Introduction

Brassica napus (oilseed rape or canola) and B. carinata (Ethiopian mustard) are important crop species worldwide. Their uses include providing oils for human consumption, forage for animal feed, biofuels and soil conditioners (Stefanski et al. 2010; Chauhan et al. 2006; Dixon 2007; Babu et al. 2013). Turnip mosaic virus (TuMV) is one of three principal virus diseases that cause economically important losses in Brassica crops worldwide (Green and Deng 1985; Walsh et al. 1989; Górecka and Lehmann 2001; Babu et al. 2013; Kehoe et al. 2010; Walsh and Tomlinson 1985). Expression of TuMV symptoms in Brassica crops depends on virus strain, host plant and environmental conditions, but typical symptoms include systemic vein clearing, mosaic, necrosis, plant stunting and, in some cases, plant death. The economic importance of TuMV varies between countries. It has a devastating effect on Brassica crops in China where infection with it is widespread (Liu et al. 1990; Yoon et al. 1993; Jiang et al. 2007). Examples of the losses it causes elsewhere include seed yield losses of up to 70 % in infected B. napus in the UK (Hardwick et al. 1994), root yield losses of c.40 % in Cochlearia arnoracia L (horseradish) in Poland (Górecka and Lehmann 2001), 50 % reduction in number and weight of marketable harvested B. oleracea capitata (cabbage) heads in Kenya (Spence et al. 2007), and 30 % losses in B. napus napobrassica (swede) in Canada (Shattuck and Stobbs 1987). It reduces numbers and sizes of seed pods, numbers of seeds, and seed size in B. napus (Walsh and Tomlinson 1985). It also causes quality defects such as internal necrotic disorders in stored B. oleracea capitata (Hunter et al. 2002).

Integrated Disease Management (IDM) is the most effective approach to manage virus diseases worldwide (Jones 2001, 2004, 2006, 2009; Coutts et al. 2010; Thresh 1982, 2006). Use of virus-resistant cultivars when available, provides an important approach to managing TuMV (Hughes et al. 2002; Walsh et al. 2002; Wang et al. 2009; Gladysz and Hanus-Fajerska 2009). However, additional resistance breaking TuMV pathotypes may arise and temperature sensitive single gene virus resistance may breakdown at higher temperatures as a result of climate change (Jones and Barbetti 2012). This is likely to affect Brassica crop productivity detrimentally as some of the world’s major Brassica producing areas are projected to experience significant increases in temperature (Barbetti et al. 2012). Phytosanitary (=hygiene) and cultural control measures should therefore be combined with host resistance to ensure effective control. Chemical control of aphid vectors in commercial Brassica plantings is unlikely to be effective with non-persistently aphid-borne viruses like TuMV, may pose a health hazard to humans and the environment, and increase resistance to pesticides in insect vectors (e.g. Jones 2004, 2006; Atreya et al. 2012).

Single dominant TuMV resistance genes TURB01 (Walsh et al. 1999), TURB03 (Hughes et al. 2003), TURB04 and TURB05 (Jenner et al. 2002, 2003) in B. napus, and TURB01b (Rusholme et al. 2007) in B. rapa give pathotype-specific TuMV resistance. Recessive TuMV resistance gene (retr01) and dominant TuMV resistance gene (conTR01) provide broad spectrum resistance to all TuMV pathotypes in B. rapa (Rusholme et al. 2007; Walsh et al. 2002). In Australia, two studies evaluated Brassica species for TuMV resistance phenotypes: Coutts et al. (2007) tested Australian B. napus accessions, breeding lines, and cultivars and Kehoe et al. (2010) evaluated a limited collection of B. carinata, B. nigra (black mustard) and B. juncea (Indian mustard) germplasm, and some crosses between B. juncea and B. napus. Their findings were encouraging and highlighted the need for more expansive studies involving greater diversity of germplasm of both B. napus and B. carinata. Further, there have been no studies examining the effect of temperature on expression of TuMV resistance phenotypes in B. napus or B. carinata. This study: (i) defines the range and occurrence of TuMV resistance phenotypes across a diverse selection of B. napus and B. carinata accessions, breeding lines, and cultivars from Africa, China, the Indian subcontinent and Australia, (ii) emphasises the dangers in only categorising B. napus and B. carinata lines at a single temperature, and (iii) highlights opportunities to improve TuMV resistance in oilseed and forage Brassicas through crosses involving TuMV resistance sources in B. napus and B. carinata. The risks associated with studies that only utilise a single TuMV pathotype in resistance screening are also examined. The paper also discusses potential future outcomes regarding TuMV epidemics in Brassica crops resulting from projected global warming scenarios.

Materials and methods

Germplasm and general plant growth conditions

Seeds of diverse accessions, breeding lines, and cultivars of B. napus [99 lines: Australia (57), China (35), India (5), France (1) and Canada (1)] were obtained from various seed sources, including from: a collection at The University of Western Australia (UWA); a completed Australian Centre for International Agriculture (ACIAR) collaborative Brassica project between Australia, India and China (Li et al. 2007, 2006, 2009); and a previous Australian study (Coutts et al. (2007)). Seeds of diverse accessions, breeding lines, and cultivars of B. carinata [32 lines: Australia (2), Malawi (5), Pakistan (4), Tanzania (16) and unknown locations (5)] were sourced via the Asian Vegetable Research and Development Centre (AVRDC), Tanzania and the seed collection at UWA. Seeds of all lines were grown in standard potting mix. Both culture hosts and experimental plants were grown in air-conditioned glasshouses or controlled environment rooms at UWA, or glasshouses at the Department of Agriculture and Food Western Australia (DAFWA) which were heated in winter and evaporatively cooled in spring to autumn. All these plant growing facilities were aphid-proofed.

TuMV inoculum and inoculation

The TuMV isolates used were WA-Ap1 belonging to pathotype 8 (Coutts et al. 2007), and NSW-1 and NSW-2 belonging to pathotypes 7 and 1, respectively [pathotyping by Walsh, cited by Kehoe et al. (2010)]. Isolates were maintained by serial subculture using sap inoculation to plants of B. juncea Tendergreen, B. rapa Evigwe 2013 (formerly called B. carinata IP 101 by AVRDC) or B. alba /B. hirta. Sap inoculation was done by grinding TuMV infected leaves from culture hosts in an extraction buffer containing disodium hydrogen phosphate (Na2HPO4) (11.5 g/L) and monosodium phosphate (NaH2PO4) (3 g/L) and the infective sap mixed with ‘celite’ (diatomaceous earth) before being rubbed onto the leaves of healthy plants. Graft inoculation was done using top-grafts made with WA-Ap1 infected scions from TuMV-infected or healthy Brassica spp. scions. After grafting, the plants were enclosed within transparent plastic bags for 1 week to maintain high humidity and allow graft unions to form. Plants were always inoculated when they had 2–3 true leaves.

Enzyme-linked immune-sorbent assay (ELISA)

Leaf samples from inoculated leaves and uninoculated tip leaves of plants were tested for TuMV by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) as described by Clark and Adams (1977). Leaf samples were extracted (1 g per 20 ml) in phosphate-buffered saline (10 mM potassium phosphate, 150 mM sodium chloride, pH 7.4), Tween 20 at 5 ml/L, and polyvinyl pyrrolidone at 20 g/L using a mixer mill (Retsch, Germany). All samples were tested in duplicate wells in microtitre plates, and sap from TuMV-infected and healthy B. juncea Tendergreen leaf samples was always included in paired wells to provide controls. The substrate used was p-nitrophenyl phosphate at 0.6 mg/ml in diethanolamine, pH 9.8, at 100 ml/l. Absorbance values at 405 nm (A 405) were measured in a Bio-Rad microplate reader (model 680; Bio-Sys, Australia). Absorbance values of positive samples were always more than three times those of the healthy sap control. The polyclonal antiserum specific to TuMV used was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany.

Responses to inoculation

Evaluation of germplasm

Plants of B. napus lines were maintained between May and October 2012 in glasshouses at c.19 °C (+/−2 °C) at UWA, or c.22 °C +/−3 °C at DAFWA. Plants of B. carinata lines were grown in the same locations from November 2012 to January 2013 in glasshouses at c.16 °C (+/−2 °C) (UWA) and c.25 °C +/−3 °C (DAFWA). Plants (7–14 per line) were inoculated with infective sap containing TuMV isolate WA-Ap1. Two plants of each line were kept uninoculated as healthy control comparisons. For 4–6 weeks after inoculation (wai), plants were inspected weekly for symptoms. Samples from inoculated and tip leaves were tested by ELISA 3–4 wai, or, if systemic symptoms were severe and developed rapidly, as early as 10–14 days after inoculation (dai). Uninoculated leaves recorded as uninfected were always re-sampled and tested again at 6 wai. Symptom reactions were classified using the phenotype codes of Jenner and Walsh (1996) and the subsequent code additions of Coutts et al. (2007) and Kehoe et al. (2010). These phenotype codes (not all of which were found) are: O, extreme resistance; +, susceptibility; RN, localised hypersensitivity; R, resistance to systemic movement without necrosis; RN/+, systemic infection with necrosis limited to inoculated leaves; +N, systemic infection with some necrosis; +ND, systemic hypersensitivity and plant death; RN/St, severe variant of RN with systemic necrotic stem streaking; RN/St/+, severe variant of RN/+ with systemic necrotic stem streaking, chlorotic mottle and leaf deformation; +N 1, mild variant of +N; and +St, severe stunting variant of +.

Effect of temperature on phenotype

Additional plants of the 18 B. napus and 14 B. carinata lines which only developed phenotype O in the germplasm evaluation study were re-tested by inoculation with isolate WA-Ap1. Procedures for classification of symptoms and testing for virus infection using ELISA were as described above under ‘evaluation of germplasm’. Two different lots of B. napus plants were inoculated at different times and maintained in glasshouses over spring to summer (November 2012 to January 2013) or summer to autumn (February to April 2013). One lot of B. carinata plants were inoculated during summer to autumn. During spring to summer, 16 plants of each line were inoculated and maintained at c.16 °C (+/−2 °C) (UWA), and seven plants at c.25 °C (+/−3 °C) (DAFWA). Two plants of each line were always left as healthy controls at each location. During summer to autumn, inoculated plants were kept at c.18 °C (+/−2 °C) (UWA) and c.25 °C (+/−3 °C) (DAFWA). Seven plants of each line were inoculated in each location and two were left uninoculated as control plants. Next, additional plants of the same 14 B. carinata and 18 B. napus lines were inoculated with isolate WA-Ap1 and maintained in controlled environment rooms at constant temperature (16 or 28 °C) with a 12 h photoperiod. Five to seven plants were inoculated per line and 1–2 plants of each left as healthy controls. Finally, six lines with uniform expression of one or other of three different TuMV phenotypes [B. napus P624 (RN), 06-6-3777 (O), Scoop (+), Ag Muster (+) and B. carinata TZ-SMN-44-6 (RN) and PI 390133 (+)] were selected from the germplasm evaluation study. A group of 18 plants of each of these lines was sub divided into two lots of nine plants, seven were sap inoculated and two served as controls. Immediately after inoculation, each lot was placed in controlled environment rooms operating at 16 or 28 °C. The experiment was repeated twice.

Temperature shift experiment

Plants (12-14/line) that developed TuMV phenotype O (B napus 06-6-3777) or phenotype + (B. carinata PI 390133) in the germplasm evaluation study, or phenotype + (B. napus Ag-Outback) in a previous study by Coutts et al. (2007), were each sub divided into two lots (6–7 plants/line) which were then sap inoculated with isolate WA-Ap1. Two uninoculated plants from each line served as healthy controls. Immediately after inoculation, plants from the first lot were placed in a controlled environment room at 16 °C with a 12 h photoperiod and kept there for 6 days. Half of these plants (3–4/line) were then exposed to 28 °C with a 12-h photoperiod for 8 days before being moved back to 16 °C and the other half were left at 16 °C throughout. Plants from the second group were placed in a controlled environment room at 28 °C and kept there for 6 days. Half of these plants (3–4/line) were then exposed to 16 °C with a 12 h photoperiod for 8 days before being moved back to 28 °C and the other half were left at 28 °C throughout. At each temperature, the procedures for classification of symptoms and testing for virus infection by ELISA were as described above under ‘evaluation of germplasm’.

Effect of graft inoculation

To establish whether the four lines which always developed phenotype O (B. napus 06-6-3777 and B. carinata IP 117, ST 18 and ST 50) in the above experiments regardless of temperature conditions had extreme resistance, seven plants of each were top-grafted with TuMV-infected scions (two scions/plant). Two healthy plants of each line were mock-inoculated by grafting with healthy scions as controls. Procedures for symptom recording and testing of leaf samples by ELISA were as described as described above under ‘evaluation of germplasm’, except that the samples tested were from tip leaves from axillary shoots that grew just beneath the graft union.

Effect of pathotype

Plants of two B. carinata and four B. napus lines that developed uniform reactions in the germplasm evaluation study were inoculated with TuMV isolates NSW-1 and NSW-2. These lines were: B. napus Ag Muster and Scoop (phenotype +), P624 (phenotype RN) and 06-06-3777 (phenotype O), and B. carinata PI 390133 (phenotype +) and TZ-SMN-44-6 (phenotype RN). Nine plants of each were inoculated and two plants of each line were left uninoculated as controls. These plants were grown in a glasshouse at c.22 °C at UWA. Procedures for symptom classification and detection of the virus in leaf samples by ELISA were as described above under ‘evaluation of germplasm’.

Results

Evaluation of germplasm

B. napus

When sap inoculated with TuMV isolate WA-Ap1, 95/99 B. napus lines developed no systemic infection (Table 1). Of these, 42 lines developed localised hypersensitivity phenotype RN (Fig. 1a) uniformly (i.e. in all plants) and 18 extreme resistance phenotype O uniformly. The lines which developed phenotype O uniformly were all grown at c. 19 °C (+/−2 °C). The 35 other lines that developed no systemic infection segregated for two phenotypes O and RN (30), R and RN (2), O and R (1) or three phenotypes O, R and RN, (2). Out of the 30 lines which segregated for phenotypes O and RN, 11 developed phenotype O in >half of the plants and the remaining 19 mainly developed phenotype RN. Two Indian lines segregated for systemic resistance phenotype +N (Fig. 1b) in combination with either phenotype RN or O or both. The +N phenotype, was not recorded in any other lines regardless of their origin. Susceptibility phenotype + (Fig. 1c) occurred in all plants of two Australian lines but never in any of the other 97 lines. Overall, phenotypes +N, R or + occurred infrequently compared to phenotypes O and RN.

Table 1 Reactions of plants of 99 Brassica napus accessions, breeding lines, and cultivars, mostly from Australia, China and the Indian sub-continent, following sap inoculation with Turnip mosaic virus
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Fig. 1
figure 1

Symptoms caused by infection of B. napus and B. carinata plants following sap inoculation with TuMV isolate WA-Ap1. a B. napus 03-p74-3 showing phenotype RN (necrotic spots in inoculated leaves without systemic infection) at 9 days after inoculation (dai). b B. napus GSL 2 showing phenotype +N (systemic infection, mosaic and leaf deformation with necrosis) at 51 dai, c B. napus Ag Muster showing phenotype + (systemic mosaic in uninoculated leaves). d B. carinata ST 27 showing phenotype R [faint chlorotic spots (arrows) in inoculated leaves] at 10 dai. e B. carinata TZ-SMN 1–1 showing phenotype RN (necrotic spots in inoculated leaves without systemic infection) at 9 dai. f a healthy leaf of B. napus ZY 008 control plant at 10 dai

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Phenotype O developed uniformly in 12 B. napus lines of Chinese origin, five from Australia, and one from France. Fourteen B. napus lines from China developed uniform RN responses and nine from China segregated for more than one phenotype, compared with 27 lines from Australia and one from Canada which developed uniform RN responses, and 23 lines from Australia which segregated for more than one phenotype. The five Indian lines all segregated for different phenotypes.

B. carinata

Inoculation of 32 B. carinata lines with TuMV WA-Ap1 failed to induce systemic infection in 18 of them (Table 2). Of these, 14 lines also developed no symptoms in inoculated leaves and no TuMV was detected in them (phenotype O), two developed chlorotic spots in inoculated leaves and TuMV was detected in them (phenotype R) (Fig. 1d) and another two developed necrotic spots in inoculated leaves (phenotype RN) (Fig. 1e). One line from Pakistan (PI 390133) developed chlorotic spots in both inoculated and uninoculated leaves and the virus was detected in both (phenotype +). Segregation for phenotypes O and R occurred in 12/32 lines and line TZ-SMN-1-1 segregated for phenotypes RN and R.

Table 2 Reactions of plants of 32 Brassica carinata accessions, breeding lines, and cultivars from Africa, Australia and the Indian sub-continent following sap inoculation with Turnip mosaic virus
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Phenotype O developed uniformly in one or more lines of B. carinata from each of three continents: Africa (11), Australia (2) and Asia (1). African lines B/C 13 A and TZ-SMN-44-6 produced phenotype RN uniformly while the others from Africa segregated for phenotypes O and R with phenotype O being commonest. In contrast, in the two lines from Pakistan which segregated for phenotypes O and R, phenotype R predominated. Line PI 390133 from Pakistan was the only one with phenotype +.

Effect of temperature on phenotype

B. napus

In the spring to summer glasshouse evaluation, 10 of the 18 B. napus lines gave phenotype O uniformly at c.16 °C in comparison to only four of these lines at c.25 °C (Table 3). These four were Chinese lines (06-6-3777, ZY 016, 03-p74-4 and 03-p74-11). Although no line developed uniform phenotypes R and RN at c.16 °C, at c.25 °C phenotypes R and RN occurred uniformly in three and one lines, respectively. Also, although only four lines segregated for each of two (O and RN) and three (O, R, and RN) phenotypes at c.16 °C, at c.25 °C nine lines segregated for two phenotypes (O and RN (6), O and R (2) or R and RN (1)), and one for three phenotypes (O, R and RN).

Table 3 Effect of different temperature regimes on the responses to inoculation with Turnip mosaic virus of 18 Brassica napus accessions, breeding lines, and cultivars with phenotype O (extreme resistance) in the initial germplasm evaluation
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In the summer to autumn glasshouse evaluation, 12 of the 18 lines developed phenotype O uniformly at c.18 °C in comparison to only two Chinese lines (06-6-3777 and 06-p71-1) at c.25 °C (Table 3). At c.18 °C, uniform phenotypes R or RN developed in one line each in comparison to three lines with phenotype R and five with phenotype RN at c.25 °C. The line Fan 028 developed phenotype RN uniformly at both temperatures. At c.18 °C, four lines segregated for different phenotypes while eight did so at 25 °C. At c.18 °C, O and RN was the only phenotype combination, whereas at c.25 °C there were three combinations of phenotypes: O and R (3), O and RN (3), and R and RN (2).

In the spring to summer and summer to autumn glasshouse evaluations, five Chinese lines (06-6-3777, ZY 016, 06-p71-1, ZY 011, ZY 013), one Australian line (RT 125) and French line Cresor developed phenotype O uniformly at c.16 °C and c.18 °C, but of these only 06-6-3777 did so at c.25 °C (Table 3). None of the lines produced phenotypes R and RN uniformly at c.16 °C or c.18 °C, but at c.25 °C Australian line Av Opal and Chinese line Ding 110 developed uniform phenotypes R and RN, respectively. When the total values for the spring to summer and summer to autumn glasshouse evaluations were compared, the same resistance phenotypes were present in seven of the 18 lines: one of these (06-6-3777) had uniform phenotype O and the other six had phenotypes O and RN (4), O and R (1), or O, R and RN (1).

In the controlled environment room evaluations, 14 lines of the 18 lines developed phenotype O uniformly at 16 °C, but only four of these (Chinese lines 06-6-3777, 06-p71-1 and 03-p74-4 and Australian line RT 117) did so at 28 °C (Table 3). None of the lines developed phenotype R uniformly at both 16 °C and 28 °C. Although no lines developed phenotype RN uniformly at 16 °C, at 28 °C phenotype RN occurred uniformly in five Chinese lines (Ding 110, Fan 028, QU 1104, ZY 007 and ZY 016). Four lines segregated for two phenotypes at 16 °C (O and RN), but at 28 °C eight lines segregated for two phenotypes [O and RN (2) or O and R (6)], and one for three phenotypes (O, RN and RN/+).

Across the individual temperature results from each evaluation cycle, four Chinese lines 06-6-3777, 06-p71-1, ZY 011, ZY 013, Australian line RT 125 and French line Cresor always developed uniform phenotype O at low temperature, but only Chinese line 06-6-3777 always did so at high temperature. At low temperature, none of the lines developed uniform phenotypes R or RN, but Ding 110 developed phenotype RN uniformly at high temperatures. When the total values of each glasshouse evaluation cycle (i.e. spring to summer and summer to autumn) were compared, seven of the 18 lines always developed the same phenotypes: 06-6-3777 only had phenotype O, while six segregated for phenotypes O and RN (4), O and R (1), or O, R and RN (1). When the total values for glasshouse and controlled environment room evaluations were compared, 11 lines always developed the same phenotypes: 06-6-3777 only had phenotype O, six segregated for phenotypes O and RN, two for O and R, and another two for O, R and RN.

The above results revealed that although only one line always developed phenotype O uniformly (06-6-3777) regardless of temperature, the proportion of plants with this phenotype was >75 % in three others (Chinese lines 06-p71-1, 06-p74-4 and French line Cresor). With phenotype RN, although Chinese line Ding 110 always developed it uniformly at high temperature, one Chinese (Fan 028) and one Australian (Hyola 42) line had >50 % of plants with this phenotype. In contrast, phenotype R was never predominant in any line.

In a further controlled environment room evaluation, B. napus line P624, which responded to TuMV inoculation with RN phenotype uniformly in the germplasm evaluation, developed the same RN phenotype uniformly 3 days earlier at 28 °C than at 16 °C (Table 5; Fig. 2a). At 28 °C, Ag Muster and Scoop, which developed phenotype + uniformly in the previous germplasm evaluation, developed phenotype + (consisting of faint local chlorotic spot lesions in inoculated leaves at 4 dai and faint systemic chlorotic rings in uninoculated leaves at 8 dai which subsequently faded away, and the virus was detected in tip leaves). At 16 °C, 1/7 plants of Ag Muster developed phenotype RN, 3/7 developed phenotype R (consisting of faint chlorotic spot lesions in inoculated leaves at 8 dai some of which turned into necrotic spot lesions as the plants grew older and 3/7 were symptomless). Systemic infection failed to develop at 16 °C in both Ag Muster and Scoop. For the first time, one plant of 06-6-3777 developed phenotype RN (at 16 °C). This single plant represented the only one of this line which did not develop phenotype O in more than 78 inoculated plants.

Fig. 2
figure 2

a Symptoms induced by TuMV isolate WA-Ap1 in inoculated leaves of B. napus P624 at 9 days after inoculation (dai). Left, large local necrotic spot lesions in a leaf from a plant grown at 28 °C. Right, small local necrotic spot lesions in a leaf from a plant grown at 16 °C. b and c, symptoms in B. carinata PI 390133 plant initially maintained at 28 °C and then shifted to 16 °C at 14 dai, 8 days after plants were moved from 28 °C: b local chlorotic rings in inoculated leaves, c small and numerous chlorotic rings in inoculated leaves. d Inoculated leaves of B. carinata PI 390133 plants at 14 dai, 8 days after shifting plants between 16 and 28 °C: (1) and (4) from plants kept at 28 and 16 °C throughout, respectively, (2) from plants shifted from 16 to 28 and (3) plants shifted from 28 to 16 °C. Insert is a close up of a leaf section: Leaf 1 has faint chlorotic rings, leaf 2 has symptomless infection, leaf 3 shows large severe chlorotic rings, leaf 4 is symptomless. e Inoculated B. carinata PI 390133 leaf shifted from 28 to 16 and then back to 28 °C showing chlorotic rings which came together to form irregular yellow patches at 23 dai

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B. carinata

During the summer to autumn glasshouse evaluation, 11 of the 14 B. carinata lines developed phenotype O at c.18 °C in comparison to only five at c.25 °C (Table 4). At c.18 °C, three lines segregated for phenotypes O and R and eight did so at c.25 °C. Although no line produced uniform phenotype R at c.18 °C, this phenotype occurred uniformly in IP 333 at c. 25 °C.

Table 4 Effect of two different temperature regimes on the responses to inoculation with Turnip mosaic virus of 14 Brassica carinata accessions, breeding lines, and cultivars with phenotype O (extreme resistance) in the initial germplasm evaluation
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In the controlled environment room evaluations, 12 lines developed phenotype O at 16 °C but only six did so at 28 °C (Table 4). None of the lines developed phenotypes R or + uniformly at 16 °C or 28 °C. At 16 °C, one line each segregated for phenotypes O and RN, or R and +. At 28 °C, eight lines segregated for phenotypes O and R (3), O and + (4), or O, R and + (1).

With the temperature results from each evaluation cycle, 10 lines always developed phenotype O uniformly at low temperature but only three (IP 117, ST 18 and ST 50) did so at high temperature. None of the lines developed uniform phenotypes R or + at low or high temperatures. When the values from each evaluation cycle were compared (Table 4), four lines always developed the same phenotypes: IP 117, ST 18 and ST 50 developed phenotype O, and ST 16 segregated for phenotypes O and R. However, although three lines (IP 117, ST 18 and ST 50) always developed phenotype O uniformly, the proportion of plants with this phenotype was >75 % in six others (Mbeya Green, 30200533, P 195923.3, ML-EM-7, ML-EM-8 and ML –EM- 1). In contrast, phenotypes R and + were never predominant within any line.

In a further controlled environment evaluation, line TZ-SMN-44-6 which responded to TuMV inoculation with phenotype RN uniformly in the germplasm evaluation, developed the same RN phenotype three days earlier at 28 °C than at 16 °C (Table 5). At 28 °C, susceptible line PI 390133 developed faint local chlorotic spot lesions in inoculated leaves at 4 dai and faint systemic chlorotic rings in uninoculated leaves at 8 dai which faded away by 14 dai. Systemic infection never developed at 16 °C in this line.

Table 5 Effect of two different temperatures on symptom development after inoculation of Turnip mosaic virus to two Brassica carinata and four B. napus accessions, breeding lines, and cultivars with uniform phenotypes
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Temperature shift experiment

When samples were tested at 14 and 23 dai, no TuMV was detected in any plants kept at 16 °C throughout or in any plants of B. napus 06-6-3777 regardless of temperature (Table 6). Both at 14 and 23 dai, the virus was detected in plants of B. napus Ag Outback kept at 28 °C throughout, shifted from 16 to 28 °C, shifted from 16 to 28 °C and back to 16 °C, or from 28 to 16 °C, or 28 to 16 °C and back to 28 °C. While, shifting plants from 16 to 28 °C, 16 to 28 °C and back to 16 °C did not induce any symptom development in plants of B. carinata PI 390133, those shifted from 28 to 16 °C developed large obvious chlorotic rings in inoculated leaves and numerous, smaller chlorotic rings in uninoculated leaves (Fig. 2b, c and d). When plants were moved from 28 to 16 °C and then back to 28 °C, the chlorotic rings coalesced to form large irregular yellow patches on leaves (Fig 2e), but TuMV was no longer detected.

Table 6 Effects of temperature shifts on development of Turnip mosaic virus symptoms in plants of one Brassica carinata and two B. napus lines with different uniform phenotypes
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Effect of graft inoculation

By 4 dai, no symptoms developed in axillary shoots of any of the graft-inoculated plants of B napus 06-6-3777, B. carinata ST 18 or ST 50 and no TuMV was detected in them. Axillary shoots developing in the graft-inoculated B. carinata IP 117 plants developed transient faint chlorotic spots, but no TuMV was detected in leaf samples from these plants. No TuMV was detected in control plants as a result of graft inoculation. The extreme resistance was therefore confirmed in all four lines by graft inoculation, the transient chlorotic spots in IP 117 being apparently non-viral in origin.

Effect of pathotype

When inoculated with TuMV isolates NSW-1 or NSW-2, all plants of B. carinata PI 390133 and B. napus Ag Muster, Scoop and 06-6-3777 developed + and/or O phenotypes uniformly which were similar to those recorded previously with isolate WA-Ap1. However, PI 390133 developed mosaic symptoms rather than the systemic chlorotic spots observed previously in this line. In contrast, B. carinata B/C 13 A segregated for RN and +N when inoculated with NSW-1 and NSW-2, both isolates giving 4/7 plants with RN and 3/7 with +N. With isolate WA-Ap1, B/C 13 A developed phenotype RN uniformly. B. napus Flinders also segregated for RN and +N when inoculated with NSW-1, but inoculation with NSW-2 induced a uniform RN response as occurred with WA-Ap1. These segregation results suggest that lines B. carinata B/C 13 A and B. napus Flinders predominantly contain plants giving the RN phenotype with a minority of plants developing the +N phenotype. No clear differences attributable to TuMV pathotype used were evident.

Discussion

This preliminary study provides new knowledge about TuMV resistance phenotypes in B. napus and B. carinata, and the influence of temperature on their expression. It found that in plants growing at low temperatures after TuMV inoculation, phenotype O occurred commonly in a diverse selection of B. napus and B. carinata lines from different continents. However, at high temperature, phenotype O was normally replaced by other phenotypes, or segregated with resistance phenotypes RN, or R, or susceptibility phenotype +. Such segregation is due to outcrossing in Brassica species, known to be up to 30 % in B. napus (Rakow and Woods 1987), which accounts for the common occurrence of heterozygous seed lots of individual lines (Wallace 2007). Phenotype O only occurred uniformly at both low and high temperatures in one Chinese B. napus line and three Tanzanian B. carinata lines. Graft inoculation with TuMV-infected scions confirmed extreme resistance in these four lines. In addition, plants of two Chinese and one French B. napus lines, and six B. carinata lines developed phenotype O predominantly and so are also likely to be useful for breeding programmes. Segregation for resistance phenotypes O, R, RN, or susceptibility phenotype +, occurred more often at higher than lower temperatures. Susceptibility phenotype + rarely occurred uniformly among lines of either species, only one B. carinata and two B. napus lines always being fully susceptible. Although resistance phenotype R developed in plants of some lines of both species at lower temperatures, when plants were grown at higher temperatures this phenotype developed in more lines. In B. carinata lines with phenotype R, TuMV sometimes moved systemically giving a susceptibility phenotype + response, indicating that R was not a true resistance phenotype. Resistance phenoptype +N occurred only rarely in B. napus lines, while resistance phenotype RN/+ developed only at higher temperature in one B. napus line. Inoculation of selected lines with three different TuMV pathotypes did not alter phenotypic responses. This study highlights the need to evaluate germplasm at higher temperatures when identifying new sources of TuMV resistance, especially for Brassica species that rarely produce virus resistant phenotypes consistently as a consequence of cross pollination. While it was demonstrated in previous studies with smaller numbers of lines of less diverse provenance that B. napus (Hughes et al. 2002; Coutts et al. 2007) and B. carinata (Kehoe et al. 2010) lines often contain different TuMV resistance phenotypes, ours is the first study to demonstrate their widespread occurrence in lines of these two Brassica species belonging to germplasm collections originating in many different parts of the world.

Occurrence of resistance phenotypes +N and RN in B. napus lines in this study presumably reflects presence of dominant TuMV resistance genes TURB01 (+N), TURB03 (+N), and TURB04 with TURB05 (RN) all of which are found on its A genome (Walsh 1989; Jenner and Walsh 1996; Walsh and Jenner 2002, 2006). However, since B. carinata lacks the A genome, the RN phenotype found in its lines likely indicates presence of unknown TuMV resistance genes present in its B or C genomes, one of which might resemble the TuMV gene TURB02. TURB02 occurs in the C genome of B. napus and is a quantitative trait locus controlling the degree of susceptibility to TuMV (Walsh et al. 1999; Walsh and Jenner 2002, 2006; Jenner et al. 2002). When additional plants of 18 B. napus and 14 B. carinata lines that developed phenotype O at lower temperature in our initial study were challenged again with isolate WA-Ap1 (three times for B. napus and twice for B. carinata), three B. napus and 10 B. carinata lines developed uniform phenotype O consistently at lower temperatures (16 and 18 °C), but this only occurred with one B. napus (06-6-3777) and three B. carinata (IP 117, ST 18 and ST 50) lines at higher temperatures (25–28 °C). The one exception was a single plant of B. napus 06-6-3777 that developed phenotype RN. However, all the other 78 B. napus 06-6-3777 plants inoculated developed phenotype O, so this one plants’ reaction probably arose due to outcrossing. Moreover, there was no indication that any phenotype O responses resulted from escapes, as plants were always re-inoculated and retested in cases where TuMV symptoms were absent, and no TuMV was ever detected in graft-inoculated plants. Thus, true phenotype O was only expressed in B. napus 06-6-3777 and B. carinata PI 117, ST 18 and ST 50. Resistance gene TURBO1 confers extreme resistance (i.e. phenotype O) to TuMV pathotype 1 in B. napus to which isolate NSW-2 belongs but not to the other two isolates used in this study, WA-Ap1 (pathotype 8) and NSW-1 (pathotype 7) (Walsh 1989; Walsh et al. 1999; Coutts et al. 2007). This suggests that another previously unknown resistance gene is responsible for the phenotype O response we found uniformly expressed in B. napus 06-6-3777 which, if present in the C genome, might also confer the phenotype O response in B. carinata. Currently, no known resistance genes correspond to phenotype R in B. napus, and our study deduced that although this phenotype developed in both species, at 28 °C the virus sometimes moved systemically in B. carinata giving a phenotype + response. This indicates that phenotype R probably reflects failure of TuMV to move systemically at lower temperatures rather than a resistance phenotype. Our study therefore highlights the need identify additional genes responsible for the different resistance and susceptibility reactions found, especially in B. carinata.

There were no previous comparative temperature studies on TuMV resistance phenotype development in B. napus or B. carinata. While Walsh (1989) reported development of phenotype + at 30 °C after inoculation of TuMV isolate UK 1 (pathotype 1) to homozygous B. napus line R7 in which it induced phenotype O at lower temperatures, in our study none of the B. napus or B. carinata lines with phenotype O at lower temperatures developed a uniform phenotype + at 28 °C. However, phenotype + developed in some plants of five B. carinata lines at 28 °C despite our initial germplasm evaluation studies at lower temperature showing only uniform phenotype O, or segregation for O and R. Similarly, Hobbs et al. (2012) found that more virus-resistant soybean accessions infected with Tobacco streak virus developed a susceptible phenotype at high temperature than at low temperature. That we found breakdown of uniform phenotype O at higher temperatures in both species apparently reflects the presence at lower temperatures of localised virus concentrations too low to be detected by ELISA in inoculated leaves expressing phenotypes RN, R or +. The finding that phenotype + is expressed more readily at higher temperatures has implications regarding future global warming scenarios resulting from climate change where B. napus and B. carinata crops continue to be grown in formerly cooler geographic regions (Jones and Barbetti 2012). It suggests that potentially damaging TuMV epidemics are more likely to occur in the future in such regions in cultivars lacking any TuMV resistances, and in cultivars that segregate for TuMV resistance and susceptibility. Although breakdown of temperature sensitive resistance at higher temperatures is often a significant factor influencing expression of virus resistance genes (Kassanis and MacCarthy 1967; Garrett et al. 2006; Fraser 1990; Fraser and Laughlin 1982; Jones and Barbetti 2012), it does not seem to be a factor here, because, as mentioned above, phenotype O remained unaffected at temperatures as high as 28 °C in one B. napus and three B. carinata lines.

In the controlled environment room evaluations involving lines which produced uniform phenotype + at higher temperatures, while B. carinata PI 390133, B. napus Ag Muster and Scoop mainly developed chlorotic spots in inoculated leaves without any systemic invasion at lower temperatures, they induced systemic infection at higher temperatures. This resembles the findings of Llamas-Llamas et al. (1998) in Nicotiana tabacum leaves inoculated with Tomato spotted wilt virus who observed chlorotic concentric ring local lesions at low temperatures but both local and systemic symptoms at high temperatures. Similarly, Schuerger and Hammer (1995) demonstrated systemic infection in pepper (Capsicum annum) plants infected with Tomato mosaic virus when plants were shifted from lower to higher temperatures. In our temperature shift study, a similar observation was made when plants were moved from 16 to 28 °C with B. napus Ag Outback, but not with B. carinata PI 390133. However, although B. carinata PI 390133 plants relocated from 16 to 28 °C developed no systemic infection, those shifted from to 28 to 16 °C developed more severe localised and systemic symptoms than plants maintained constantly at 28 °C. The chlorotic rings that formed were similar to temperature-shift or heat shock-induced viral lesions reported previously by others (e.g. Harrison and Jones 1971; Jones 1973; Foster and Ross 1975; Lozoya-Saldana and Dawson 1982). When kept at 16 °C after initially being held at 28 °C, an increased rate of virus replication and movement within infected leaves may have occurred corresponding to a more efficient redistribution of virus movement protein (Boyko et al. 2000), and hence to increased numbers of infected cells resulting in severe ring symptoms. In contrast, when plants were maintained at 28 °C or moved back from 16 °C to 28 °C, virus replication and movement may not have been stimulated so the plants only developed mild TuMV symptoms that subsequently faded possibly due to heat masking.

When Kehoe et al. (2010) inoculated two B. carinata lines with the same three TuMV isolates used in our study, similar resistance phenotypes developed with all three isolates in both lines. Their findings resembled ours at 22 °C for B. napus Ag Muster and Scoop (phenotype +) and B. napus 06-6-3777 (phenotype O). However, there were minor differences in expression of phenotype + in B. carinata PI 390133: isolate WA-Ap1 (pathotype 8) induced chlorotic spots or rings in both inoculated and non uninoculated leaves, but isolates NSW-1 (pathotype 7) and NSW-2 (pathotype 1) induced occasional chlorotic spots in inoculated leaves and systemic mosaic in uninoculated leaves. Although a few plants of B. carinata BC 13/A and B. napus Flinders segregated for phenotype +N , they developed phenotype RN predominantly so the presence of two different phenotypes was not attributable to pathotype.

The low incidence of TuMV found previously in B. napus crops in Australia (Coutts and Jones 2000) was attributed to widespread occurrence of TuMV resistance phenotypes in Australian B. napus cultivars (Coutts et al. 2007). Such differences were also noted by Sánchez et al. (2007) in Spain where there was a lack of TuMV outbreaks in vegetable and forage Brassica crops compared to significant outbreaks reported in Italy, Greece and France. Our research confirmed the widespread occurrence of TuMV resistance phenotypes in Australian B. napus cultivars. As mentioned in the Introduction, in China, TuMV causes widespread economic losses in Brassicas species (Jiang et al. 2007; Liu et al. 1990; Yoon et al. 1993). Possibly, the Chinese lines we tested, in which we found TuMV resistance phenotypes to be widespread, may not be representative of widely grown Chinese cultivars. Differences in the predominantly occurring TuMV pathotypes between Australia and China might explain its widespread occurrence in China. Further pathotyping of TuMV populations would help to clarify this.

Our study is the first to highlight the inherent dangers in only categorising B. napus and B. carinata resistance phenotypes in lines grown a single glasshouse temperature. It also serves as a reminder of the risks associated with only utilising a single isolate or pathotype for TuMV resistance screening. Moreover, this is the first study to identify uniform phenotype O resistance against TuMV isolate WA-Ap1 (pathotype 8) in B. carinata (lines IP 117, ST 18 and ST 50) and B. napus (line 06-6-3777) through sap inoculation with repeated cycles at both low (16 and 18 °C) and high (25 and 28 °C) temperatures complemented by graft inoculations. It also demonstrated for the first time that in plants developing phenotypes RN, R and +, low temperature retards virus multiplication in inoculated leaves to below the level at which it can be identified using ELISA. Promising lines which displayed phenotype O in >75 % of inoculated plants include three for B. napus (Chinese lines 06-p71-1 and 06-p74-4 and French line Cresor) and six for B. carinata (Mbeya Green, 30200533, P 195923.3, ML-EM-1, ML-EM-7 and ML-EM-8). We also report five promising B. napus lines segregating for hypersensitivity (phenotype RN) especially at higher temperatures (Ding 110, Hyola 42, Fan 028, ZY 007 and Qu 1104). These new resistance sources should be studied under field conditions for their reactions to different TuMV pathotypes to obtain information on their overall durability as TuMV-resistant lines for use as parents by plant breeding programs producing new virus-resistant cultivars of B. napus and B. carinata.