin’ barley (KSU-ARCH seed source), ‘Mace’ wheat (provided by Gary Hein, Univ. Nebraska) (‘Mace’ was not tested with virus sets 4 and 5) and ‘Tomahawk’ wheat (KSU-ARCH source) were planted into separate soil-filled (as above) flats (30- by 50-cm). The N28Ht maize was planted 2 days prior to the barley and wheat so that plants would be at the two-leaf stage of growth when the barley and wheat plants were at the single-leaf stage when mechanically (finger-rub) inoculated. Following planting, the flats were held in a greenhouse under natural light.

To establish if the plants could be infected with a given isolate, a single 14-row flat for the maize, a 28-row flat for the barley and ‘Mace’ wheat (14 rows of barley and 14 rows of ‘Mace’ wheat) and a 28-row flat for ‘Tomahawk’ wheat were planted identically and not randomized in each experiment. This was done because we were interested only in determining if the plants of maize, barley and ‘Mace’ wheat could be infected with a given isolate. These latter plants, and 14 rows of ‘Tomahawk’ wheat plants, were inoculated with extracts with a relative titre of 1:10 (w/v) of the appropriate isolate. The remaining 14 rows of ‘Tomahawk’ wheat were inoculated with an extract with a relative titre of 1:300 or 1:600 (v/v) (prepared from the 1:10 extract) of the appropriate isolate. The relative titre of an extract as used here is defined as the numbers of infected assay plants resulting from mechanical inoculation with a 1:10 dilution of plant extract when compared with the 1:300 or 1:600 dilutions prepared from the 1:10 dilution, so that the more dilute the plant extract is, the fewer virions are present in the extract, and therefore, fewer infected plants are expected. Although the 1:300 is in the linear-response range for determining relative concentrations of virions or titre in wheat infected with TriMV (Seifers et al. 2013), the 1:600 was used for most comparisons because isolates causing high infection rates at the 1:300 dilution were observed in the initial studies. Isolates were randomly assigned to rows prior to inoculation. Isolate sets used were 14 or fewer due to space requirements for planting and efforts were made to keep sets of isolates from the same geographic state; however, this was not possible in all cases. Following inoculation, the plants were held in a greenhouse under natural light and the percentage of symptomatic plants was recorded at 21 days after inoculation. Temperature ranges in the greenhouse for a given virus set are provided in each table. The experiment was conducted three times for virus sets 4 and 5 (Supplementary Tables 4 and 5) and two times for the other virus sets, and each experiment was considered a replication-in-time for the analysis of variance (ANOVA). Because differences among treatments exceeded 40 percentage points, the data were arcsine-transformed before conducting the ANOVA (Little & Hills 1978). The 1:10 and 1:300 and 1:600 infectivity data presented in the tables are untransformed. The infectivity data from the experiments for each virus set were subjected to ANOVA (SAS, version 8, SAS Institute, Cary, NC) and significant treatment effects were identified according to Fisher’s protected least significant difference LSD test (P = 0.05). In the following paragraphs, ‘means’ refers to mean percentages of symptomatic plants as determined from ANOVA and the use of ‘significant differences’, ‘differences’, ‘differ’ or ‘differed’ is in reference to the LSD at P = 0.05.

Results

None of the TriMV isolates or the 06-123 control isolate caused symptoms on N28Ht maize (data not shown). All isolates of TriMV in the 15 sets tested systemically infected ‘Gallatin’ barley. However, only 26 of the isolates had infection means that were significantly different from the respective 06-123 isolate control for a given virus set (Table 2). All means except that for the N11-2618 isolate (set 15) were lower than the mean for the 06-123 control for a respective set. Thirteen of the isolates were collected from Colorado, 7 from Kansas and 6 from Nebraska.

Table 2. Mean^A percentages of symptomatic ‘Gallatin’ barley plants 21 days after mechanical inoculation with a 1:10 w/v dilution of extracts from different sets of Triticum mosaic virus (TriMV) isolates compared with the Kansas 06-123 isolate of TriMV.

Set^B	Isolate^C	N^D	% Symptomatic^E	Set	Isolate	N	% Symptomatic
1	C10-386	18	55.6k-o	10	C11-898	15	53.5i-l
1	C10-492	18	36.8m-p	10	C11-899	16	56.3h-l
1	C10-524	19	57.2i-o	10	C11-902	19	52.6h-l
1	C10-1371	19	52.7l-o	10	06-123	19	84.4b-f
1	06-123	18	88.9a-e
				11	C11-926	16	56.2e-h
6	K11-261	19	63.2g-j	11	C11-927	16	56.2e-h
6	K11-262	23	68.4f-j	11	06-123	17	88.9a-d
6	K11-264	21	72.0e-j
6	K11-265	21	75.5c-i	12	N11-1596	15	52.6e-h
6	K11-266	21	76.3d-i	12	06-123	16	87.5a-d
6	K11-276	21	72.2d-i
6	K11-277	18	77.8b-i	13	N11-1667	16	48.4f-h
6	06-123	18	100a	13	N11-1676	18	61.1c-h
				13	06-123	18	94.4ab
8	C11-776	19	21.1j-m
8	C11-782	18	33.3i-l	15	N11-2608	17	40.2kl
8	C11-783	20	30.0i-m	15	N11-2609	17	42.3kl
8	C11-784	18	11.1lm	15	N11-2618	17	94.4ab
8	06-123	17	70.1d-h	15	06-123	16	68.7d-j

^A Means are not arcsine transformed.

^B Numbers of isolates of TriMV in each of the 15 sets were: 13, 13, 12, 11, 11, 11, 10, 11, 12, 11, 10, 13, 10, 11 and 11, respectively. Virus sets 2, 3, 4, 5, 7, 9 and 14 did not have any isolates whose means were different from the 06-123 control; therefore, the data were not included.

^C See Table 1 for information concerning the isolates.

^D N = Total number of plants mechanically inoculated. The temperature range for the experiments was 9–36 °C.

^E Treatments not having the same letter within each set (not across sets) in common are significantly different according to Fisher’s protected least significant difference (LSD; P = 0.05). The probability associated with the F value for each set was <0.0001.

Each isolate of TriMV from the 13 sets tested caused systemic infection of ‘Mace’ wheat. Twenty-three of the isolates had means significantly different from the mean of the respective 06-123 control (Table 3). Eleven isolates, 5 from Colorado (C10-492, C11-782, Cll-783, C11-784 and C11-785), 2 from Kansas (K11-277 and K11-292) and 4 from Nebraska (N11-1521, N11-1536, N11-1667 and N11-2600) had means lower than the mean for the 06-123 control, while all other means were higher. For all isolates, the symptoms induced in ‘Mace’ were a faint mosaic, while ‘Tomahawk’ wheat inoculated with the same isolates had a prominent mosaic.

Table 3. Mean^A percentages of symptomatic ‘Mace’ wheat plants 21 days after mechanical inoculation with a 1:10 w/v dilution of extracts from different sets of Triticum mosaic virus (TriMV) isolates compared with the Kansas 06-123 isolate of TriMV.

Set^B	Isolate^C	N^D	% Symptomatic^E	Set	Isolate	N	% Symptomatic
1	C10-492	13	23.1p	10	C11-897	15	100a
1	06-123	15	92.3a-c	10	C11-904	14	100a
				10	C11-908	16	100a
3	N10-4685	14	93.7ab	10	06-123	17	88.2b-e
3	N10-4712	18	83.3a-d
3	N10-5071	18	88.9a-c	12	N11-1521	15	47.3f-h
3	N10-5249	17	93.7ab	12	N11-1536	15	40.1gh
3	06-123	18	61.1f-j	12	06-123	13	83.3a-e
6	K11-277	21	84.0b-h	13	N11-1667	15	58.0d-h
6	K11-292	18	84.0b-h	13	06-123	16	100a
6	06-123	21	100a
				14	N11-2597	15	100a
8	C11-782	17	81.9b-g	14	N11-2599	17	100a
8	C11-783	17	88.2b-e	14	N11-2600	17	35.2lm
8	C11-784	18	33.3i-l	14	06-123	17	82.3b-f
8	C11-785	19	84.2b-f
8	06-123	18	100a	15	N11-2611	16	94.4ab
				15	N11-2612	15	94.4ab
				15	N11-2618	13	91.6a-c
				15	06-123	14	70.8d-j

^A Means are not arcsine transformed.

^B Numbers of isolates of TriMV in each of the 13 sets were: 13, 13, 12, 11, 10, 11, 12, 11, 10, 13, 10, 11 and 11, respectively. Virus sets 2, 7, 9 and 11 did not have any isolates whose means were different from the 06-123 control; therefore, the data were not included. Virus sets 4 and 5 were not tested for infection of ‘Mace’ wheat.

^C See Table 1 for information concerning the isolates.

^D N = Total number of plants mechanically inoculated. The temperature range for the experiments was 9–36 °C.

^E Treatments not having the same letter within each set (not across sets) in common are significantly different according to Fisher’s protected least significant difference (LSD; P = 0.05). The probability associated with the F value for each set was <0.0001.

All isolates of TriMV from the 15 sets tested systemically infected ‘Tomahawk’ wheat at the 1:10 and 1:300 or 1:600 dilutions. Using the 1:10 extracts, all isolates except K11-82 (96.3%), K11-259 (96.3%), C11-926 (92.6%), N11-1528 (94.4%), N11-1667 (80.05) and N11-2601 (75.5%), infected 100% of the ‘Tomahawk’ plants (Supplementary Tables 4, 6, 11, 12, 13 and 14, respectively). Except for the N11-2601 isolate, the means were not different from the means of the other isolates at this dilution. Three instances occurred where the means for the 1:10 dilution did not differ from that of the 1:300 extracts (06-123 and K11-259; supplementary Tables 4 and 5, respectively) and at the 1:600 dilution (C10-1412; supplementary Table 1). At the 1:300 or 1600 dilutions, 40 isolates had means that were different from the respective mean of the 06-123 control (Table 4). Of these, 15 isolates were collected from Colorado, 14 from Kansas, and 11 from Nebraska. Only the Colorado isolate C10-1412 had a mean higher than the respective 06-123 control.

Table 4. Mean^A percentages of symptomatic ‘Tomahawk’ wheat plants 21 days after mechanical inoculation with either 1:300 or 1:600 v/v dilutions of extracts from 15 different sets of Triticum mosaic virus (TriMV) isolates compared with the Kansas 06-123 isolate of TriMV.

Set^B	Isolate^C	N^D	% Symptomatic^E	Set	Isolate	N	% Symptomatic
1	C10-492	17	23.6o-p	10	C11-897	18	50.0i-l
1	C10-524	15	33.0n-p	10	C11-898	17	52.9i-l
1	C10-1412	17	94.4a-c	10	C11-899	19	42.2kl
1	06-123	16	68.7e-m	10	C11-900	19	37.2kl
				10	C11-901	17	35.3l
2	C10-1447	16	33.3g	10	C11-908	19	47.1j-l
2	06-123	17	76.5b-f	10	06-123	16	81.2b-h
4	K11-81	29	69.0d-f	11	C11-916	16	25.0h
4	K11-83	29	49.1f-i	11	C11-926	17	40.2gh
4	K11-85	26	57.7e-h	11	C11-928	17	40.9f-h
4	K11-94	31	60.7e-g	11	06-123	17	81.9b-e
4	K11-100	31	52.2f-i
4	K11-118	28	53.6f-i	13	N11-1672	17	36.1h
4	K11-136	30	73.3d-f	13	06-123	16	80.1b-g
4	06-123	25	92.0a-c
				14	N11-2598	16	43.7i-m
5	K11-138	33	72.8d-j	14	N11-2599	18	38.8k-m
5	K11-143	30	66.7e-k	14	N11-2600	17	41.2j-m
5	K11-258	30	70.0d-j	14	N11-2601	17	29.4m
5	06-123	32	90.9a-c	14	N11-2603	19	36.8k-m
				14	N11-2604	17	41.2j-m
6	K11-261	19	21.2l	14	N11-2605	16	43.7j-m
6	K11-262	20	30.1kl	14	06-123	19	78.9b-h
6	K11-263	19	53.0l-k
6	K11-276	19	41.6j-l	15	N11-2610	19	41.6kl
6	06-123	20	85.0b-h	15	N11-2613	15	41.0kl
				15	N11-2619	16	31.2l
8	C11-776	16	12.5m	15	06-123	17	70.7d-j
8	C11-784	17	12.5m
8	06-123	19	57.9f-j

^A Means are not arcsine transformed.

^B Numbers of isolates of TriMV in each of the 15 sets were: 13, 13, 12, 11, 11, 11, 10, 11, 12, 11, 10, 13, 10, 11 and 11, respectively. Virus sets 3, 7, 9 and 12 did not have any isolates whose means were different from the 06-123 control; therefore, the data were not included. Virus sets 4 and 5 were at a 1:300 v/v dilution.

^C See Table 1 for information concerning the isolates.

^D N = Total number of plants mechanically inoculated. The temperature range for the experiments was 9–36 °C.

^E Treatments not having the same letter within each set (not across sets) in common are significantly different according to Fisher’s protected least significant difference (LSD; P = 0.05). The probability associated with the F value for each set was <0.0001.

Discussion

Triticum mosaic virus (06-123 isolate) was first identified in Kansas in 2006 (Seifers et al. 2008). Since then, it has been reported from many states in the Great Plains (Burrows et al. 2009; Byamukama et al. 2013). The 06-123 isolate of TriMV from Kansas infects barley (Hordeum vulgare L.), rye (Secale cereale L.) and oats (Avena sativa L.), but not maize (Zea mays L.) (Seifers et al. 2008, 2010). The Red Willow isolate from Nebraska also infects barley, oat and rye, but not maize (Tatineni et al. 2010). The C10-492 and C11-775 isolates from Colorado have been shown to have biological diversity (Seifers et al. 2013). When these two Colorado isolates of TriMV were compared with the 06-123 isolate, they reacted weakly against antiserum prepared to the 06-123 isolate, caused significantly lower relative virus concentration or titre that was manifested as lower infection means, and caused significantly less reduction in wheat dry matter accumulation compared with 06-123. Based on these findings with the C10-492 and C11-775 isolates, the question arose as to how much diversity in biological behaviour might be present in an extensive array of TriMV isolates. We report characteristics of isolates of TriMV collected in 2010 and 2011 from Colorado, Kansas, Nebraska and South Dakota for infection of maize, barley, wheat with temperature-sensitive resistance to WSMV (Graybosch et al. 2009), and relative virus concentration or titre in ‘Tomahawk’ wheat.

In previous studies, the 06-123 and Red Willow isolates of TriMV did not infect maize but infected barley (Seifers et al. 2008, 2010; Tatineni et al. 2010). In this study, we have shown that all isolates and the 06-123 control failed to infect N28Ht maize. In addition, all isolates and the 06-123 control infected ‘Gallatin’ barley, which has been shown to be a host for TriMV but not WSMV (Seifers et al. 2010). Thus, both N28Ht maize and ‘Gallatin’ barley should serve as diagnostic hosts when working with TriMV isolates as has been previously reported (Seifers et al. 2010).

‘Mace’ wheat has temperature-sensitive resistance to WSMV (Graybosch et al. 2009). This cultivar is susceptible to infection with the Red Willow isolate of TriMV (Tatineni et al. 2010; Byamukama et al. 2012). We also observed that ‘Mace’ became infected by all isolates that were used to inoculate it in this study. In the present study, the symptoms expressed by ‘Mace’ in the greenhouse consisted of a faint mosaic, while ‘Tomahawk’ infected with the same isolates had a moderate mosaic, possibly indicating that TriMV replicates more slowly in ‘Mace’. Previous work has shown that the Red Willow isolate replicates poorly in ‘Mace’ (Tatineni et al. 2010) and mild symptoms have been noted for ‘Mace’ under greenhouse conditions when inoculated with the Red Willow isolate, indicating that the resistance to WSMV may also be effective to a lesser extent for TriMV (Byamukama et al. 2012).

For a virus that infects wheat to be considered as a threat, it is necessary to demonstrate that significant reductions in yield occur following infection. Infection of wheat by the 06-123 isolate of TriMV caused significant yield losses in replicated field trials at Hays, Kansas in the cultivars ‘Danby’, ‘RonL’ and ‘Jagalene’ but not the wheat line KS96HW10-3 (Seifers et al. 2011). Similarly, in a 2-year field study, the Red Willow isolate of TriMV caused significant reductions in yield and yield components (spikes/m², kernels/spike, 1000-kernel weight) in ‘Millennium’ but not ‘Mace’ (Byamukama et al. 2014). In addition, when the C10-492 and C11-775 isolates from Colorado were compared with the 06-123 isolate in a growth chamber experiment, they caused significantly less reduction in dry matter accumulation and this was associated with low relative virus concentration (Seifers et al. 2013). We observed significantly lower relative virus concentration manifested as lower infection means in ‘Tomahawk’ wheat for some of the isolates from Colorado, Kansas and Nebraska based on low infection percentages for the 1:300 and 1:600 treatments. However, it remains to be demonstrated that the low relative virus concentration observed for these isolates is associated with less reduction in dry matter accumulation and yield reduction in replicated trials in the field in diseased plants. Based on the observations for C10-492 and C11-775 (Seifers et al. 2013), it would be appropriate when selecting field isolates for use in studies or resistance screening efforts to determine if they have a low relative virus concentration in wheat because these isolates may provide conflicting results when compared with results obtained from an isolate such as 06-123 (Seifers et al. 2011) and the Red Willow isolate (Byamukama et al. 2012).

In summary, none of the TriMV isolates infected N28Ht maize but infected ‘Gallatin’ barley and ‘Mace’ wheat (among those isolates tested). Analysis for relative virus titre in ‘Tomahawk’ showed that some of the isolates from Colorado, Kansas and Nebraska had low relative virus concentrations in wheat as was observed for the Colorado C10-492 and C11-775 isolates of TriMV (Seifers et al. 2013). Thus, future work should be directed towards determining if such isolates cause yield reduction in replicated field trials when compared with the 06-123 isolate (Seifers et al. 2008) or the Red Willow isolate from Nebraska (Tatineni et al. 2009). This information is valuable for critical selection of TriMV isolates for use in greenhouse and field studies and resistance screening programmes.

Acknowledgements

We thank Jeff Ackerman for his valuable assistance during all phases of these studies and Clayton Seaman for data analyses, and Ned Tisserat and all persons at Colorado State University involved in sample collection. This research has been assigned contribution No. 13-279-J from the Kansas Agricultural Experiment Station. Funding for this work was provided by the Agriculture and Food Research Initiative Competitive Grants Program Grant No. 2010-85605-20546 from the National Institute of Food and Agriculture. The findings and conclusions in this paper do not necessarily reflect the view of the US Department of Agriculture.